Debasish Kar

Sagartirtha Sarkar Editors

Genetics
Fundamentals
Notes
Genetics Fundamentals Notes
Debasish Kar • Sagartirtha Sarkar
Editors

Genetics Fundamentals
Notes
Editors
Debasish Kar Sagartirtha Sarkar
Department of Biotechnology Department of Zoology
M S Ramaiah University of Applied University of Calcutta
Science Kolkata, West Bengal, India
Bengaluru, Karnataka, India

ISBN 978-981-16-7040-4 ISBN 978-981-16-7041-1 (eBook)

https://doi.org/10.1007/978-981-16-7041-1

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Contents

Part I Classical Genetics

1 Fundamentals of Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Shweta Panchal
2 Mendelian Principle of Inheritance . . . . . . . . . . . . . . . . . . . . . . . . . 53
Dhruti Patwardhan
3 Extension of Mendelism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Rohini Keshava
4 Chromosome Mapping in Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . 165
Rohini Keshava
5 Study of Chromosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Dhruti Patwardhan, S. A. Varshini, and Latha Galoth
6 Genetic Study of Bacteria and Bacteriophage . . . . . . . . . . . . . . . . . 299
Nidhi Sharma

Part II Molecular Genetics I: Analysis of Gene

7 Replication of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
Tanushree Banerjee
8 Chromosomal Organization of DNA . . . . . . . . . . . . . . . . . . . . . . . . 411
Payal Gupta
9 DNA Mutation, Repair, and Recombination . . . . . . . . . . . . . . . . . . 433
Atish Ray
10 RNA Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
Manasa G. Sharma
11 Protein Translation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537
Tanushree Banerjee
12 Regulation of Gene Expression in Prokaryotes . . . . . . . . . . . . . . . . 569
Tanushree Banerjee

v
vi Contents

13 Regulation of Gene Expression in Eukaryotes . . . . . . . . . . . . . . . . 597

Aathmaja Anandhi Rangarajan

Part III Molecular Genetics II: Analysis of Genomes

14 Techniques of Molecular Genetics . . . . . . . . . . . . . . . . . . . . . . . . . 635
Nidhi Sharma and Shrish Tiwari
15 Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 699
Sai Krishna AVS, Sonali Patle, Parampreet Kaur
Shama Omkumar, and Aarti Sharma
16 Application of Molecular Genetics . . . . . . . . . . . . . . . . . . . . . . . . . 761
Dhruti Patwardhan and Nidhi Sharma
17 Genetic Analysis of Development . . . . . . . . . . . . . . . . . . . . . . . . . . 803
Tapodhara Datta Majumdar and Atrayee Dey
18 Molecular Genetics of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 871
Bhawna Chuphal

Part IV Population Genetics

19 Developmental Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 955
Divya Vimal and Khadija Banu
20 Quantitative Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1029
Anindo Chatterjee
21 Population Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077
Payal Gupta
22 Evolutionary Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1105
Ankita Dua and Aeshna Nigam
About the Editors

Debasish Kar is an Assistant Professor in the Department of Biotechnology at

Ramaiah University of Applied Sciences, Bangalore. He pursued his MSc in Bio-
technology from Utkal University, Bhubaneswar, India, followed by an MTech in
Biotechnology from West Bengal University of Technology, Kolkata, India. Later,
he completed his PhD in Biotechnology from IIT Kharagpur, India. Dr. Kar has
several publications in international peer-reviewed journals. Dr. Kar has also
published several books for competitive entrance examinations, viz. CSIR-NET,
GATE, DBT-JRF, and ICMR-JRF, in the domain of life sciences and biotechnology.

Sagartirtha Sarkar is a Professor in the Genetics and Molecular Cardiology

laboratory, Department of Zoology, Calcutta University, India. He completed his
PhD from the University of Calcutta, India. He has worked at the Cleveland Clinic
Foundation, Cleveland, Ohio, USA, as a postdoctoral fellow. Dr. Sarkar has also
served as visiting scientist in the Department of Medicine, University of California,
San Diego, CA, USA. He has more than 40 publications in peer-reviewed journals.
He has supervised 18 doctoral and 5 MPhil students.

vii
 Part I
Classical Genetics
Fundamentals of Genetics
1
Shweta Panchal

Genetics is a dynamic and rapidly advancing field of biology that has found
applications in all dimensions of human life. Breakthrough genetic discoveries are
reported regularly in all major newspapers. Genetic tools are developing at a rate that
has never been witnessed before in history. While understanding genetics and
applying it is mostly driven for human and environmental betterment, it can also
be misused and can raise ethical and moral concerns. For example, first half of the
year 2019 witnessed two important discoveries that were reported by all major
newspapers across the globe. One of the discoveries was genetic modification of a
fungus, Metarhizium, to produce a spider toxin that kills malaria-causing mosquitoes
in large numbers (Lovett et al. 2019). Malaria, being the biggest contributor of
mortality worldwide, seeks rapid ways for disease prevention, especially in
sub-Saharan countries. Studies like these help us to get a step closer to this goal
clearly benefiting human society enormously. The other research news that got
worldwide attention was the announcement of the birth of the world’s first gene-
edited babies by Chinese scientist He Jiankui (Cyranoski 2019). He was condemned
by the scientific community for being irresponsible and reckless. Jiankui used a latest
technology for genome editing called as CRISPR-Cas9 to edit specific genes in
human embryos and allowed the babies to be born. The condemnation is mainly
because scientists still don’t know everything about this technology. CRISPR-Cas9
is based on a mechanism that some bacteria use to defend themselves against viruses
by using an enzyme Cas9. This enzyme can be directed to make cuts in the DNA by
providing a small RNA sequence for your site of interest in the DNA. This technol-
ogy has revolutionized genetic manipulation in research which is exemplified by the
2020 Nobel Prize in Chemistry awarded to the discoverers of this powerful tool,
Emmanuelle Charpentier and Jennifer Doudna. However, it has been shown that
there are off-target effects too, meaning that the enzyme can cut into other sites in the

S. Panchal (*)
Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 3

Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_1
4 S. Panchal

DNA and can potentially inactivate a gene essential for proper functioning of the
cell, like a tumour suppressor gene and thus lead to health problems. So, while most
of the discoveries made in genetics have been extensively useful for humans and the
environment, we are in an age where such scientific challenges need to be addressed
by assembling an international community of experts while taking into consideration
public opinions. It is indeed an interesting era for genetics.
While genetics has been used by civilizations for thousands of years for selective
animal and plant breeding as well as for fermentations used in brewing and baking,
the journey of understanding the underlying mechanisms in genetics begins in
nineteenth century with prominent scientific giants in the field, Gregor Mendel and
Charles Darwin. The understanding of inheritance, genetic changes and their role in
evolution is critical to understanding all life.
Some fundamental concepts as a prelude to the understanding of genetics:

• The tree of life indicates that all organisms can be divided into three domains:
Archaea, Bacteria and Eukarya. Bacteria and Archaea are prokaryotes, meaning
that their cells lack a nuclear membrane and possess no membrane-bound
organelles. All other organisms are eukaryotes belonging to Eukarya which
have more complex cellular organization with membrane-bound organelles like
mitochondria and chloroplasts, as well as membrane-bound nucleus.
• The gene is the basic unit of heredity. A gene is a unit of information in the DNA
that encodes for a functional product and is involved in the expression of a trait or
phenotype or characteristic.
• Genes occur in multiple forms that are called alleles. For example, a gene for the
height of a pea plant can exist as an allele for tall plants or another allele for short
plants.
• Genes confer phenotypes. Genes are inherited, and expression of these genes
along with environmental effects determines the trait or the phenotype. The
genetic information of an organism is called the genotype and the expressed
trait is called the phenotype.
• The macromolecules of the cell that carry genetic information are
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids are
polymers of repeating units called as nucleotides. A nucleotide is made of a
sugar, a phosphate, and a nitrogen base. The nitrogen bases are of four types:
adenine (A), cytosine (C), guanine (G) and thymine (T). In RNA, thymine is
replaced with uracil (U). The sequence of these bases determines the DNA
sequence or the genetic information of an organism. DNA is made of two
complementary double strands of nucleotides.
• Genetic information is transmitted from DNA to mRNA to protein.
• Genes are located on the chromosomes. Large strings of DNA sequence are
compacted in the form of a chromosome with the help of DNA-associated
proteins. Each species possesses a specific number of chromosomes. For exam-
ple, humans have 46 chromosomes, while bacteria possess only one
chromosome.
1 Fundamentals of Genetics 5

• During mitosis and meiosis in the process of cell division, DNA replication takes
place, the chromosomes separate, and the genomic content is divided equally into
two daughter cells. Mitosis occurs in the somatic cells and meiosis occurs in the
sex cells to form gametes.
• Mutations are permanent changes in the sequence of the DNA that can be passed
on the future generations.
• Evolution is a process of genetic changes over a period of time in a population.

1.1 From Mendel Theory to Discovery of DNA

Genetics was being applied by our ancestors even before scientists started thinking
about heredity. Human civilization was possible owing to application of genetics to
domesticate plants and animals. The domesticated dog of today (Canis domesticus)
is a result of years of artificial selection using controlled mating of parents of
ancestral wolves (Canis lupus). Same is true for plants that have been selected for
favourable traits from their wild ancestors. Thus, years of selective plant and animal
breeding had indicated that useful traits can be selected by controlled mating.
However, for centuries, people wondered how traits are transferred from one gener-
ation to the other and why some traits skip generations and appear later. However, it
was not until 1865 that people began to understand the mechanism underlying this
phenomenon, which is largely credited to the painstaking and extensive work of an
Austrian monk Gregor Mendel (Fig. 1.1). Prior to Mendel, two misleading theories

Fig. 1.1 Photograph of Gregor Mendel (left) and his garden (left) as a part of the monastery’s
property in Brno where he conducted seminal experiments that laid the foundation of genetics
6 S. Panchal

of inheritance were proposed. The first concept suggested that one parent contributes
mostly to an offspring’s inherited characteristics. Aristotle believed it to be a male by
proposing a diagram of a fully formed homunculus inside the sperm. The second
concept for inheritance was blending, suggesting mixing of parental characteristics
in the progeny. This was more of an opinion rather than scientific concept, but it fit in
the observations of human and animal features like skin colour, which is often a
combination of both parents. However, blending was never observed in Mendel’s
experiments and both these theories were put to rest by his experiments. In contrast,
by rigorous analysis, Mendel in 1865 discovered that individual traits are determined
by discrete “factors” which we now know as genes, and are inherited from parents.
His precise work on garden pea plants, which has now developed as a field of
biology called as Mendelian genetics, was published in 1866 and went largely
ignored, only to be rediscovered after his death in the 1900s by geneticists like
Hugo de Vries and Thomas Hunt Morgan. Soon, Mendel’s results were confirmed in
a wide range of eukaryotic organisms suggesting that Mendelian principles of
inheritance were a general theme in nature. In 1902, Sir Archibald Garrod became
the first person to utilize Mendel’s laws to explain the basis of inheritance in a human
disease. He, along with a strong Mendel advocate, William Bateson, established that
alkaptonuria was a recessive genetic disorder. This study paved the way for several
future milestones with scientists providing evidences for molecular basis of inheri-
tance in human diseases. The chromosome theory of inheritance by William Sutton
and Theodor Boveri reaffirmed Mendel’s laws of inheritance. In 1920, Thomas
Morgan isolated the first genetic mutant of fruit flies and went on to use the fruit
fly model to dissect and discover several details of classical genetics. Ronald
A. Fisher, John B.S. Haldane and Sewall Wright established the field of population
genetics in the 1930s by combining Mendelian genetics and evolutionary theory.
Use of bacteria and viruses as simple genetic systems led to detailed study of the
structure and function of genes. Elegant, breakthrough experiments by Frederick
Griffith, Oswald Avery, Colin MacLeod, Maclyn McCarty, Alfred Hershey and
Martha Chase proved that DNA is the molecule of heredity. Seminal papers
published in the year 1953 by Rosalind Franklin, Maurice Wilkins, James Watson
and Francis Crick established the three-dimensional double helical structure of DNA
which ushered the field into the era of molecular genetics. Advances in techniques
like recombinant DNA, gene cloning, polymerase chain reaction (PCR) and Sanger
sequencing were a huge impetus to molecular genetics. This paved the way for
development of gene therapy and launch of Human Genome Project leading to a new
wave of genomics. Today, hundreds of genomes are being sequenced on a daily
basis owing to immense improvement in sequencing technologies and analysis
platforms. All this started with Mendel’s detailed and robust experiments over a
period of 8 years that paved the way for the golden era of research breakthroughs in
life sciences and hence he is rightly known as the Father of Genetics.
1 Fundamentals of Genetics 7

1.1.1 Mendel’s Work

Mendel served at an Augustinian monastery in the city of Brünn, Austria (now in

Czech Republic), where natural sciences were taught. There, he embarked on a
research project on plant hybridization in the monastery’s own garden where he
grew thousands of pea plants and provided the basis of genetics. Students should
appreciate the meticulous scientific approach he used that resulted in robust data. He
chose a plant which could be studied for the hypothesis he proposed. In addition, he
designed the experiments carefully, and collected large amount of data, which was
subjected to mathematical analysis and was highly reproducible. Mendel chose the
humble pea plant, Pisum sativum, mainly for two reasons. First, peas had distinct
observable traits that could be easily identified, and second, peas can both self-
pollinate (pollen from the same flower) and be cross-pollinated (pollen from differ-
ent flower from different plant), thus allowing the experimenter to generate specific
pure lines as well as hybrid lines (Fig. 1.2). In addition, the plants were inexpensive
and could produce large number of offspring. Using these pea plants, Mendel
conducted experiments for about 8 years and consequently proposed the principles

Stigma
Anther

Stamen
Filament

Pollen Anthers
transfer removed

Fig. 1.2 Mendel’s experimental model—the garden pea plant. Anatomy of the pea flower is shown
(upper cartoon) indicating the male and female parts of the flower. The pollen is produced in the
anthers, which land on the stigma, which contains the ovary, which later becomes the pea pod with
the progeny (seeds). Lower cartoon: method for cross-pollinating pea plants is shown. To prevent
self-fertilization, the anthers are removed from the female parent. Pollen from another plant is
transferred to the stigma of this female plant using a paint brush. Each fertilized egg or ovule
becomes an individual seed that can grow into a new pea plant. (Image credit: University of
Waikato, www.biotechlearn.org.nz)
8 S. Panchal

of inheritance. So whether it is pea plants or humans, genetic traits that follow these
principles are known to be following Mendelian inheritance.

1.1.2 Mendelian Theory of Inheritance

Mendel studied seven different traits in the pea plant, each of which had two
alternate forms. These traits were height (tall or short), seed colour (green or yellow)
and seed shape (smooth or wrinkled), among others (Table 1.1). These were “either-
or” traits, meaning there were no intermediate forms. This allowed the tracking of the
traits in the subsequent generations by simple observations. Such traits are identified
as discrete, as compared to continuous traits like skin colour in humans that show
several intermediate forms. He collected lines with these discrete features and let
them self-pollinate for several generations until he could confirm that they were pure
breeding lines, meaning tall plants always produced tall progeny for several
generations. These were the parental (P) generation. He then crossed these pure
breeding lines of alternate forms and meticulously counted the hybrid plants to
observe how the trait was inherited. These were monohybrid crosses, meaning
mating was carried out between individuals that differ in only one trait. He carefully
controlled the mating by making sure that no foreign pollen landed on the stigma of
the flowers he chose. He found striking patterns of inheritance for all the traits

Table 1.1 Results of Mendel’s garden pea hybridization experiments. Mendel’s experiments with
seven pea plant traits are shown here, with the results obtained for the F1 and F2 generations.
[Adapted from “The results of Mendel’s garden pea hybridizations” by OpenStax College, Biology
(CC BY 3.0)]
F1 offspring traits F2 offspring F2 trait
Characteristic Contrasting P0 traits (dominant trait) traits ratios
Flower Violet vs. white 100% violet 705 violet 3.15:1
colour 224 white
Flower Axial vs. terminal 100% axial 651 axial 3.14:1
position 207
terminal
Plant height Tall vs. dwarf 100% tall 787 tall 2.84:1
277 dwarf
Seed texture Round vs. wrinkled 100% round 5474 round 2.96:1
1850
wrinkled
Seed colour Yellow vs. green 100% yellow 6022 yellow 3.01:1
2001 green
Pea pod Inflated vs. constricted 100% inflated 882 inflated 2.95:1
texture 299
constricted
Pea pod Green vs. yellow 100% green 428 green 2.82:1
colour 152 yellow
1 Fundamentals of Genetics 9

Fig. 1.3 Mendel’s monohybrid cross. Cross-breeding of pure lines having yellow and green seeds
(P generation) gives rise to plants with only yellow seeds (F1 generation). Self-pollination of
F1 plants gives rise to F2 generation with individuals resembling the parent generation in the
ratio of 3:1

individually studied. On crossing two pure bred lines of alternate traits, for example,
plants with yellow versus green seeds, he observed that the first generation (F1; first
filial generation) always had plants with yellow seeds. He called this trait as the
dominant trait and the trait with green seeds as recessive, which was “hidden” in this
generation. This recessive trait reappeared in the F2 generation when the F1 plants
were allowed to self-pollinate, although only in minority. However, the ratio of
plants with yellow to green seeds observed in this generation was always 3:1
(Fig. 1.3).
Mendel’s actual counts were 6022 yellow:2001 green seeds in this generation
(3.01:1 ratio). This ratio was observed for all the traits that he studied (Table 1.1).
These observations clearly refuted the theory of blending inheritance. Mendel was
also able to perform reciprocal crosses, in which he studied whether a particular trait
was transmitted via egg or sperm by reversing the traits of male and female parents.
For example, he could use pollen from a plant with green seeds to fertilize the eggs of
a plant with yellow seeds and vice versa. He observed that the progeny of these
crosses was always similar in both cases, thus proving that the inherited factors are
contributed by both parents equally.
He studied nearly 30,000 pea plants which provided robust statistical merit, and
he communicated his results in 1865 to the scientific community. Thus, he
concluded that traits like plant height and seed colour are controlled by a pair of
10 S. Panchal

Fig. 1.4 Monohybrid

cross (“Figure 12 02 02” By
CNX OpenStax – (CC BY
4.0) via Commons
Wikimedia). In the P
generation, true-breeding pea
plants (homozygous) for the
dominant phenotype of
yellow seeds and the recessive
green seed phenotype are
crossed. This cross produces
F1 heterozygote generation
with all individuals having
yellow seeds. Hence, yellow
is the dominant trait here. On
self-pollination, F2 generation
produces has a mix of yellow
and green seed individuals, in
the ratio of 3:1. Punnett square
analysis can be used to predict
the genotypes of the F2
generation as shown in
the box

heritable factors, which we now know as genes, received one from each parent.
These genes have two alternate forms, known as alleles. For example, plant height
has two alleles, tall and short alleles. The set of alleles carried by an organism is
known as its genotype. The genotype determines the phenotype, which is the
observable trait or feature, in this case, height of the plant. The individual is said
to be homozygous when the two alleles for a trait are identical (AA or aa) and
heterozygous if the alleles are different (Aa). Dominant and recessive alleles are
designated by capital and small letters, respectively. In heterozygous situation, the
dominant allele can mask the effect of the recessive allele. These two alleles
segregate randomly during gamete formation such that each gamete (sperm or
egg) randomly receives just one form. A Punnett square provides a simple method
for tracing the gametes produced and determining the possible genotypes of the
progeny (Fig. 1.4). Thus, according to the law of segregation, during meiosis, the
two alleles for specific traits segregate randomly in individual gametes (egg or
sperm) such that each gamete carries only one allele. During fertilization, these
1 Fundamentals of Genetics 11

alleles unite and the genotype of the progeny is determined depending on the alleles
received from the two parent gametes.
While monohybrid crosses established the law of segregation, Mendel had
devised experiments involving dihybrid crosses to study the inheritance of two or
more unrelated traits. Mendel questioned that if you have a dihybrid plant, which is
heterozygous for two genes at the same time, how those alleles would segregate. He
created a dihybrid by mating a pure breeding line of two alleles with a pure breeding
line of the alternate forms of the alleles, for example, breeding plant with yellow
round peas (YYRR) with green wrinkled peas (yyrr). The dihybrid of F1 would have
the genotype of YyRr displaying the dominant phenotype of yellow round peas.
When Mendel allowed these dihybrids to self-fertilize, he found that plants of all
combinations were produced, including parental type of yellow round and green
wrinkled as well as new recombinant phenotypes of yellow wrinkled and green
round. Mendel suggested that this was possible because the genes for both the traits
assort independently. Thus, if a gamete has Y, it has equal probability of having R or
r, and so a gamete can have four types of genotypes, viz. YR, yr, Yr or yR. Thus, on
fertilization, any four kinds of eggs can be fertilized by any four kinds of sperms,
thus leading to 16 possible outcomes for the zygote genotype. Four types of
phenotypes can observed from these 16 combinations in a ratio of 9:3:3:1, as
shown in Fig. 1.5. This is the basis for law of independent assortment, which states

Fig. 1.5 Dihybrid cross (“Figure 12 03 02” By CNX OpenStax – (CC BY 4.0) via Commons
Wikimedia). Pure breeding parent for two traits are crossed with pure breeding lines with recessive
phenotype. F1 generation has heterozygous plants with the dominant phenotype for both traits, and
F2 generation has plants with all combinations in the ratio of 9:3:3:1
12 S. Panchal

that each pair of alleles segregates independently of other allele pairs during gamete
production and subsequent fertilization from random union of gametes determines
the observed phenotype. Hence, the inheritance of seed colour did not influence the
inheritance of seed texture. This was held true for all the seven traits he studied,
which gave the same phenotype ratios during dihybrid crosses.

1.1.3 Genetic Variation

Unlike the pea plant traits that Mendel examined, most traits, like skin colour, that
we observe do not fall into the “either-or” category, meaning they have more than
two forms. Some traits do not display clear-cut dominance or recessiveness, while
some traits are multifactorial. Such traits do not follow the Mendelian ratios. These
extensions to Mendelian inheritance can be thus divided into two categories, viz.
single-gene inheritance and multigene inheritance.
Single-gene inheritance:

1. Incomplete dominance: In nature, sometimes a mixed or intermediate phenotype

is observed in the presence of both the alleles, indicating that neither displays
dominance to generate a phenotype. This was never encountered in Mendel’s
work because in a heterozygous situation, the dominant allele determined the
phenotype. A typical example of incomplete dominance is the cross between a red
snapdragon and a white snapdragon plant. According to Mendel’s laws, the
offspring flower should be either red or white. But in reality, the flowers are all
pink suggesting that neither allele is completely dominant.
2. Multiple alleles: For a given gene, it is possible to have more than two alleles that
determine a particular trait within a given population. For example, human blood
types are A, B, AB and O. Combinations of three possible alleles give rise to the
four blood types. While one individual can have only two alleles for the gene, in
the population all four alleles are present which result in the different blood types
observed. This results in genetic variation within the population.
3. Codominance: Two alleles may be simultaneously expressed with both alleles
determining the phenotype.
4. Pleiotropy: In some cases, one gene may contribute to several observable
phenotypes. For example, many men of the Maori tribe of New Zealand develop
frequent respiratory problems and are also sterile. It was discovered that the
problem occurs in an instance when a defective protein is produced by one
gene necessary for the action of cilia and flagella. Men who are homozygous
recessive for this particular gene have abnormally functional cilia for respiratory
functions and abnormal flagella for sperm motion. Thus, one gene directly affects
two separate functions in an individual.
5. Lethal alleles: In Mendel’s experiments, all allelic combinations produced viable
progeny. However, that is not the case for certain genes where homozygosity or
heterozygosity affects the survival of the individual. Individuals homozygous for
sickle cell anaemia allele die in childhood or early adulthood due to problems in
the circulatory system.
1 Fundamentals of Genetics 13

Multigene inheritance: Several variations from Mendel’s laws involve

interactions between two and more genes to give rise to specific trait. In some
cases, one gene’s alleles can mask the effects of other gene’s alleles, known as
epistasis. In addition, several genes that lie physically close to each other on the
chromosome and are genetically linked do not assort independently. Such genes are
called linked genes and they do not follow Mendel’s law of independent assortment
and are expressed together as a cluster. In humans, the autosomal genes for red hair,
freckles and a fair complexion are linked genes. Many characteristics like skin
colour, human height and several diseases are controlled by multiple genes, several
times in combination with environmental factors.

1.2 Model Organisms

Some organisms have been used as model organisms for several years to help
scientists understand complex biological processes that cannot be easily studied in
the organism of interest. Model organisms can be bacteria, fungi, rodents or plants.
Use of humans in biomedical sciences is limited due to practical and ethical issues,
and hence most of the understanding in cell biology and molecular biology is due to
studies done in model organisms.
The important criteria that define model organisms include rapid growth and short
generation times, high reproduction rates, ease of genetic manipulation, production
of large number of offspring and ease of maintenance in standard laboratory
conditions. Over the years, model organisms have been instrumental in helping
scientists understand complex biological processes to discover the fundamental
causes of a disease, production of better varieties of crops, understanding drug
resistance in pathogens and so on. Since all organisms have a common point of
origin in evolution, the basic molecular mechanisms and processes are the same, and
hence studies in model organisms can be extrapolated to the organism of interest. For
example, research in the single-celled eukaryote Saccharomyces cerevisiae has led
to deep understanding of the cell cycle and has helped scientist develop drugs to
target cell cycle of tumour cells. Studies with model organisms stand strong even in
this era of next-generation sequencing where hundreds of genomes are sequenced
daily. Complex processes of behaviour, disease and pathology require the need for a
simplistic reductionist approach that is only possible in model organisms.

1.2.1 Escherichia coli

This model prokaryote is a Gram-negative, rod-shaped bacterium with size approxi-

mately 1 μm in length to 0.35 μm in width (Fig. 1.6), and sizes vary only slightly
between strains. This bacterium was discovered by the German paediatrician and
bacteriologist Theodor Escherich in the 1800s. He was investigating the causative
agents for diarrhoea in babies which led to the identification of this bacterium in the
lower intestine of humans, which he called Bacillus communis coli. It was later
14 S. Panchal

Fig. 1.6 Scanning electron

micrograph of E. coli, grown
in culture and adhered to a
cover slip. Credit: Rocky
Mountain Laboratories,
NIAID, NIH

named Escherichia coli in his honour in 1958. Several strains of E. coli are normal
inhabitants of the lower gastrointestinal tract of humans and other warm-blooded
animals. While most strains are non-pathogenic, some strains like E. coli O157:H7
are infamous for causing food poisoning and bloody diarrhoea, which can be fatal.
However, most strains of E. coli are classified as biosafety level 1, which makes this
organism useful for teaching and demonstrations in undergraduate and high school
levels. This bacterium belongs to a large family of Gram-negative bacteria called as
Enterobacteriaceae which also includes some well-known pathogens like Salmo-
nella, Shigella, Yersinia and Klebsiella.
E. coli has been considered as a molecular biologist’s toolbox or a workhorse of
molecular biology owing to its amazing properties. This bacterium can grow in
minimal media with only one carbon source and in rich media where it can divide
every 20 min. This incredible fast generation time makes it suitable to study rare
genetic events in a relative short time. Fast generation time makes it possible to scale
up the culture volume to industrial production making it extremely useful for
biotechnological applications. E. coli is a facultative aerobe, which means it can
grow in the presence as well as absence of oxygen. There is extensive knowledge
about its genome, transcriptome, proteome as well as metabolome. The ~4.6 Mb
genome of the bacterium is arranged in a closed circular double-stranded DNA.
During bacterial cell division, the circular DNA undergoes replication, and then the
cell undergoes binary fission such that each daughter cell receives one copy of the
DNA. This entire process is highly regulated to ensure correct replication and
segregation of the DNA. Since the haploid bacterial genome consists of one chro-
mosomal DNA, a genetic mutation will directly lead to expression of a phenotype
since a second, wild-type allele is absent. This makes understanding gene functions
very straightforward. The phenotypes that can be observed for screening genetic
mutations in E. coli are changes in colony morphology, resistance to antibacterials or
bacteriophages, auxotrophic mutants and conditional temperature-sensitive
(ts) mutations for essential genes. Hundreds of studies done using E. coli have led
1 Fundamentals of Genetics 15

to amazing discoveries, several of which have been awarded the Nobel Prize. This
includes work of Joshua Lederberg for his discoveries of genetic recombination and
the organization of the genetic material of bacteria; Francois Jacob, André Lwoff and
Jacques Monod for their discoveries on the genetic control of enzyme and virus
synthesis; Max Delbruck, Alfred Hershey and Salvador Luria for their discoveries
on the replication mechanism and the genetic structure of viruses; Arthur Kornberg
for processes of DNA replication; and Jacob and Monod for gene regulation, among
many others. This organism has been critical for development of recombinant DNA
technology. Genetic engineering of E. coli plasmids was achieved to harbour and
amplify transgenes or for transfer of the gene to another organism. Due to fast
generation times, genetically engineered E. coli culture can be scaled up to large
volumes and grown in bioreactors for production of useful compounds like insulin,
growth hormones and drugs.

1.2.2 Saccharomyces cerevisiae

Yeasts have been exploited for thousands of years for the purpose or brewing and
baking. Initially, the impetus to research on yeasts was mainly provided by its
industrial applications. In modern science, budding yeast has been used extensively
in laboratories and has provided understanding of several molecular and genetics
questions in eukaryotic biology. Saccharomyces cerevisiae or baker’s yeast is a
single-celled eukaryote with short generation time and with a simple life cycle,
which alternates between haploid and diploid phases. Like all eukaryotic cells, yeast
cells also regulate cellular processes to determine the fate of the cell in a particular
condition. The molecular basis of cellular processes in yeasts can be easily extended
to multicellular eukaryotes. Yeast cells express and regulate genes, perform
biological functions and differentiate using processes similar to the cells of multicel-
lular organisms. However, unlike multicellular organisms, yeasts have experimental
advantages like fast growth rate and ability to survive as haploids and diploid, thus
making functional characterization of genes and pathways easier and faster.
S. cerevisiae is indeed the first eukaryotic organism sequenced (1996), and genome
analysis has indicated that it has more than 30% protein-coding genes homologous
to humans. In addition, this budding yeast has also been utilized as a model to study
drug resistance in pathogenic fungi, a concern that is growing at an alarming rate
worldwide.
S. cerevisiae contains membrane-bound organelles like nucleus, mitochondria
and endoplasmic reticulum. Yeast cells divide by budding in which the daughter bud
pinches off the mother cell (Fig. 1.7). Yeast cells can divide once every 90 min under
optimal laboratory conditions. Even though they are eukaryotes, yeast cells can be
cultured like bacteria in agar plates and liquid growth media and can be stored for
years at 80  C by freezing in glycerol. They are inexpensive to grow and easy to
maintain with hundreds of mutants that can be obtained for screening. One of the
most widely used experimental strains of S. cerevisiae is S288C. This strain, isolated
by Robert Mortimer, is now a standard laboratory strain that has been widely used as
16 S. Panchal

Fig. 1.7 Scanning electron

micrograph of S. cerevisiae
cells (Murtey and Ramasamy,
2016). Buds and bud scars can
be seen here

the parental strain for the isolation of biochemical mutants, which continues even
today in laboratories around the world.
The yeast life cycle is simple with alternating haploid (n) and diploid (2n) stages
(Fig. 1.8). Haploid cells occur in two mating types, a and α. Both mating types can
divide asexually by mitosis wherein with each round of cell cycle, a cell produces a
daughter bud which pinches off from the mother bud at the end of cytokinesis, and
thus maintain as stable haploids. However, opposite mating types can also engage in
sexual reproduction, in which a and α cells communicate via pheromones that
induce fusion of the cell and then the nucleus to give a diploid progeny. The diploid
cell can divide mitotically and survive as a diploid. But in certain conditions like
starvation, diploids undergo meiosis and sporulation to form an ascus with four
haploid spores. The mating type is determined by mating type locus (MAT) present
on chromosome III.
The cell cycle of S. cerevisiae has been a subject of intensive research over
several decades and has provided valuable insight into human cell cycle and diseases
related to it. It is easy to visualize different stages of yeast cell cycle by simple light
microscopy by observing the yeast cell bud size. Unbudded cells are in G1 stage,
cells with small buds are in S phase, and cells with large buds are in G2/M (Fig. 1.9).
Scoring budding index has been a powerful tool in characterizing the changes in cell
cycle due to changes in external or internal variables.
In 2001, three scientists, Leland Hartwell, Paul Nurse and Tim Hunt, shared the
Nobel Prize for their independent studies on cell cycle regulation in yeast and
humans. Specifically, Leland Hartwell uncovered the genetic basis of cell division
in S. cerevisiae and contributed significantly on our understanding of the eukaryotic
cell cycle with broad implications for human health and prevention and treatment of
diseases like cancer. Several other discoveries using yeast genetics have been
awarded prestigious prizes including the Nobel Prize. One such recent example is
the 2016 Nobel Prize given to Yoshinori Ohsumi for his work on autophagy in
budding yeast, which was used to identify several homologous autophagy genes in
mammalian cells.
1 Fundamentals of Genetics 17

Fig. 1.8 Representative life cycle of S. cerevisiae (Duina et al. 2014). Haploid yeast cells (a cell or
α cell) undergo mitotic cell division through budding to produce daughter cells. The two cell types
(a cell and α cell) release pheromones, initiating the formation of schmoos and subsequent mating,
which leads to the formation of a stable diploid (a/α cell). Diploid cells also divide mitotically by
budding to produce genetically identical daughter cells. Under starvation conditions, diploids are
induced to undergo meiosis, forming four haploid spores, which can germinate into two a cells and
two α cells

Fig. 1.9 Cell cycle of S. cerevisiae. (adapted from Hanson 2018) During asexual reproduction, cell
cycle stage can be observed based on bud size as indicated here
18 S. Panchal

Haploid S. cerevisiae genome has a size of around 12 Mb packed in 16 linear

chromosomes in the nucleus which encode around 6000 polypeptides. The genome
has relatively low number of intron-containing genes in S. cerevisiae. Due to this, the
gene density of protein-coding genes is much higher than the gene density in human
genome.
Several techniques have been developed over the years which make S. cerevisiae
easy to genetically manipulate. Transformation of DNA in budding yeast is highly
efficient. A plasmid constructed with a marker and a functional yeast gene of interest
can be readily taken up by the cells from the external environment. Two kinds of
plasmids can be transformed in yeast: Yeast integrative plasmids (Yips) are circular
plasmids that are unable to replicate on their own and integrate readily at a region of
homology by genetic recombination. Autonomously replicating plasmids can be
yeast episomal plasmids (YEps) and yeast centromeric plasmids (YCps). These can
exist independently in the cells and are useful as shuttle vectors to carry genes from
other organisms and be replicated in yeast.
Yeast cells can be transformed with engineered DNA that will readily integrate
into homologous region of interest for construction of deletion mutants, epitope
tagging or any other genetic modification. Only a homology region of 40 bp is
enough for efficient integration. This homologous recombination-based technology
is one of the most useful tools that made S. cerevisiae an ideal model organism.
Owing to the ease of genetic manipulation, entire libraries have been constructed and
are available in depositories for research purposes. Examples are deletion library
(collection of deletion mutants of non-essential genes), GFP clone library (collection
of more than 4000 genes tagged with GFP for microscopy-based studies),
TAP-tagged ORF library (more than 4000 genes tagged with TAP for protein
purification studies) and several others. Important technological innovations in
yeast have been widely useful for study of several fields of biology. Techniques
like yeast two-hybrid screening for study of protein-protein interactions have led to
significant discoveries before the era of next-generation sequencing.

1.2.3 Drosophila melanogaster

The common fruit fly, Drosophila melanogaster, has been used extensively as a
model organism for over a century and continues to remain a useful system for
studying genetics and cell biology. It has proved to be an ideal organism to study
human genetic diseases, animal behaviour, development and neurobiology, evolu-
tion and pathogenesis. Rapid life cycle, small chromosome number and genome size,
giant salivary chromosomes, ease of maintenance and genetic manipulation are the
important experimental advantages that make it an ideal model organism.
Drosophila genetics mainly began in the lab of Thomas Hunt Morgan in the early
1900s, in what was famously called as “fly-room” (Fig. 1.10). Several crucial and
historic experiments were performed in the fly-lab that laid the foundation of modern
fly genetics. The sex-linked white eye mutation was discovered by Morgan in 1910.
A PhD student of Morgan, Calvin Bridges, proved the chromosome theory of
1 Fundamentals of Genetics 19

Fig. 1.10 Image of an adult D. melanogaster (a) (Jennings 2011) and Morgan’s “fly-room” at
Columbia University (Allocca et al. 2018) (b)

inheritance by showing that the nondisjunction of the sex-linked white eye gene
correlates with the nondisjunction of the X chromosome. An undergraduate student
in Morgan’s lab, Alfred H. Sturtevant generated the first chromosome map by
calculating the recombination frequencies. The mutagenicity of X-rays was
demonstrated by Herman J. Muller. Several other discoveries continued to be
made in the fly-room that added to the knowledge of transmission genetics. Indeed
several Nobel prizes have been awarded to studies in Drosophila biology, with the
most recent one awarded in 2017 to Jeffrey C. Hall, Micheal Rosbash and Michael
W. Young for their discoveries of the molecular mechanisms controlling circadian
rhythm.
A crucial factor for being a model organism is having similarity of cellular
mechanisms with humans. D. melanogaster has homologues of approximately
75% genes involved in human diseases. Study of these genes can be extrapolated
to humans, thus bypassing the ethical issues of biomedical research involving human
subjects. Drosophila genome is about 137 Mb arranged in four pairs of
chromosomes (one pair of sex chromosomes and three pairs of autosomes) with
around 15,500 genes. Chromosome 1 is the X chromosome and chromosomes 2–4
are autosomes. The X chromosome is large and acrocentric, chromosomes 2 and
3 are large and metacentric, while chromosome 4 is a tiny acrocentric “dot”
chromosome. A fly is a female if it has two X chromosomes, whereas an X and Y
will designate a male. The small chromosome number and small genome size as
compared to humans make it ideal for genetic studies by simplifying genetic
manipulations. In addition, the fruit fly’s salivary glands possess giant polytene
chromosomes, features of which can be easily viewed through a light microscope by
adding a chemical dye that gives unique banding patterns. These large chromosomes
were the key tools for development of fly genetics and are still used today.
D. melanogaster is also very easy to grow and maintain and has a very short life
cycle of 10 days. The fruit flies are regularly grown on a corn meal and sugar
medium at room temperature. The reproduction rates are also very high, with several
20 S. Panchal

Fig. 1.11 Life cycle of D. melanogaster (Hales et al. 2015). D. melanogaster are cultured in glass
vials closed using a cotton plug and containing food. The vial in the picture contains flies at different
stages of growth. Depending on the growth stage, the organism can be in the food area or on the
walls of the vial. The life cycle is completed within 9–10 days in laboratory conditions

hundred progeny produced on each mating. A female can produce around 3000
progeny in her lifetime and a male can sire over 10,000 offspring. The eggs laid by a
female are about half millimetre long and can self-sustain embryonic development,
which usually takes 24 h to complete. Embryogenesis ends with formation of first
instar larva that grows in body size and moults to produce second and third instar
larvae. After the third instar larva completes its growth, it crawls out of the food and
pupates. It undergoes metamorphosis inside the protective pupal case. The metamor-
phosis involves radical change in the fly body plan where most adult structures like
wings, eyes and genitalia develop. After the pupal development is complete, adult
flies emerge out of the case in a process called as eclosion and become sexually
mature in 8–12 h, thus repeating the life cycle (Fig. 1.11).
A range of genetic tools are available for genetic manipulations in Drosophila. A
number of resources for the research community including stock centres and pub-
licly available genome databases are available for Drosophila like FlyBase. In
Drosophila, crossing-over occurs only on females making it possible to maintain
gene linkage through male inheritance. This simplifies genetic manipulations and
1 Fundamentals of Genetics 21

allows for a variety of genetic screens. Balancer chromosomes created by X-ray

mutagenesis have multiple overlapping inversions, which are useful for stock-
keeping of lethal alleles. Transgenes can be easily made in this organism using P
element transposons, homologous recombination and more recently CRISPR/Cas9.
Indeed, thanks to the abundance of genetic manipulation tools and publicly available
fly stocks and databases, Drosophila will continue to be used as a key model
organism for understanding the molecular basis of human diseases.

1.2.4 Arabidopsis thaliana

All of science, whether curiosity based or having direct application, is ultimately

done for the benefit of human society and the environment we live in. Despite this
understanding, model organisms appear out of obscurity leaving one wondering
about its usefulness. One such model organism is the small stature plant, Arabidopsis
thaliana, which surpasses all the agriculturally important plants like rice, wheat and
beans in terms of the extensive research done. A small angiosperm, A. thaliana, is a
weed and member of the Brassicaceae (mustard) family of plants which includes
economically important species like cabbage and radish. Even though it has little
direct usefulness to the human society, it has paved the way for breakthrough
research in plant molecular biology and genetics. This plant was first described by
Johannes Thal and he called it Pilosella siliquata. The name was later changed to
Arabidopsis thaliana in his honour by Linnaeus. The credit for bringing this plant to
genetic research limelight is given to Friedrich Laibach, who established its chro-
mosome number during his PhD in 1907 and made seminal contribution to establish
Arabidopsis as a fundamental model for plant genetics research by 1943.
A. thaliana, commonly known as thale cress, has a wide geographical distribution
with different ecotypes growing in Europe, Asia and Africa. The Arabidopsis seed
stock centres like Arabidopsis Biological Research Centre have been established to
store seeds of different ecotypes (accessions) from natural populations to be made
available for scientific research. The Columbia and Landsberg ecotypes are used
widely as standards in genetic and molecular biology research in Arabidopsis.
The entire life cycle, starting from seed germination to maturation to produce
seeds, is completed in around 6 weeks (Fig. 1.12). This is a significant advantage for
using this plant when compared to crops and trees with much longer lifespan. The
size of this plant is also small, with fully mature plant reaching about 15–20 cm in
height and flowers which are about 2 mm in size. On seed germination, the seedling
develops into a rosette plant which on maturation gives rise to an inflorescence stalk
which produces flowers. The flowers can self-pollinate as the bud opens, but can also
be cross-pollinated. The seeds from the flowers are produced in encases called as
siliques. Often a single plant can produce more than 4000 seeds.
Due to this short and simple life cycle and high reproduction rates, genetic
manipulation and isolation of large number of mutants is possible. Hundreds of
small seeds can be cultivated in petri dishes for germination, and the small stature of
the adult plant makes it easy to be grown in growth chambers or greenhouses in large
22 S. Panchal

Fig. 1.12 Images of Arabidopsis life cycle (Woodward and Bartel, 2018). The small seed of the
plant germinates with the radicle seen within 3 days to give rise to a seedling. The seed can be
germinated till the seedling stage in petri dish with soft agar and growth medium. The seedling is
then transferred to pots with soil and fertilizer. The seedling grows under fluorescent light to form a
rosette plant with 25–28 days giving rise to stalks of seed pods called as siliques

numbers. Unlike several other plants, A. thaliana can grow indoors under weak
fluorescent light and does not require co-culture with symbiotic organisms allowing
growth in aseptic conditions with maximal control of biotic and abiotic factors. It is
therefore not surprising to see hundreds of variables being tested individually and
together to study the role of biotic and abiotic environment in plant physiology, plant
defence and ecology. A. thaliana has a diploid genome of about 135 Mb with a
haploid chromosome number of five. This is a very small genome as compared to
other plant genomes like maize (2500 Mb) and rice (430 Mb) which are difficult to
1 Fundamentals of Genetics 23

genetically manipulate. Whole-genome sequencing of Arabidopsis was completed in

2000 which was followed by availability of other resources like Affymetrix
microarrays, identification of mutants through forward genetics and availability of
precise insertion mutants of nearly all genes through reverse genetics. This led to a
humungous rise in the numbers of papers published improving the pace of
Arabidopsis research drastically. A useful approach in Arabidopsis research has
been the use of reverse genetics to generate thousands of modified T-DNA inser-
tional mutants. This is possible due development of simple method of transformation
using genetically engineered Agrobacterium tumefaciens culture. The transferred
DNA (T-DNA) is inserted into the plant genome by Agrobacterium naturally
resulting in insertion of T-DNA in precise locations as designed. This method has
added greatly to its use in molecular biology and led to amazing breakthroughs.
Most importantly, the studies in A. thaliana can be extrapolated in other agricultur-
ally and economically important plants like tomato, soybean, rice and so
on. Research advancements in understanding plant hormone and defence pathways,
circadian rhythm and environmental response have led to many applications in crops
and other plants useful for humans. One important area of research which is highly
relevant with increased need for food security is disease resistance. Plants are
exposed to all kinds of pathogens, from bacteria, fungi, viruses and even nematodes.
Developing plants that are genetically resistant to its pathogen is a major goal of
most plant-breeding programmes. Research in Arabidopsis had led to identification
of genes and genetic pathways important for plant immunity and disease resistance.
These studies are useful for generating plants that have genetic resistance to
pathogens, thus improving crop yield and output.

1.2.5 Mus musculus

When Mendel’s laws were rediscovered in 1900, many in the scientific community
questioned if using the pea plant and Mendel’s laws of inheritance was enough to
study inheritance of traits in humans. Soon the need to have a mammalian model for
biomedical research became obvious. It was required that like other model
organisms, the ideal mammal for genetic studies should be able to breed quickly
producing large number of offspring and should display many easily scored, variable
traits and that could be housed in large numbers in a laboratory space. These
attributes were found in the common house mouse, Mus musculus. The most
advantageous feature of this model organism for genetic analysis is the availability
of hundreds of single-gene mutations. These mutations have arisen during its course
of evolution for domestication during the “fancy-mouse” trade where mice were
bred for different fur coats and other visible phenotypes. Early on researchers made
use of these single-gene mutations to explain Mendel’s laws and proved that they
can be extrapolated to humans as well. One such scientist that used these trade mice
was Clarence Cook Little, who became the father of modern lab mouse. He founded
the Jackson Laboratory where he mated closely related mice for generations, creat-
ing the first inbred strains (Fig. 1.13).
24 S. Panchal

Fig. 1.13 The common house mouse, Mus musculus, is widely used as the mammalian model
system in biology (a) (Phifer-Rixey and Nachman, 2015). Clarence Cook Little who created the first
inbred mouse strain at Jackson Laboratory (b)(Clarke 2002)

Several other features make the mouse a mammal of choice of genetic studies.
Mice have a short generation time of 8–9 weeks. They can breed in captivity, are
docile and have a large little of right or more pups. They are small, are easy to handle
and take up less laboratory space. While model organisms like fruit flies are used for
several important genetic investigations, mice have an advantage of being a mammal
and hence sharing complex traits with humans that are unavailable in fruit flies,
worms and other model organisms. The mouse genome is around 3000 Mb arranged
on 19 autosomes and 2 sex chromosomes (X and Y). This is similar to human
genome of around 3000 Mb compacted in 22 autosomes and 2 sex chromosomes
(X and Y). Almost every gene in the human genome has a homologue in mouse
genome. Another important aspect is the conservation of synteny between the two
genomes. This means that the genes that are closely linked on a locus in one species
are also closely linked in the other species. Conserved synteny implies similar
evolutionary trajectories of these genomes. Synteny also allows researchers to map
homologous genes rapidly in the human genome.
The mouse’s life cycle is similar to humans and other placental mammals only
differing with respect to the timing for each step. The development stages both
before and after birth are remarkably similar in all mammalian species. The male
haploid sperm cell produces sperms via spermatogenesis, which it passes on during
copulation. The females are born with all the haploid egg cells that they will have
over their lifetime. After ovulation, the egg is fertilized by the sperm, the fusion of
which activates the pathway of animal development (Fig. 1.14). From here on,
mouse development is divided into two stages. First is the preimplantation stage
where the zygote freely floats within the female body and mostly remains undiffer-
entiated. This stage is beneficial for scientists because embryo at this stage can be
removed from the female, cultured in a petri dish, manipulated genetically and then
1 Fundamentals of Genetics 25

Fig. 1.14 Events in mouse development from fertilization to birth. The fertilized egg undergoes
division and differentiation through various stages to develop into an embryo that undergoes
organogenesis and development to form an adult mouse. The period from fertilization to birth
lasts for about 21 days (Source: Dr. Brian E. Staveley, Department of Biology, Memorial University
of Newfoundland)

placed back in the female body for further development. This cannot be done in the
next stage, postimplantation, where the embryo grows and develops tissues and
organs.
Advanced genetic techniques and tools have been developed which are helping
scientists discover and characterize increasing number of human diseases. Powerful
techniques for analysing the mouse genome include transgenic technology of addi-
tion of particular genes by nuclear injection into the germline in order to determine
gene function and regulation. Creation of “knockout” mouse by homologous recom-
bination and targeted mutagenesis advanced the field rapidly. New genetic
techniques like transcription activator-like effector nucleases (TALENs) or the
CRISPR/Cas9 system that use guided endonucleases allow precise genetic
manipulations in the mouse genomes (Kaczmarczyk and Jackson, 2015). These
and related techniques have led to invaluable mouse models of several number of
human diseases. These mouse models include thousands of unique inbred strains and
26 S. Panchal

genetically engineered mutants that are available to the research community. There
are mice strains prone to specific diseases like Lou Gehrig’s and Huntington’s
disease, to different types of cancers, to lifestyle diseases like diabetes and obesity
and even to behavioural and neurological disorders like anxiety, aggression, alco-
holism and drug addiction. Immunodeficient mice are also available which are useful
for research in AIDS and cancer.

1.3 Genetics and Evolution

From December 1831 to October 1836, Charles Darwin travelled across the globe as
a naturalist in his HMS Beagle (Fig. 1.15). He studied and collected samples of
hundreds of species that he encountered in varied environments that he visited. The
famous finches were collected from different islands of Galapagos. About 23 years
later, he published On the Origin of Species in 1859. In the book, Darwin makes
remarkable observation about the specimens and fossils he collected. “The similar
framework of bones in the hand of a man, wing of a bat, fin of a porpoise, and leg of
the horse—the same number of vertebrae forming on the neck of the giraffe and of
the elephant—and innumerable other such facts, at once explain themselves on the
theory of descent with slow and successive modifications”. He concluded that “all
organic beings which have ever lived on this earth may be descended from some one
primordial form”. Darwin proposed that species undergo “descent with modifica-
tion”, which means that species evolve, and that all living organisms can trace their
descent to a common ancestor. He suggested that the mechanism of evolution was by
natural selection. Darwin’s ideas had revolutionary impact on the way questions in
biology were being asked. He proposed that there is variation of expression of a trait
among the individuals of a population of a particular species. These trait variants can

Fig. 1.15 Charles Darwin (a) voyaged on the HMS Beagle (b) to study and collect specimens from
around the globe to propose one of the biggest ideas in biology, which he published titled On The
Origin of Species by Means of Natural Selection (c)
1 Fundamentals of Genetics 27

be passed down to future generations. In addition, he suggested that the some

individuals are considered the fittest because of the possession of some of these
variant traits and the variants are selected to be expressed more in the population.
Darwin developed this idea not just by his own collection of species, but also from
observations from artificial breeding of plants and animals. He noted: “The key is
man’s power of cumulative selection: nature gives successive variations; man adds
them up in certain directions useful to himself”. He applied this to selection by nature
and wrote: “Can it, then, be thought improbable, seeing that variations useful to man
have undoubtedly occurred, that other variations useful in some way to each being in
the great and complex battle of life, should sometimes occur in the course of
thousands of generations? . . . This preservation of favourable variations and the
rejection of injurious variations, I call Natural Selection”. Even though these revo-
lutionary insights gave a huge impetus to the scientific thought process, Darwin was
unable to explain the source of this visible variation on which natural selection acts.
Mendel’s experiments on plant hybridizations were published in 1866, just 7 years
after Darwin’s publication of On the Origin of Species, and it is believed that Darwin
received a copy of Mendel’s paper, but he probably never read it. Several years later,
scientists were able to merge genetics and the theory of evolution. After the
discovery of DNA as the heritable material and deciphering the structure of DNA,
scientists could finally understand that evolution is a process that begins at the
molecular level, in the double helical molecule, DNA. A new field of evolutionary
genetics emerged which dealt with study of genetic variation leading to evolutionary
change. It is worth mentioning that Alfred Russel Wallace is jointly credited for
coming up with the theory of evolution, at the same time as Darwin, wherein they
co-published it in 1858, demonstrating their independent observations leading to the
same conclusions. However, since the publication of On the Origin of Species by
Darwin, Wallace was generally overshadowed.

1.3.1 Natural Selection

While on this globe-trotting voyage, Darwin made remarkable observations about

birds called as finches on Galapagos Islands, a cluster of islands about 600 miles
from mainland Ecuador. He observed that these finches closely resembled each other
but had formed a graded series of beak sizes and shapes (Fig. 1.16). In 1860, he
wrote, “seeing this gradation and diversity of structure in one small, intimately
related group of birds, one might really fancy that from an original paucity of
birds in this archipelago, one species had been taken and modified for different
ends”. With the help of his ornithologist friend, John Gould, Darwin realized that
these variations in beaks could have evolved for specific functions. For example, the
little warbler finch has a very fine needle-like beak that is perfect for picking insects.
Another finch, called as woodpecker finch, has a harder robust beak that is used for
beetle and termite larvae, while the cactus finches use long beaks for probing into
cactus flowers. So basically, the variation of this trait occurred for the function
required in the environment that the population was exposed to. Darwin called this
28 S. Panchal

Fig. 1.16 Variation of beak shape in Darwin’s finches. Darwin hypothesized that the beak of the
ancestor species for these finches had adapted over time to the food source available, leading to
formation of completely new species. This illustration shows the beak shapes for four species of
ground finch: 1. Geospiza magnirostris (the large ground finch), 2. G. fortis (the medium ground
finch), 3. G. parvula (the small tree finch) and 4. Certhidea olivacea (the green warbler-finch)
(Source: The Galapagos Finches and Natural Selection. (2020, August 15). https://bio.libretexts.
org/@go/page/13415)

mechanism as natural selection. Over time, the ancestral population of the finches
had adapted to the food source available by acquiring changes in the beak and
evolved. Different groups from the ancestral population would have become isolated
from one another by geographical barriers or by other mechanisms. Once isolated,
the groups would not be able to interbreed and were exposed to different
environments. In each environment, natural selection acted as a drive to favour
different traits. Over many generations, these changes in heritable traits accumulated
in each isolated group such that the groups became a separate species. Hence, natural
selection can also act as a mechanism driving speciation.
Natural selection is one of the core mechanisms of evolutionary change which
leads to the evolution of adaptive traits. These selected traits are inherited and passed
on to the next generation. The species that are better adapted have higher chances of
survival in their environment. Evidence to support evolution and natural selection
has accumulated over time, and now evolution is accepted as a robust scientific fact.
Another example of natural selection was discovered among peppered moths near
industrial cities in England. The moth population had varieties that varied in wing
and body coloration. Most of the insects were pale in colour as compared to the dark
1 Fundamentals of Genetics 29

coloured minority, so that they could easily camouflage on the birch trees to prevent
being seen by their predators, the birds. In the nineteenth century, pollution of sooty
smoke from the coal furnaces killed the lichen on the trees making the tree bark dark
coloured. On account of this, the pale majority population of moths became visible
when they landed on the blackened tree surfaces and were predated upon by the
birds, while the dark coloured ones survived as they became camouflaged. The dark
moths passed on the alleles for dark wing colour leading to offspring with the dark
wing colour phenotype. Over several generations in continued environmental con-
dition, the darker moths became more common and as many as 98% of the moth
population became dark coloured. In today’s world, one of the most relevant
examples of natural selection or adaptive evolution is antimicrobial resistance,
which is currently a global crisis. Pathogenic bacteria and fungi are able to evolve
resistance to antimicrobials on account of repeated exposure to drugs such that the
drugs are no longer able to control the infection.
While we understand now that evolution occurs over a long period of time as
heritable traits change according to the environment, but what exactly changes? It
had already been established that the unit of heredity is the DNA. Specifically,
changes in the genes that affect the phenotype or the trait lead to evolution. Along
with natural selection, there are other mechanisms that drive genetic variation and
hence evolution. These are random mutations, gene flow and genetic drift. While
mutation is actually the original source of any genetic variation, the mutation rate is
generally less, except when driven by a strong selective force. Random and rare
mutations that are desirable can be fixed in a population. Gene flow refers to
movement of genes into or out of a population, mainly by movement of organisms
to a different location. Genetic drift leads to changes in allele frequency due to
chance events. This may lead to loss of some alleles completely.

1.3.2 Evolutionary Lineage

Biological evolution, as Darwin proposed, is descent with modification by genetic

inheritance. This modification includes changes in genes or alleles as well as descent
of different species from a common ancestor over many generations. The central
idea thus is that all life on earth shares a common ancestor. Through a continuous
process of descent with modification, the last universal common ancestor (LUCA)
gave rise to the amazing diversity of species that we see today. The evolutionary or
phylogenetic tree is a branching diagram depicting the relationships of species or
other entities based on their similarities or differences of physical and genetic
characteristics. The tree of life refers to such a phylogenetic tree of all the species
tracing the relationship back to the common ancestor. Evolutionary lineage is a
series of organisms connected by a continuous line of descent from ancestor to
descendant over a period of time. Thus, evolutionary lineages are a part of the
phylogenetic tree that allows us to study the evolutionary history of a species.
Figure 1.17 depicts a hypothetical phylogenetic tree showing relationships
between species A, B, C, D and E. The study of the branches and from where they
30 S. Panchal

Fig. 1.17 A hypothetical

phylogenetic tree displaying
relationships between
species A, B, C, D and E is
shown. The common
ancestors are indicated by
arrow. The pink highlight
indicates the evolutionary
lineage of species A which
can be traced to the last
common ancestor

arise helps us to determine their evolutionary relationship. Each branch point or the
internal node depicts a divergence event, where a single group of species split into
two different species. Thus, the lineage of species A and B can be traced to the
branch point from where they emerged, which is the common ancestor for A and
B. This also indicates that A is more closely related to B than to any other species.

1.4 Genetics in Biological Research

The understanding of inheritance and its relation to evolutionary concept had

resulted in development of a plethora of techniques which help scientists answer
the most basic and important questions in biology. Development of tools for genetic
manipulation has revolutionized the study of genetics and has been useful in
development of recombinant products like insulin secreted by bacteria, pest-resistant
crops and gene therapy to treat diseases in humans, among several other
applications. Moreover, every day we are able to learn more about the mechanisms
underlying cellular functions and organismal behaviour by applying genetic analysis
in laboratories worldwide. Powerful techniques for isolating, recombining and
analysing DNA techniques have allowed us to ask important questions in biological
research. Most of the time to understand a biological pathway or a process, a scientist
1 Fundamentals of Genetics 31

embarks on identifying and deducing the function of the gene(s) involved in that
pathway. There are two main approaches for analysing gene function, viz. forward
genetics and reverse genetics. Both these approaches are based on the traditional
methodology of isolation of a mutation in a particular gene to identify the gene
function, and both approaches are widely used by scientists today.

1.4.1 Forward Genetics

In forward genetics, genetic screens are developed by inducing mutations in the

population using different means, described below. The aim is to induce mutation in
every gene in the population by saturation mutagenesis. Then, the scientist begins
studying an aberrant phenotype (a mutant in the population) to identify the gene that
is responsible for producing this phenotype. For example, consider that a scientist is
interested in identifying the gene responsible for cell division in budding yeast. First,
the scientist will try to identify mutant individuals that are either slow growing or are
arrested in their cell division cycle. On finding such mutant strains of budding yeast,
the mutations can be mapped, thus allowing identification of the genes involved. The
genes can be cloned and sequenced. The proteins formed by the genes can be
isolated and their role in cell division can be biochemically studied. Hence, in
forward genetics, the mutant phenotypes are known and are available before the
corresponding genes are identified. One can find the names of many genes as per the
phenotype that was used to identify them. One example is the rosy gene in Dro-
sophila that encodes the enzymes xanthine dehydrogenase, mutation in which leads
to lack of red pigmentation in the eye of the flies.
In early studies of genetics, scientists looked at naturally occurring variants or
mutants in a population and studied them to identify and understand the gene
function. However, discovery of mutagenic agents—chemicals that cause genetic
mutations—allowed scientists to have large number of mutants with varied
phenotypes. The earliest example of this is the use of X-rays by Hermann Muller
in 1927 to induce mutations in Drosophila melanogaster. In addition to X-rays, there
are different types of mutagen agents that are used today. X-rays cause breaks in
double-stranded DNA, but may not be accurate for single genes and may lead to
alterations in large pieces of the DNA. For finer mutations, scientists use chemicals
like ethyl methanesulfonate (EMS) that cause point mutations and change at one
nucleotide position. These mutations can be within a coding sequence of the gene or
the regulatory region of the gene. However, such mutations are difficult to map.
Another way for inducing mutations is by the use of transposons. Transposons, or
transposable elements (TE), are DNA sequences that have the ability to move
(or transpose) to different sites within the genome. If a transposon from a transcrip-
tionally inactive site like heterochromatin moves into a genic region, it results in
functional inactivation of the gene. This is called as insertional mutagenesis. Trans-
poson mutagenesis leads to higher mutation frequency as compared to chemical
mutagenesis, and availability of using a selectable marker for confirmation of
32 S. Panchal

transposition makes confirmation of mutants easy. The TE insertion can be easily

mapped and the region can be cloned and sequenced. With this ability to generate
mutations, scientists can develop genetic screens to understand gene function and
dissect biological pathways. Forward genetic screens are hence unbiased with no
prior knowledge of the gene product.

1.4.2 Reverse Genetics

Reverse genetics is an alternative to forward genetics, wherein the scientist begins

with a genotype, alters its expression or sequence and then studies the effects of that
alteration in the phenotype. A gene with an unknown function can be mutated, and
its phenotype can be studied to understand its function in the cellular biology of an
organism. The method of inducing mutations at specific locations in the genome is
called site-directed mutagenesis. One strategy for site-directed mutagenesis is to use
short oligonucleotides containing the desired mutated base. These oligonucleotides
can anneal to single-stranded DNA template, and then are extended by DNA
polymerase by polymerase chain reaction (PCR). The products of this amplified
mutation are introduced into the cells and then mutants can be screened for the
presence of the mutated gene.
Another method that is widely used in reverse genetics is transgenics. In this
method, gene of interest is added to an organism that naturally lacks that gene, and
the effect of the function of this gene in this organism is studied. This organism
whose genetic makeup is now altered is called a transgenic organism and the foreign
DNA added is called a transgene. Often, genes from the human genome are added to
mice to obtain transgenic mice because of the ease of genetic manipulation of these
genes in this model organism.
“Knockout” libraries of model organisms, made by using reverse genetics
approach, are widely used in research today. Knockout mutant of a particular gene
indicates complete inactivation of the gene resulting in loss of function. Knockout
libraries are collections of mutants of a species in which almost all genes have been
inactivated. In this library collection, each strain has a single mutation in a different
gene. Such library can be generated by random mutation of a population using
transposon insertions or T-DNA insertions (for plants). These libraries are then
screened by PCR using a primer specific to the gene of interest and a primer specific
to the inserted DNA. Such libraries are an invaluable resource for large-scale reverse
genetics experiments where scientists aim to study the function of all genes in a
model organism.
RNA interference (RNAi) is a gene silencing mechanism in different organisms.
The 2006 Nobel Prize was awarded to Andrew Fire and Craig Mello for their work
on RNAi which they used as a genetic approach to dissect biochemical pathways in
C. elegans. RNAi is a naturally occurring cellular mechanism in which an
organism’s own gene is switched off by double-stranded RNA (dsRNA) in a process
called as gene silencing. During RNAi, long dsRNA are cut into small fragments by
1 Fundamentals of Genetics 33

an enzyme called as Dicer. These fragments called as small interfering RNAs

(siRNA) bind to a family of proteins called as Argonaute proteins. On binding,
one strand of the dsRNA is removed, and the single-stranded siRNA binds to the
complementary mRNA (according to base pairing rules). At this stage, the
Argonaute can cleave this target mRNA or regulate it in such a way that it is
non-functional. The primary role of this process in the cell is to silence repetitive
DNA of the cell or invading double-stranded RNA viruses. This natural process of
the cell is exploited by scientists to design siRNAs complementary to the mRNA for
the gene of their choice. The phenotypes obtained on RNAi silencing show partial
loss-of-function phenotypes, called as knockdowns. The advantage of this approach
is the ease and rapidity of the method and is especially useful for studying essential
genes.

1.4.3 Manipulation of DNA

The above-mentioned approaches for genetic manipulation have been possible

owing to the development of robust techniques for manipulation of DNA. Under-
standing gene function requires precise techniques to be able to isolate DNA, find the
desired gene and isolate it or mutate it, insert the mutated gene in a cell and study the
consequent effects. While the development of these techniques was not straightfor-
ward, they are now routinely used labs worldwide as basic molecular biology
techniques.
The first step in any molecular genetics analysis is obtaining the DNA from a cell
in isolation of other cellular components. Several methods are available for isolation
of DNA and most of them rely on the same principle. The first step involved
breakdown of the cell wall and membrane using lysis buffer which mostly contains
a detergent. Macromolecules other than DNA (like proteins and RNA) are broken
down using enzymes. The DNA is then precipitated with alcohol. DNA extracted
from large number of cells can be seen as white stringy mass that can be spooled
around a glass rod (Fig. 1.18).
A key development in molecular genetics was the discovery of restriction
endonucleases, commonly known as restriction enzymes. These enzymes recognize
specific short DNA sequences and make double-stranded cuts in the DNA at specific
sites. These enzymes are naturally present in bacteria and act as an immune mecha-
nism against bacterial viruses. Bacteria’s own DNA is protected from its enzymes
because it is modified by addition of methyl groups. Several types of restriction
enzymes have been isolated, and the most commonly used in molecular biology are
the type II restriction enzymes, most of which are commercially available. The
restriction enzymes are named according to the species from where they were
isolated. For example, EcoRI was isolated from Escherichia coli. Usually, restriction
enzymes recognize 4–6 bp long DNA sequence. Some enzymes cut the DNA in such
a way that the ends are staggered generating single-stranded overhangs. For
34 S. Panchal

Fig. 1.18 White spool of

strawberry DNA extracted
from strawberry
fruit (Teacher’s manual by
Carolina Biological Supply
Company, USA). Total DNA
was extracted by mashing
strawberry fruit in an
extraction buffer and
precipitating the DNA with
ethanol

example, the enzyme HindIII recognizes the following sequence and cuts the sugar-
phosphate backbone of each strand at the point indicated by the arrow:

This generates fragments with the overhangs as shown below:

Such staggered ends are called cohesive ends or sticky ends because they have
sequence complementarity and can be easily paired or glued together. Hence, any
two DNA fragments that are cut by this enzyme will give such complementary ends
allowing us to join two different fragments together. This is called cutting and
joining (ligating) DNA fragments. Some enzymes generate ends that are not sticky,
but are blunt ends. PvuII is an enzyme that cleaves in the following way:
1 Fundamentals of Genetics 35

A variety of recombinant DNA molecules can thus be generated by this process of

cutting and joining at ambient conditions required for the enzyme action.
These changes made in the DNA have to be confirmed by visualization of the
DNA. If the DNA fragment is cut by one enzyme that cuts at a site that occurs only
once in your DNA fragment, then you should be able to observe two cut fragments
with sizes that add up to the original fragment. To observe this, DNA fragments can
be visualized by a technique called as agarose gel electrophoresis (Fig. 1.19).
Agarose is a polysaccharide obtained from seaweed which is used to make a porous

POWER SUPPLY

CATHODE

ELECTROPHORETIC
BUFFER

WELL ANODE

SAMPLE

AGAROSE
GEL

POWER SUPPLY

CATHODE

HIGH MOLECULAR ANODE

WEIGHT SPECIES

LOW MOLECULAR
WEIGHT ANALYTES

Fig. 1.19 Agarose gel electrophoresis (Drabik et al. 2016). A DNA sample is loaded into wells
towards the cathode side in the agarose gel which is immersed in a buffer. The gel tank is connected
to a power supply which passes electric current through the buffer. Due to this, DNA migrates from
cathode towards anode with smaller DNA fragments migrating faster than larger fragments
36 S. Panchal

gel matrix through which DNA molecules can move. DNA molecules are negatively
charged ions at neutral or basic pH in an aqueous environment. In gel electrophore-
sis, DNA fragments get separated on the basis of their size, which is expressed in
terms of number of base pairs present in that fragment. DNA samples are loaded into
a well or a slot near the negative electrode of the gel matrix and drawn towards the
positive electrode at the opposite end of the gel by applying electric current. Smaller
molecules move through the pores in the gel faster than larger molecules, and this
difference in the rate of migration separates the fragments on the basis of size.
Standard DNA samples with known sizes are usually run alongside the molecules to
provide a size comparison. DNA can be visualized by using fluorescent dyes like
ethidium bromide that can intercalate between the DNA strands and fluoresce on
exposure to UV. Distinct nucleic acid fragments appear as bands at different
distances from the top of the gel depending on their size. DNA samples can also
be probed using certain complementary short sequences. The short fragments called
as probes are designed and labelled with radioactive or fluorescent dyes for detec-
tion. After running the DNA sample on agarose gel for separation, the DNA
fragments are transferred onto a nylon membrane, and this process is called as
blotting. This membrane with the DNA fragments can now be probed with the
designed probes and visualized by X-ray or fluorescence. This technique is called
Southern blotting, and it is used for confirmation of the DNA manipulation or
mutation that has been induced in your sample.
On obtaining the required recombinant DNA, it is critical to amplify the product
or have several copies of the DNA. This can be achieved by placing the recombinant
DNA fragment in a bacterial cell and allowing the cell to replicate the DNA. This
process is called as gene cloning as large number of identical copies or clones can be
generated. Bacteria and yeasts have plasmids (also known as a vectors), small
circular DNA molecules that can replicate independently of the cellular DNA.
Plasmids occur naturally and have genes that can contribute to favourable properties
to the organism carrying it, like antibiotic resistance. These plasmids can be
engineered using restriction enzymes. Usually, plasmids used in molecular biology
have multiple cloning site (MCS). The MCS is a short DNA sequence containing
multiple sites that can be cut with different commonly available restriction
endonucleases. This property makes plasmids suitable vectors for carrying the
DNA sequence of interest. One cell can have multiple number of plasmids, which
amplifies the clones that can be obtained. On introduction within a host cell, such
plasmids replicate to make several copies, thus amplifying your DNA of interest as
well (Fig. 1.20). A transgene like a human gene can thus be placed within the
bacterial cell on a plasmid and allowed to amplify with the bacterial cell. Plasmids as
vectors have been used for several biotechnological applications with large-scale
production of economically important products like insulin and human growth
hormone.
Any fragment of DNA can be amplified from the genome using a technique called
as polymerase chain reaction (PCR). This technique was first developed by Kary
Mullis and allows DNA fragments to be amplified billion times in just a few hours.
1 Fundamentals of Genetics 37

Fig. 1.20 Steps in molecular gene cloning (OpenStax College, Biotechnology. October 16, 2013.
Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44552/latest/Figure_17_01_06.
png). This diagram shows the steps involved in molecular cloning of lacZ gene required for lactose
metabolism. This gene is ligated into the plasmid using restriction enzymes. The bacteria that have
the correct recombinant plasmid are screened by blue-white screening using the chemical X-gal

Even a single molecule of DNA can be used as a starting point to obtain several
million copies by PCR. It is a robust and most widely used technique in molecular
biology. The critical factor in a PCR reaction is the enzyme DNA polymerase. To
replicate DNA, the parent or template DNA should be single-stranded. To achieve
this, the temperature of the reaction is increased to 90–100  C so that the hydrogen
bonds between the two strands of the double-stranded DNA break. Primers or short
complementary sequences are added to the reaction that binds to the single-stranded
DNA at a particular temperature between 30 and 65  C when the reaction is cooling
from 90 to 100  C. DNA polymerase is able to synthesize a complementary DNA
strand starting from the site where the primer attaches. Thus, two new strands from
two parent strands are produced. The whole cycle is then repeated several times and
38 S. Panchal

Polymerase chain Reaction (PCR)

Cycle 2
The PCR cycle consists of three steps denaturation,
annealing, and DNA synthesis that occur at high, low, 5’ 3’
and intermediate temperatures, respectively. The cycle
is repeated again and again, resulting in a doubling of
DNA molecules each time. After several cycles, the vast
majority of strands produced are the same length as the
distance between the two primers. 3’ 5’

Cycle 1
5’ 3’
5’ 3’
3’ 5’
Step 1: denaturation 3’ 5’
The sample is heated to
a high temperature so
the DNA strands separate.
Cycle 3
5’ 3’
5’ 3’
3’ 5’

Step 2: annealing
The sample is cooled so
3’ 5’
the primer can anneal to
the DNA .
5’ 3’
5’ 3’

3’ 5’

3’ 5’ 5’ 3’
Step 3: DNA synthesis
The sample is warmed.
Taq polymerase
Synthesizes new strands 3’ 5’
of DNA.

5’ 3’
5’ 3’

3’ 5’ 3’ 5’

Fig. 1.21 PCR amplification (OpenStax College, Biotechnology. October 16, 2013. Provided by:
OpenStax CNX. Located at: http://cnx.org/content/m44552/latest/Figure_17_01_04.jpg). PCR is
used to amplify a specific sequence of DNA using thermostable DNA polymerase, primers and
deoxynucleotides

the number of strands produced increases exponentially (Fig. 1.21). The critical
discovery for this technique to work was the discovery of a DNA polymerase that
was active such high initial temperatures at every cycle. This thermostable DNA
polymerase was isolated from the bacterium Thermus aquaticus from the hot water
springs of Yellowstone National Park, USA, and is known as Taq polymerase. In
addition to amplification of DNA, PCR can also be used for amplifying sequences
complementary to RNA. For this, the RNA is first converted to its complementary
DNA (cDNA) using a viral enzyme called as reverse transcriptase. The cDNA is then
subjected to regular cycles of PCR. This method is known as reverse-transcription
PCR.
1 Fundamentals of Genetics 39

1.5 Pathway of Genetic Analysis

The study of genetics consists of three major sub-disciplines: classical genetics (also
called transmission genetics), molecular genetics and population genetics
(Fig. 1.22). Classical genetics includes the basic principles of heredity and inheri-
tance of traits. The focus of study is an individual organism—how the organism
inherits traits from the parents and then transmits traits to the next generation.
Molecular genetics deals with the nature of the actual genetic information under-
lying the inherited traits. This study includes the chemical nature of the gene and
cellular processes that lead to the phenotype, including DNA replication, transcrip-
tion, translation and gene regulation. The organization, structure and function of the
gene are studied under molecular genetics.
Population genetics explores inheritance of traits and the underlying genetic
mechanism in groups of individuals of the same species, which is called as a
population. How the genetic composition and hence the trait changes spatially and

Fig. 1.22 Categories of genetics. The field of genetics can be subdivided into three different
types—transmission genetics, molecular genetics and population genetics. Image source: top,
@IngoDiBella via Flickr; bottom left—Livescience.com; bottom right—Time.com
40 S. Panchal

temporally is studied. Hence, population genetics is fundamental to the study of

evolution in which changes over time are studied.

1.5.1 Classical Genetics

When Gregor Mendel described the fundamental laws of inheritance through his
rigorous experiments on garden pea plants, a new era of understanding in biology
had commenced. His work and the work that followed gave rise to a field in biology
that came to be known as Mendelian genetics, a synonym for classical or transmis-
sion genetics. The understanding for the mechanism for variation in traits within a
population and inheritance of trait by next generations was mostly vague before
Mendel’s study. Mendel’s hereditary experiments with pea plants led to the forma-
tion of law of segregation and law of independent assortment. The law of segregation
describes how a pair of gene variants (alleles) is segregated in the reproductive cells
(gametes). Mendel crossed two heterozygous plants (each with a different allele for a
trait) and found that the trait in the offspring did not always match the trait of the
parents, indicating that the alleles for the trait had segregated during the formation of
gametes leading to different possible outcomes for the offspring’s phenotype.
Depending on the parental genotype, he could predict consistent ratios of phenotype
in the offspring that can be produced. The law of independent assortment predicts the
inheritance of two or more traits. Non-Mendelian inheritance patterns discovered
later are also widespread in nature.

1.5.2 Molecular Genetics

Several lines of discoveries and inventions led to the formation of this field of
genetic analysis. Identification of the unit of heritance, the gene, as a biochemical
molecule, one gene-one enzyme theory, use of mutagens for making heritable
changes in the genes and identification of the nature and structure of the inheritable
molecule, the DNA, allowed for tremendous development of techniques to study the
structure and function of genes that lead to the traits that were studied by classical
genetics. Several techniques described previously for DNA manipulation using
either forward or reverse genetics approach are used in the study of molecular
genetics. Gene cloning employing plasmids, restriction enzymes and ligases, DNA
amplification by PCR, hybridization methods and gel-based separation of DNA
fragments are the basic tools of today that are used to answer questions in molecular
genetics. Construction of whole-genome libraries and identification of mutations
using PCR-based methods also helps to answer questions in molecular genetics. The
advent of genome sequencing technologies in the last two decades has opened newer
avenues and methods to answer basic questions in molecular genetics. Conventional
sequencing methods are being constantly updated in order make genome sequencing
rapid and easy. Today, we have technologies like Oxford Nanopore sequencing and
Illumina sequencing, using which genome sequencing can be accomplished in a
1 Fundamentals of Genetics 41

matter of few days. This is proven by hundreds of genomes being sequenced every
day, and the technology being refined with newer and newer methods. Large-scale
genome data (genomics) has opened up newer methods of computational analysis
(bioinformatics). Different software and analysis platforms are continuously being
updated for rapid and robust analysis of large quantities of “omics” data. Such data in
collaboration with experimental data can answer major questions in biology that
were not possible earlier. For example, if one wishes to know the binding regions of
a protein on the genomic DNA of an organism, one can employ a technique called as
“chromatin immunoprecipitation sequencing (ChIP-seq)” wherein the regions of the
DNA that are bound by the protein are isolated and a library of the DNA fragments is
made and then sequenced. Such whole-genome experiments are widely used by
scientists and are accelerating the rate at which science is progressing.

1.5.3 Population Genetics

Population genetics is a branch of genetics that deals with the genetic composition
and variation among individuals of a population within a species. In nature, often we
observe individuals of a population displaying a variety of phenotype due to
expression of different alleles of a gene, called as polymorphisms. This expression
in a polymorphic population depends on the genetic structure as well as the environ-
ment. In population genetics, scientists try to understand the sources of such
phenotypic variation in a population and predict how that population will evolve
over time in the presence of different evolutionary factors. Organisms studied in
population genetics are interbreeding, are sexually reproducing and have a common
set of genes, known as the gene pool. Due to changes within the gene pool over time,
the population evolves. The evolutionary forces that lead to these changes are also
studied in population genetics. Genotypic and allelic frequencies are used to describe
the genetic composition of a population. G.H. Hardy and Wilhelm Weinberg
independently formulated a law, called as Hardy-Weinberg equation, that describes
how reproduction and Mendelian principles affect these allelic and genotypic
frequencies within a population. Allelic frequencies can be changed by several
operational factors like mutations, migration, genetic drift and natural selection.
Mutations can directly induce changes in the base composition of the DNA
sequence. In natural selection, alleles that confer beneficial traits are selected and
the ones that are deleterious are removed over time. Migration or large-scale
movement of organisms from a population to another location leads to gene flow
causing changes within the older as well and the new forming population. Genetic
drift is a chance occurrence when some individuals have more offspring than others
in the population, thus increasing the representation percentage of that allele.
Usually, population geneticists develop mathematical models to study the patterns
of genetic variation with a population. Modern population genetics however
comprises of theoretical aspects, lab and field work.
42 S. Panchal

1.6 Genetic Database

Decades of scientific research and the explosion of sequencing technologies have led
to development of large number of databases which help connect scientific
discoveries worldwide (Bianco et al. 2013). A biological database is an organized
computer-based storage of information and data generated from scientific
publications, experiments in research laboratories (in vitro and in vivo) and bioin-
formatics analysis (in silico). The information stored on a database is well-organized
and easy to use. Databases are essential for continuous storing, sharing and updating
data to keep the scientific community apprised of the latest research. Sharing and
open data access to large-scale experimental projects has also led to large number of
collaborative research projects. This is vital for rapid progress of scientific research
which is ultimately beneficial to the human society. These databases are bioinfor-
matics resources and tools that are open to public for information dissemination.
Large number of sequenced genomes has led to development of not just an informa-
tion storing database, but an interactive browser on which a user can view as well as
analyse the sequence information deposited there. Such browsers are often linked to
various bioinformatics tools for this purpose. In addition to genome sequence, other
“omics” data like transcriptomics, proteomics and metabolomics collect experimen-
tal data which can be visualized and analysed on the databases.
Literature databases were one of the first scientific databases generated to collect
and store all scientific publication in once place. Literature search is the first step of
any scientific project that allows one to formulate a hypothesis based on the research
already done in that particular field. The oldest scientific article database for bio-
medical research is PubMed, developed by National Center for Biotechnology
Information (NCBI) which includes abstracts of the articles and links to the journal
website. PubMed is the most widely used and updated site for bibliographic research
in biomedical research.
NCBI also serves as an integrated and one of the largest and oldest platform for
sharing and utilizing sequence-based resources in the scientific community. It
provides an integrated data system for almost all existing genetic resources. It
links the data to its original source as well as to a number of analytical tools that
allow a researcher to obtain an in-depth valuable knowledge at the same location.
Search on this website is extremely user-friendly with terms like gene symbol, gene
name, marker name, text work or phrase related to the gene can be searched. The
output displays the availability of your search term in different NCBI databases. It
provides the options for a refined search depending on the user need. For example,
search term “tnf” gives an interface as shown in Fig. 1.23. TNF (tumour necrosis
factor) is a gene superfamily that regulates several cellular processes including
immune response, cell proliferation and differentiation. As seen in the figure, the
search gives links to several databases housed within NCBI, including genomes,
proteins and genes that carry this search term. The user can easily navigate to the
database of choice and visualize the data required. NCBI also houses several
bioinformatics tools like Basic Local Alignment Search Tool (BLAST), conserved
domain search tool, multiple sequence alignment tools and several others.
1 Fundamentals of Genetics 43

Fig. 1.23 Interface of NCBI website on search of the tern “tnf”. The search term is found in several
databases in the website. Depending on the question to be asked, the user can navigate to the
databases and view and analyse the gene or protein of interest

Ensembl database (from the French word “ensemble” and “EMBL” European
Molecular Biology Laboratory) database is a software system created by the
EMBL-European Bioinformatics Institute (EBI) and the Wellcome Trust Sanger
Institute (WTSI) to handle genome annotations of eukaryotic organisms. Like other
databases, its aim is to provide genome sequence annotations as a free resource to the
scientific community while integrating and linking other biological data. Several
other databases exist and more are coming up as the bioinformatics data for biology
keeps increasing at a rapid pace. Some of the important databases for model
organisms are shown in Table 1.2.
Other important databases are listed here:

• National Center for Biotechnology Information (GenBank)—http://www.ncbi.

nlm.nih.gov
• Ensembl—http://www.ensembl.org
• Human Gene Mutation Database (HGMD)—http://www.hgmd.cf.ac.uk/
• International Sequencing Consortium—http://www.intlgenome.org
• Kyoto Encyclopedia of Genes and Genomes (KEGG)—http://www.genome.ad.
jp/kegg/
44 S. Panchal

Table 1.2 Genetic databases for model organisms

Common name Scientific name Genetic database URL
Baker’s yeast Saccharomyces Saccharomyces Genome www.
cerevisiae Database (SGD) yeastgenome.org
Fruit fly Drosophila FlyBase www.flybase.org
melanogaster
Thale cress Arabidopsis The Arabidopsis Information www.
thaliana Resource (TAIR) arabidopsis.org
Common house Mus musculus Mouse Genome Informatics www.
mouse (MGI) informatics.jax.
org
Worm/ Caenorhabditis WormBase www.wormbase.
nematode elegans org
Zebrafish Danio rerio The Zebrafish Information www.zfin.org
Network (ZFIN)

• NCBI Entrez Web site—http://www.ncbi.nlm.nih.gov/Entrez/

• National Human Genome Research Institute (NHGRI)—http://www.genome.gov
• Online Mendelian Inheritance in Man (OMIM)—http://www.ncbi.nlm.nih.gov/
omim
• Protein Data Bank (PDB)—http://www.pdb.org
• University of California at Santa Cruz (UCSC) Genome Browser—http://
genome.ucsc.edu
• TIGR Gene Indices—http://www.tigr.org/tdb/tgi

1.7 Application of Genetics

Genetics has been exploited for thousands of years, even before the underlying
mechanisms were unknown. Fermentation is used widely in the brewing and bakery
industries since a very long time. Artificial selection of animals and plants by cross-
breeding benefitted humans by providing improved products of dairy and increased
yield of plant-based food products. In the recent past, understanding the inheritance
mechanisms and development of genetic tools has opened myriad of opportunities
for using genetic engineering in a variety of field that has benefitted humans directly
as well as the environment. Some of the significant applications have been described
below.

• Pharmaceutical products: Using recombinant DNA technology, easy to grow

organisms like bacteria and yeast have been used as small “biofactories” for
production of several compounds useful in medicine. Bacterial or yeast cells are
transformed with a vector containing the gene for the product of choice derived
from the human genome. These cells are grown in large bioreactors where they
overexpress the transgene and produce large quantities of product that can be
harvested and purified. One of the earliest examples of such recombinant
1 Fundamentals of Genetics 45

technology was the production of human insulin. Insulin is essential for the
control of blood sugar levels, and when the body cannot produce insulin, it
leads to the disease diabetes mellitus. In such cases, patients have to take insulin
from external sources to control blood sugar levels. Recombinant insulin is
produced either in yeast or E. coli. The first genetically engineered, synthetic
“human” insulin was produced using E. coli in 1978 by Arthur Riggs and Keiichi
Itakura at the Beckman Research Institute in collaboration with Herbert Boyer at
Genentech. Genentech in 1982 started selling the first commercially available
biosynthetic human insulin under the brand name Humulin. Other medically
useful products like growth hormones, vaccines, blood clotting factor, monoclo-
nal antibodies as well as drugs are being produced using this technology. Recent
advances have also led to use of plants for production of recombinant pharma-
ceutical products (Ma et al. 2003).
• Specialized microorganisms: Several microorganisms are also being used to
recover oil from oil wells, break down toxic chemicals like oil spills and other
pollutants and solubilize minerals from ores. Bioremediation is a field of biology
where microorganisms like bacteria and fungi or their products are used to
degrade or remove toxic compounds from the ecosystem.
• Agricultural products: Most of the crops that are agriculturally important today
are quite different from their wild progenitors. Artificial breeding has caused
several genetic modifications in these crops to select for desirable traits like high
yield, disease and pest resistance, high nutritional value and so on. The Green
Revolution led by Norman Borlaug relied heavily on genetic methods to develop
high yielding crops. Norman Borlaug was awarded the Nobel Peace Prize because
his revolution fed malnourished populations across the globe by introducing these
techniques in the agricultural system of poorer countries. Such methods of
conventional breeding also involve changes in the DNA sequence of the organ-
ism, just like the relatively recent genetic modification (GM) technology. How-
ever, genetic changes brought about by the GM technology are small in number,
well defined, precise and targeted as compared to classical breeding methods
where several genes of an organism may be involved. Today, a significant
proportion of food products like corn and soybeans are genetically modified.
However, the rules for modifying crops by genetic engineering are different
across different countries with some concerns about their safety. One of the first
transgenic plants produced by GM technology was Bt cotton. A gene from the
bacteria Bacillus thuringiensis (Bt) that encodes for an insect toxin was cloned
into the cotton genome, which made the plant resistant to the common pest
bollworm. Bt cotton has gained tremendous success in countries like India as
the cotton production boosted when transgenic plants were used.
• Genetic testing for diagnosis: Several human diseases have been found to have an
underlying genetic and heritable component. Genetic disorders like sickle cell
anaemia, Huntington disease and breast cancer are some of the examples. If there
is a family history of a particular genetic disease, then identifying the genetic
mutations and the genes involved in these diseases allows diagnosis of the
disorder before it occurs and predicts the person’s predisposition to that disease
46 S. Panchal

so that prophylactic measures can be taken if necessary. For example, mutations

in the human genes BRCA1 and BRCA2 notably increase the risk of breast and
ovarian cancers in women at younger age. These two genes produce tumour
suppressor proteins that help repair damaged DNA and thus maintain stability of
the genome. Mutations that lead to non-functional proteins or proteins with
altered function prevent this process of repair and hence lead to cancer. The
deleterious BRCA1 or BRCA2 mutation can be inherited from mother or father
such that each child with a parent carrying the mutation has 50% chance of
inheriting the mutation. The effects of mutations in BRCA1 and BRCA2 are seen
even when a person’s second copy of the gene is normal. Identification of the
mutation by sequencing the DNA can help patients in taking risk-reducing
measures before the full-blown disease occurs.
During an infection, the causative pathogen can be identified using genetic tools.
Several diagnostic kits are available for identification of specific species of the
pathogen to allow for targeted treatment.
• Gene therapy: Gene therapy is the direct modification of the genes of a patient’s
cells to treat a disease. Gene therapy is mostly an experimental technique and is
used only when no treatment options are available. However, it works well in
certain kinds of diseases like haemophilia and severe combined immunodefi-
ciency (SCID). The modifications in the genes can be carried out by introducing
an engineered virus inside the cell or by collecting cells from the bone marrow or
blood, editing them externally and using them to replace patient’s cells. This
method is currently at its infancy with no gene therapy products available on the
market, but is promising and requires more in-depth research on the side effects
and aspects of safety.

1.8 Promise of Genetics

The completion of the Human Genome Project paved the way for intense research on
human genetic composition, identification of genes involved in diseases and study of
cellular pathways that get altered in a disease. In the future, the area of functional
genomics to study the function of genes in normal conditions will continue to be an
active area of research as scientists will categorize more and more genes involved in
human health. Gene therapy as a technique is likely to gain major advancements that
will allow scientists to gain more success in treatment of diseases with mutations in a
single gene. Medicinal genetics is headed towards comprehensive study of a disease
by investigating multiple genes, pathways, systems, the effects of environment as
well as genetic variations within a population. Newer methods will lead to study of
complex diseases with such comprehensive outlook. Such studies are likely to utilize
large sample sizes of thousands of patients whose genome data can be analysed.
While robust, relatively cheap and easy to use sequencing platforms are already
available today for analysis for large datasets like genomes, transcriptomes and
proteomes, analysis tools, algorithms and software for handling such datasets are
likely to be developed soon. These studies will allow doctors to predict
1 Fundamentals of Genetics 47

predisposition of a patient to diseases like cancer, diabetes, cardiovascular diseases

and so on. Another area of research that has benefitted greatly by advancements in
genomics is the field of pharmacogenomics, the study of how genes are involved in a
patient’s response to drugs. While there have been only small studies so far, the
future for these studies is promising. All these research areas will boost the arrival of
personalized medicine. While the real-life implementation of personalized medicine
still has a long way to go, there are good indications of major advancements in the
steps leading to it. It can be predicted that precise genome editing techniques
developed recently like meganucleases, zinc finger nucleases (ZFNs), transcription
activator-like effector nucleases (TALENs) and clustered regularly interspaced
palindromic repeats (CRISPR)/Cas will be improved and utilized in generation of
genetically modified organisms like pathogen-resistant or drought-tolerant crops.
While the promise of genetics in the future is likely to be fulfilled, major steps will be
required for regulation of these methods to prevent misuse. The sad events of human
experimentation during World War II exemplify how advancement of these
technologies, especially genome editing, can be exploited. It will be the collective
responsibility of the scientific community, policy makers and governments to ensure
that does not happen and the science of genetics can be utilized for betterment of the
human society.

Box 1.1 Scientific Concept: Forward and Reverse Genetic Approaches

for the Analysis of Vertebrate Development in the Zebrafish—Nathan
D. Lawson and Scot A. Wolfe
Forward and reverse genetics approaches have been widely used in bacterial,
fungal and invertebrate models to study gene function and dissect cellular
pathways. Study of developmental processes using these approaches has been
successful in organisms like C. elegans and D. melanogaster, but poses major
challenges for use in mammalian models like mouse because of complex
internal development. As an alternative, utilizing zebrafish (Danio rerio)
was proposed as a model system to study vertebrate development. This
organism possesses several characteristics that make it suitable for such
studies. For example, its clutch size per mating pair is large (>100), and it is
externally fertilized with embryos developing rapidly and synchronously
ex vivo. An important factor was the availability to perform detailed micro-
scopic observations of the nearly transparent embryo at early stages of devel-
opment. Zebrafish are small and easy to handle, and relatively less expensive
to maintain as compared to other mammalian models. These factors have made
it an established model organism to study vertebrate developmental pathways.
As with other systems, forward genetics approach was initially utilized to
identify gene functions in zebrafish. Several mutagens have been utilized to
obtain genetic lesions with observable phenotypes. Initially, gamma rays were
used which largely gave gross chromosomal aberrations which made accurate

(continued)
48 S. Panchal

Box 1.1 (continued)

gene function prediction difficult. Later, N-ethyl-N-nitrosourea (ENU) was
used which gave high mutagenic loads with phenotypes that can be linked to
discrete genes. It is easy to expose individuals to the mutagen by simply
adding the compound to the water, and ENU is currently the choice of
mutagen for forward genetics in zebrafish. Other methods include using
replication-deficient pseudotyped retroviruses or transposons as insertional
mutagens. Methods to identify phenotype include serial microscopic observa-
tion of morphology and behaviour during embryo development, using cell- or
tissue-specific molecular markers using whole mount in situ hybridization and
immunostaining, and generating transgenic lines with tissue-specific fluores-
cent makers. Several studies employing these forward genetics techniques
have resulted in identification of genes and pathways important for gastrula-
tion and mesoderm induction, cilia formation, progression of infectious dis-
ease, regeneration and cardiovascular development. In many of these studies,
pathways and genes conserved in diseases affecting humans have been
revealed. However, given the large size of zebrafish genome, forward genetics
approaches alone are unlikely to provide functions of all the genes. Addition-
ally, availability of whole-genome sequence assembly and extensive expres-
sion analysis has provided the opportunity to develop tools to dissect gene
function using reverse genetics approaches. These approaches include
morpholino-mediated gene knockdown, Targeting Induced Local Lesions In
Genome (TILLING), retroviral- and transposon-mediated mutagenesis and
targeted gene inactivation via zinc finger nucleases. Of these, zinc finger
nuclease (ZFN) is an exciting recent advance in genome editing in several
organisms. ZFNs are a class of engineered DNA-binding proteins that can
create double-strand breaks at specific locations leading to precise genome
editing. Each ZFN consists of two functional domains, viz. a DNA-binding
domain and a DNA-cleaving domain. The DNA-binding domain is designed
to bind specific target loci, while the DNA-cleaving domain consisting of
Fok1 nuclease creates a double-stranded break on dimerization. This fusion of
the DNA-binding and DNA-cleaving domains creates a highly specific molec-
ular scissors for precise gene editing. Use of ZFN in zebrafish is shown in
Fig. 1.24. ZFN-mediated gene inactivation is sufficiently robust in zebrafish
and causes minimal off-target damage in the genome. Despite several
advances in the past few years, ZFN technology in zebrafish has limitations
owing to the lack of knowledge of sequence-specific DNA recognition by
ZFNs in the zebrafish genome. Technological advancements should allow use
of this as well as other technologies in zebrafish rapidly in the coming years to
be able to create several human disease models in this vertebrate model
organism.
1 Fundamentals of Genetics 49

Fig. 1.24 Overview of ZFN-based gene inactivation. (a) A pair of ZFNs are designed to bind
neighbouring sequences within the target gene of interest. DNA recognition is mediated by the
ZFA, while the attached FokI nuclease domain generates a double-stranded break (DSB) upon
dimerization. (b) mRNAs encoding each ZFN are prepared and then injected into one-cell embryos.
Putative founders from these injections are raised to adulthood and out-crossed to identify carriers
and the mutant alleles they transmit. Founders harbouring interesting alleles are out-crossed to
generate an F1 population, and heterozygous F1 carriers are identified and then in-crossed to
provide homozygous mutant embryos for phenotyping

1.9 Summary

• Genes confer phenotypes. Genes are inherited, and expression of these genes
along with environmental effects determines the trait or the phenotype. The
genetic information of an organism is called the genotype and the expressed
trait is called the phenotype.
• Mendel studied seven different traits in the pea plant, each of which had two
alternate forms. These traits were height (tall or short), seed colour (green or
50 S. Panchal

yellow) and seed shape (smooth or wrinkled). These were “either-or” traits,
meaning there were no intermediate forms.
• Monohybrid cross: In the P generation, true-breeding pea plants (homozygous)
for the dominant phenotype of yellow seeds and the recessive green seed pheno-
type are crossed. This cross produces F1 heterozygote generation with all
individuals having yellow seeds. Hence, yellow is the dominant trait here. On
self-pollination, F2 generation produces has a mix of yellow and green seed
individuals, in the ratio of 3:1.
• Dihybrid cross: Pure breeding parents for two traits are crossed with pure
breeding lines with recessive phenotype. F1 generation has heterozygous plants
with the dominant phenotype for both traits, and F2 generation has plants with all
combinations in the ratio of 9:3:3:1.
• Natural selection is one of the core mechanisms of evolutionary change which
leads to the evolution of adaptive traits. These selected traits are inherited and
passed on to the next generation.
• In forward genetics, genetic screens are developed by inducing mutations in the
population using different means.
• Reverse genetics is an alternative to forward genetics, wherein the scientist begins
with a genotype, alters its expression or sequence and then studies the effects of
that alteration in the phenotype.

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Mendelian Principle of Inheritance
2
Dhruti Patwardhan

Genetics is the study of genes and their variation and heredity among organisms.
Long before DNA was recognised as the genetic material, Gregor Mendel through
his studies predicted the presence of such a factor responsible for heredity. Heredity
had been observed in nature for centuries, but Mendel studied this phenomenon in a
scientific manner, performed experiments and put forth his hypothesis that has
withstood the test of time. Although some variations to principles of Mendelian
inheritance have been observed, the basic framework of genetic inheritance initially
proposed by him in essence remains true.

2.1 Mendelian’s Monohybrid Cross

Gregor Mendel is widely regarded as the founder of genetics. He was a priest in an

abbey in Brno where he conducted his experiments on pea plants. Mendel carried out
his experiments on breeding of pea plants for about 7 years from 1856 to 1863 and
presented his findings at the Brno Natural Science meetings in 1865. His paper
which underlined the basic principles of inheritance was published in 1866. How-
ever, Mendel’s work remained unnoticed until 1900 when other scientists like
botanists Hugo de Vries, Erich von Tschermak and Carl Correns obtained similar
results while working independently with plant breeding. They interpreted their
results in the context of Mendel’s theories and published their work supporting
and drawing attention to Mendel’s original work.
Mendel was successful in obtaining and interpreting his results due to a number of
factors. One of the most important factors was his scientific approach and analytical
reasoning. Others before him had crossed plants and described their results. Mendel,
however, was able to formulate a hypothesis based on initial observations and design

D. Patwardhan (*)
Indian Institute of Science, Bangalore, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 53

Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_2
54 D. Patwardhan

suitable experiments to test them. He also recorded the number of plants with
different features across various crosses and tried to interpret these observations to
fit a single framework of inheritance. One of the other reasons for his success was the
choice of garden pea as his experimental model.

2.1.1 The Garden Pea

Pisum sativum, commonly known as the garden pea, was the ideal choice for genetic
breeding experiments. Since Mendel worked in a monastery at Brno, he could easily
access the monastery garden and greenhouse. Pea plants are easy to cultivate and can
grow relatively rapidly with a life cycle of 1 year. He therefore invested years in
following several generations of the plants. Pea plants are also able to produce
numerous seeds which allowed Mendel to calculate mathematical ratios in the traits
of offspring (seeds). Different varieties of peas are available and Mendel was able to
choose those that differed in various traits and were purebred. He also chose to study
features that were present in two easily distinguishable forms/traits like round seeds
versus wrinkled seeds. He avoided those features which had a range of variable
traits. He chose to study seven features which are shown in Fig. 2.1. Apart from
these, some references also mention an eighth feature: seed coat colour which can be
either green or white. Mendel noticed that a coloured seed coat always gives rise to
plants bearing purple flowers, while the white seed coat gave rise to plants having
white flowers. Seed coat colour is therefore sometimes mentioned instead of flower
colour as the traits studied by Mendel.
With advances in molecular biology, studies have been performed to identify the
genes and particular mutations responsible for the traits studied by Mendel. Genes
can be classified into groups based on the structural or functional similarities of
proteins they produce. They may also be grouped together if they all participate in a

Fig. 2.1 The traits or plant characteristics studied by Mendel in his experiments on pea plants:
Mendel studied seven different traits in pea plant for conducting his experiments. Traits being
studied were present in either one of the two forms in the different varieties of pea plant (Pierce
2010)
2 Mendelian Principle of Inheritance 55

Table 2.1 Group symbols and their functions for the traits studied by Mendel: Molecular studies to
understand the traits studied by Mendel have resulted in identification of the genes responsible for
the trait and their function. Of these R, LE, A and I have been cloned and well-studied. Less is
known about the other genes
Trait Group symbol Gene function
Seed shape R Starch branching enzyme 1
Stem length LE GA3 oxidase 1
Flower colour A bHLH transcription factor
Pod colour GP Chloroplast structure in pod wall
Pod form V Sclerenchyma formation in pods
Position of flowers FA Meristem function
Seed colour I Stay-green gene

particular process. Gene group symbol and their functions for the traits studied by
Mendel are given in Table 2.1.
The seed shape can be either round or wrinkled. The seeds differed in their starch,
sugar and lipid content. Wrinkled seeds possess a higher amount of fructose, glucose
and sucrose resulting in higher water retention due to osmotic pressure. A mutation
in the R gene which codes for a starch branching enzyme 1 affected starch biosyn-
thesis. This also further affected the protein and lipid biosynthesis in the seed
ultimately changing its shape. Stem length was controlled by the LE gene. This
gene codes for one of the GA3 oxidase genes which convert the gibberellin to an
active form GA1. Gibberellin is a plant hormone which regulates the development of
the plants including its length. Seed colour is influenced by the I gene which is
important in chlorophyll degradation. Mutation in this gene leads to the appearance
of green seeds. The flower colour is influenced by a gene which exhibits pleiotropic
effects. This means that mutations in the gene can affect multiple traits. The gene
codes for a basic helix-loop-helix (bHLH) transcription factor. It regulates multigene
family-chalcone synthesis (CHS) genes which are responsible for flavonoid produc-
tion. Flavonoids are secondary metabolites produced in plants and are responsible
for pigmentation in plants, thus governing the flower colour. Other genes associated
with the traits studied by Mendel have not been cloned and studied in molecular
detail (Reid and Ross 2011).
To perform his experiments, Mendel crossed different varieties of pea plants. In
order to understand how he achieved this, we need to know a little bit about the plant
reproductive system. The male reproductive organ in a plant is called a stamen. It is
composed of the filament and anther. The filament holds up the anther where pollen
is produced. This pollen is carried by either wind, water or wildlife to the female
reproductive organ. The female reproductive organ is called a pistil which consists of
the stigma, style and ovary. The style holds the sticky stigma at the distal end, while
the ovary is present at its proximal end. Stigma captures the pollen and allows it to
germinate. Sperm carried in the pollen reaches the ovary through tubes formed
during germination. Fertilisation occurs and an embryo is formed which is stored
in the seed capsule. The seed remains dormant until favourable environmental
conditions allow it to develop into a plant.
56 D. Patwardhan

Pea plants often undergo self-pollination. This means that pollen from the flower
will fall on the stigma of the same flower due to its close proximity. This happens
even before the flower has opened. This type of pollination reduces genetic
variability as the pollen and egg come from the same plant allowing them to maintain
their characteristics. Plants which always pass on a specific trait to their offspring are
called purebred varieties. Mendel grew pea plants for around 2 years in this manner
to obtain purebred varieties for each trait. He also wanted to cross plants with
different traits to see what traits were seen in the offspring. To achieve this, he
opened the flowers and removed their anthers to prevent self-pollination. He then
manually dusted pollen from the desired plant on the stigma of a flower from a
different variety. This is called cross-pollination and resultant offspring are called
hybrids. He obtained seeds from these cross-pollinated plants and observed their
traits. He also grew these seeds through the next season to observe the traits of the
hybrid plants (Fig. 2.2).

Fig. 2.2 Figure illustrating the male and female reproductive organs in a plant: To cross different
varieties of plants, Mendel removed anthers from the flower to prevent self-pollination. He then
dusted pollen from desired plant onto the stigma of this flower (Griffiths et al 2011)
2 Mendelian Principle of Inheritance 57

2.1.2 Concept of Dominant and Recessive Traits

Mendel crossed different varieties of plants to study the traits inherited by the
resultant offspring. He started by conducting monohybrid crosses, i.e. crosses
between plants which differed by a single trait. Let us take the example of seed
colour which can be either green or yellow. When Mendel crossed plants having
green seed colour with those having yellow seed colour (referred to as the parental
generation—P), he found that all the offspring called the first filial generation
(F1) had yellow seed colour. He also carried out reciprocal crosses where instead
of taking pollen from yellow seed plant and dusting it on the stigma of a green
seeded plant, he took pollen from a green seeded plant and dusted it on the stigma of
a yellow seed plant. In both cases, he found that plants in F1 generation had yellow
seeds. Similarly, in crosses for the rest of the traits, he found that F1 generation
always showed a single parental trait. It wasn’t a mix of the parental traits, nor did the
outcome change with various repetitions. This trait which was observed in the F1
generation was called the dominant trait and the trait which was lost was called the
recessive trait. Mendel took this experiment one step further and allowed the plants
from F1 generation to undergo self-pollination to create the F2 (second filial)
generation. Most of the plants in the F2 generation had yellow seed colour, but
surprisingly, there were a few plants in which seed colour was green. He counted the
number of these plants and found that the number of plants having yellow seed
colour was roughly thrice the number of plants having green seed colour.
Based on these results, Mendel made certain assumptions and put forth a hypoth-
esis. Although the F1 generation always showed a single parental trait, the second
parental trait reappeared in the F2 generation. This led him to assume that the F1
generation might have received genetic factors for both parental traits. Unless the F1
generation inherited genetic factors from both parents, it is impossible to explain the
appearance of both parental traits in F2 generation. He hypothesised that offspring
must inherit genetic factors from both parents and there must be two genetic factors
in the plant for a single trait. The two genetic factors described here are what we now
know as alleles. Alleles are, simply put, different forms of the same gene and are
designated by a single letter. In this case, the allele for yellow seed colour is
designated as Y and that for green seed colour is designated as y. Since the parental
generation was purebred, the parental generation with yellow seed colour would
have the alleles YY and the one with green seed colour would have the alleles
yy. This composition of the alleles (YY or yy) is referred to as the genotype, and the
trait physically expressed by the plant is called the phenotype (green or yellow seed
colour).
He next assumed that the alleles separate when forming gametes and each allele
gets segregated into one gamete. So the parental yellow seed coloured plants formed
the gametes having allele Y and those from green coloured seed formed the gamete
with allele y. In the F1 generation, these two gametes united and they had the
genotype Yy. All F1 generation only had yellow coloured seeds. This trait was
called the dominant trait. Although the allele for green coloured seed was present, it
was masked and not expressed in the presence of Y. This was called the recessive
58 D. Patwardhan

trait. He concluded that, of the two parental traits, one trait is the dominant and the
other is recessive. Only the dominant trait gets expressed even in the presence of the
recessive trait.

2.1.3 Segregation of Alleles

He further hypothesised that the F1 generation having genotype Yy forms gametes

having Y and y with equal probability. Therefore, half the gametes will have allele Y
and the other half will have allele y and these will get paired randomly in the F2
generation. The resulting progeny might have the genotypes YY, Yy, yY and
yy. Since yellow coloured seed is the dominant character, YY, Yy and yY will
have yellow coloured seeds, and only yy will have green coloured seeds. This
explains the 3:1 ratio of phenotypes observed by Mendel in his experiments. This
ratio can only be obtained if we assume that the alleles get segregated with equal
probability while forming gametes. When the genotype of a plant consists of the
same alleles, they are called homozygous (YY, yy), and when the alleles differ they
are called heterozygous (Yy) (Fig. 2.3).

2.1.4 Mendel’s Analytic Approach

The findings from Mendel’s monohybrid cross are formally stated in two laws
known as law of segregation and law of dominance. Law of segregation states that
during the formation of gametes, two alleles in an individual will separate such that
each gamete will have one allele. Law of dominance states that hybrids of different
alleles will express only one of the parental traits called the dominant trait. Mendel
was able to draw meaningful insights from his work due to the analytic approach
towards his experiments. The ratios Mendel obtained from his experiments were not
perfect. Plants may die or wither before their characteristics can be noted. Some
plants may fail to germinate. Therefore, the ratio of monohybrid cross that Mendel
obtained was almost but not exactly 3:1. However, Mendel obtained numbers for
multiple experiments and noted that the ratios were approximately 3:1 in all cases.
He further went on to self-pollinate plants obtained from F2 generation to confirm
his findings.
Let us take the example here of round vs wrinkled seeds. When plants having
round seeds are crossed with those having wrinkled seeds, the resulting F1 genera-
tion has all round seeds. Here, round seeds is the dominant trait and represented by
the allele R, while wrinkled seeds are represented by the allele r. We can therefore
say that the parental generation had a genotype of RR and rr and the F1 generation
has the genotype Rr. When these F1 plants are self-pollinated, 1/4 seeds are wrinkled
(rr) and 3/4 are round (RR and Rr) in the resulting F2. On further self-pollinating the
F2 generation, he observed that all the wrinkled seeds gave rise to wrinkled seeds on
selfing. This can be explained by the fact that as the genotype of wrinkled seeds is rr,
they will always form gametes carrying the allele r. Therefore, on self-pollination,
2 Mendelian Principle of Inheritance 59

Fig. 2.3 Figure illustrating

monohybrid cross between
plants having yellow and
green seed colour: The
parental generation is
homozygous and produces
only one type of gametes.
These get united in the F1
generation resulting in all
plants having yellow coloured
seeds. The F1 hybrids on
selfing give rise to yellow and
green seeds in a ratio of 3:1.
The genotypic ratio is 1:
2:1 (The Punnett Square
Approach for a Monohybrid
Cross 2020)

they will always give rise to wrinkled seeds. Among the round seeds, 1/3 of the
plants gave rise to only round seeds on self-pollination. The remaining 2/3 gave rise
to a mix of round and wrinkled seeds in the ratio of 3:1. It follows that the seeds
always giving rise to round seeds had the genotype RR. The remaining 2/3 seeds had
the genotype Rr which, similar to the F1 generation, gives rise to the seeds in a ratio
of 3:1. The results of these F3 generations give further evidence to support Mendel’s
hypothesis. These analytic approaches followed by Mendel were one of the strongest
reasons why Mendel was able to come up with a reasonable hypothesis and support
his claims through further experimentation (Fig. 2.4).

2.1.5 Test Cross: One Character

Homozygotes for the dominant allele as well as heterozygotes will display the
dominant trait. To verify if the plant was homozygous or heterozygous for the
dominant allele, Mendel crossed it with a plant showing the recessive trait and
observed their progeny. This cross between plants showing dominant phenotype
60 D. Patwardhan

Fig. 2.4 Monohybrid cross

till F3 generation shows
Mendel’s analytic approach:
Mendel crossed plants with
round seeds and wrinkled
seeds and obtained F1
generation having all round
seeds. On selfing the F1
generation, he obtained the F2
generation in a ratio of
3 round:1 wrinkled as
expected. He further allowed
the F2 to undergo self-
pollination to obtain the F3
generation. He found that
wrinkled seeds always gave
rise to wrinkled seeds. Of the
round seeds, 1/3 always gave
rise to round seeds and the
remaining 2/3 gave rise to
seeds in the ratio of 3 round:1
wrinkled like the F2
generation. This provides
further evidence to support
Mendel’s theory of
segregation and
dominance (Pierce 2010)
2 Mendelian Principle of Inheritance 61

of unknown genotype with a plant of homozygous recessive genotype is called a test

cross. Following Mendelian laws, if the plant being examined is homozygous for the
dominant allele, all its progeny with the homozygous recessive parent will display
the dominant phenotype. In the example below, if the purple flower is homozygous
(having the genotype PP), it will produce gametes having P, and so all the F1 plants
will show purple flowers. If, on the other hand, the purple flower is heterozygous
(having the genotype Pp), it will produce two types of gametes having P or p. These,
on crossing with p from the recessive parent, will give progeny with either purple or
white flowers in a ratio of 1:1.
One of the recessive alleles will be provided by the parent showing the recessive
phenotype as it will only produce gametes with the recessive allele. If a plant appears
in the progeny displaying the recessive phenotype, it is clear that the other parent was
carrying the recessive allele implying that the parent was heterozygous. If all the
progeny show the dominant trait, the other parent has only provided gametes having
the dominant allele implying that the parent was homozygous for the dominant
allele. Test cross is a powerful method to examine unknown genotypes of an
organism showing the dominant trait. This information is useful for breeders who
would wish to choose homozygous plants for further breeding but cannot estimate
the genotype based on observation of phenotype alone (Fig. 2.5).

2.2 Mendelian Dihybrid Cross

We have seen the results of crosses between plants differing in one trait. Mendel’s
next step was to study the pattern of inherited traits in crosses of plants differing in
two traits. Let us take two traits in the pea plant: round vs wrinkled seeds and green
vs yellow seeds. When Mendel crossed green round seeds with yellow wrinkled
seeds, he observed that all the F1 progeny were yellow and round. In the monohybrid
cross for each of the above traits, the F1 progeny expressed the dominant trait which
was yellow colour and round shape. In the dihybrid cross too, the F1 hybrids
expressed the two dominant traits. On selfing the F1 hybrids, he observed four
phenotypes in the F2: yellow and round, green and round, yellow and wrinkled and
green and wrinkled. On counting the number of plants in each category, he surmised
that the plants were approximately in a ratio of 9:3:3:1 for the above combination of
traits.
To make sense of the ratio that he obtained, Mendel made some logical
deductions. He counted the number of yellow vs green and round vs wrinkled
seeds and observed that they were in a ratio of 3:1 similar to the monohybrid
cross. Mendel deduced that of all the F2 plants, 3/4 had yellow seeds and the
remaining 1/4 had green seeds. Of the 3/4 yellow seeded plants, 3/4 had round
seeds and 1/4 had wrinkled seeds. Similarly, 3/4 of the green seeds had round seeds
and 1/4 had wrinkled seeds. This calculation gives the 9:3:3:1 ratio seen above. It
appeared therefore that the dihybrid cross was a combination of 3:1 ratio for two
traits. This will be easier to understand in the branched diagram in Fig. 2.6.
62 D. Patwardhan

Fig. 2.5 Test cross involves crossing of a plant of unknown genotype with a plant showing the
recessive trait: If the parent plant is homozygous, all its progeny will show the dominant trait. If the
parent plant is heterozygous, half of its progeny will show the dominant trait and the other half will
show the recessive trait. In the above illustration, test cross of a homozygous purple flower plant
will result in all its progeny showing purple flowers. Test cross of a heterozygous purple flower
plant will give flowers in the ratio of 1 purple:1 white (Reece et al 2011)

2.2.1 Independent Assortment

Mendel performed the dihybrid cross for a number of combinations of traits and
always got a phenotypic ratio of 9:3:3:1. Let us now understand this ratio in
biological terms. When a plant having yellow wrinkled seeds with the genotype
YYrr is crossed with a plant having green round seeds with the genotype yyRR,
hybrids with yellow round seeds having the genotype YyRr are produced. These
hybrids can produce four gametes having four different combinations: YR, Yr, yR
and yr. Each gamete carries one allele for each trait. On self-pollination, these
gametes can merge in a variety of different combinations giving a phenotypic ratio
of 9:3:3:1 as seen in the figure below (Fig. 2.7).
The fact that the dihybrid cross ratio is a combination of 3:1 ratio for each trait
tells us that the gametes for each trait can assort independently. It means that allele Y
has equal probability of pairing with either allele R or r to form a gamete. If one of
the alleles in the cross above assorted preferentially with another allele, we would
2 Mendelian Principle of Inheritance 63

¾ round seeds ¾ X 3/4 = 9/16 yellow round

seeds
¾ yellow seeds
¼ wrinkled seeds ¾ X ¼ = 3/16 yellow
wrinkled seeds

¾ round seeds ¼ X ¾ = 3/16 green round

seeds
¼ green seeds
¼ wrinkled seeds ¼ X 1/4 = 1/16 green
wrinkled seeds

Fig. 2.6 Phenotypic ratios obtained in a dihybrid cross: Each of the traits gives a 3:1 ratio. In the
example above, we obtain seeds in a ratio of 3 yellow:1 green seeds. Each of these phenotypes also
shows a ratio of 3 round:1 wrinkled seeds. This gives the overall 9:3:3:1 phenotypic ratio seen in a
dihybrid cross

not obtain a phenotypic ratio of 9:3:3:1. This is called the law of independent
assortment which states that different gene pairs can assort independently during
gamete formation. However, genes which are close to each other on the same
chromosome do not assort independently because they are held together on the
same chromosome. In this case, alleles for different genes which are on the same
chromosome always assort together during meiosis. The modified law of indepen-
dent assortment can therefore be stated as ‘Gene pairs present on different
chromosomes assort independently of each other during formation of gametes’.
The tendency of genes which are close to each other to be inherited together is
called linkage. Genes which get inherited together are classified into a single linkage
group. Therefore, if any of the genes studied by Mendel belonged to same linkage
group, their phenotypic ratios would have differed from the ones defined by Mendel.
Mendel did not observe linkage between the genes that he studied and hence put
forth the law of independent assortment. Mendel’s work has been criticised on the
basis that his data fits too well with his hypothesis and does not show as much
variation due to chance as expected. Mendel’s critics also cite the lack of evidence of
linkage as one of the reasons to doubt Mendel’s work. Recent work has, in fact,
shown that the seven traits that Mendel studied belong to five different linkage
groups of which only stem length and pod form show strong linkage. Mendel might
have been lucky in his choice of traits for dihybrid cross. He might not have
performed dihybrid crosses for this particular combination of stem length and pod
form, or he would have been surprised by the resulting phenotypic ratios. Seed shape
and pod colour show weak linkage, and all other traits are not linked allowing
Mendel to obtain the same ratio for most of his dihybrid crosses. The debate about
the validity of Mendel’s work is discussed in detail in Box 2.1.
64 D. Patwardhan

Fig. 2.7 Genotypes of progeny obtained from a dihybrid cross for wrinkled yellow seeds with round
green seeds: The F1 progeny has yellow round seeds and can produce four types of gametes. These
are shown on the top row and first left column of the square on the right. The various combinations of
these gametes give a progeny with a phenotypic ratio of 9:3:3:1 (Griffiths et al. 2011)
2 Mendelian Principle of Inheritance 65

2.2.2 Test Cross: Two Characters

Similar to the monohybrid cross, Mendel performed test cross for the dihybrid cross
to verify his conclusions. As stated above, test cross involves crossing a plant of
unknown genotype with a plant homozygous recessive for the traits under consider-
ation. If we were to perform a test cross for the F1 produced from the dihybrid cross
above, the tester (homozygous recessive individual) would be a plant having green
and wrinkled seeds. In this case, we would expect the F1 to form gametes with all the
combinations, RY, Ry, rY and ry, according to the law of independent assortment.
This when fertilised with ry gametes from the tester would give the following
different combinations: RrYy (round, yellow), Rryy (round, green), rrYy (wrinkled,
yellow) and rryy (wrinkled, green). A phenotypic ratio of 1:1:1:1 would be expected.
This is the result that Mendel obtained from his test cross for two characters
providing evidence for his law of independent assortment.
The F1 hybrid obtained above is heterozygous for both traits. Let us see how the
results would differ if the plant being tested was homozygous for either of the traits.
If we take a plant having yellow, round seeds, its genotype could be either YyRr
(discussed above), YYRr, YyRR or YYRR. If it is YYRR, it will produce only one
type of gamete YR which when crossed with yr will produce all plants of YyRr
genotype and single phenotype of yellow round seeds. If it is YYRr, two types of
gametes will be produced: YR and Yr. This will give two phenotypes on test cross:
yellow, round seeds (YyRr) and yellow, wrinkled seeds (Yyrr) in a ratio of 1:1.
Similarly, if the genotype is YyRR, it will produce two phenotypes on performing
test cross: yellow, round seeds (YyRr) and green, round seeds (yyRr) in a ratio of 1:
1. In this manner, we can detect the genotype of the individual based on the number
and ratio of phenotypes produced (Fig. 2.8).

2.3 Mendelian Trihybrid Cross

Mendel’s laws can be extended to obtain genotypic and phenotypic ratio of a

trihybrid cross, i.e. a cross between plants differing in three traits. As the number
of traits increase, the complexity of the ratio increases and several different methods
can be used to predict the results. Let us take the example of three genes A, B and C
having recessive alleles a, b and c. If a homozygous dominant plant with genotype
AABBCC is crossed with a homozygous recessive plant aabbcc, the F1 hybrid will
have a genotype of AaBbCc. When the F1 plant is further self-pollinated, it will
produce eight different gametes as shown below (Fig. 2.9).

2.4 Application of Mendelian Principles

Mendel’s principles of segregation, dominance and independent assortment can be

applied to analyse inheritance of multiple traits. Various methods like Punnett
square, forked line method or probability method can be utilised to obtain
66 D. Patwardhan

YYRr YyRr YyRR

YYRr yyrr YyRr yyrr YyRR yyrr

yr yr yr

YR YyRr YR YyRr YR YyRr

Yr Yyrr Yr Yyrr yR yyRr

yR yyRr

yr yyrr

Fig. 2.8 Test cross for a dihybrid cross can reveal the parental genotype: Yellow round seeds with
three different genotypes (YYRr, YyRr and YyRR) give rise to different phenotypes and pheno-
typic ratios in the progeny when subjected to a test cross. This allows us to estimate the genotype of
the unknown individual. (Adapted from Klug et al. 2012)

Fig. 2.9 Gametes produced during a trihybrid cross: Eight different gamete combinations of alleles
and therefore eight different types of gametes may be produced by an individual heterozygous for
three gene pairs (Klug et al. 2012)
2 Mendelian Principle of Inheritance 67

phenotypic and genotypic ratios of cross between multiple traits. We will obtain the
genotypic ratios for the trihybrid cross mentioned above using both Punnett square
and forked line method.

2.4.1 The Punnett Square Method

In the Punnett square method, a grid is created with the gametes from one parent on
the upper side and those from the other parent on the left side. Each cell or block
within the grid contains a combination of alleles from both parents giving the
genotype of the offspring resulting from the combination of the respective gametes.
It is named after Reginald C Punnett who devised this method. It is a tabular
representation of all possible combinations between the maternal and paternal
alleles.
In the image below, gametes produced from F1 hybrid of a trihybrid cross are
represented on the top row and left column of the grid. Since this is a self-pollination,
the gametes on both sides are identical. In case the parents differ in genotype,
gametes from one parent will be in the top row and will differ from gametes
produced by the other parent which will be on the left column. The first box in the
grid shows the genotype AABBCC which is produced if the gametes ABC and ABC
(corresponding parental gametes in first row and first column) were to combine. In
this manner, all possible genotypes of the progeny can be represented in the Punnett
square in a simplified manner. The genotypes showing the same phenotypes are
represented by the same colour of the cell in the grid. From this, we can infer that the
phenotypic ratio of a trihybrid cross will be 27:9:9:9:3:3:3:1 (Fig. 2.10).

Fig. 2.10 Punnett square showing genotypes produced from a trihybrid cross: The genotypic and
phenotypic ratio for a cross between individuals of genotype AaBbCc and AaBbCc is shown in the
Punnett square above. The gametes produced from each parent are shown in the top row and left
column. Each grid represents a combination of the gametes in the respective row and column. The
individuals showing the same phenotype are in the same colour. We can see that a phenotypic ratio
of 27:9:9:9:3:3:3:1 is obtained
68 D. Patwardhan

2.4.2 The Forked Line Method

Although useful, Punnett square can be too cumbersome to use when more than
three traits are being analysed. In such instances, forked line method, also called the
branch diagram, can be very useful. In this method, the genotypic or phenotypic
outcome for one gene pair is first predicted. Then, outcome of the next gene pair is
computed in conjunction with earlier gene pair. This method is followed for all the
remaining gene pairs. In the figure below, phenotypic ratio of trihybrid cross is
predicted using the forked line method. Here, three traits are considered: round vs
wrinkled seeds, green vs yellow seeds and grey-brown vs white seed coat. We know
the dominant phenotype for the other traits except seed coat where grey-brown is
dominant over white. According to Mendel’s law of segregation and random
fertilisation, a monohybrid cross between round and wrinkled seeds will result in
3/4 seeds being round and the remaining 1/4 being wrinkled. This is the outcome for
the first trait. Now, of the round seeds, 3/4 seeds would be yellow and 1/4 green,
which is the outcome for the second trait. Similarly, 3/4 of the wrinkled seeds will be
yellow and 1/4 green. Next, of the round and yellow seeds, 3/4 will have grey-brown
coat and 1/4 will have a white coat. This gives us a total of 27 round, yellow and
grey-white coat seeds (3/4 round
3/4 yellow
3/4 grey-brown coat ¼ 27/64).
Similarly, proportion of round, yellow and white seed coat will be 3/4
3/4

1/4 ¼ 9/16. We can calculate the proportion for all phenotypes in this manner and
obtain a phenotypic ratio of 27:9:9:9:3:3:3:1.
Calculations for genotypic ratio can be done in a similar manner. Although the
phenotypic ratio for a monohybrid cross is 3:1, we need to bear in mind that the
genotypic ratio is 1:2:1 (1 AA, 2 Aa and 1 aa). The genotypic calculation for a
trihybrid cross using forked line method is illustrated below. There are a few rules of
thumb that can be used to cross-check your calculations. First, count the number of
heterozygous gene pairs in the cross. In a cross AaBbCc X AaBbCc, heterozygous
gene pairs 3. 2n will be the number of different gametes that can be produced from
each parent. 3n will be the number of different genotypes produced after fertilisation,
and 2n will be the number of different phenotypes produced in this cross. In the
above example, therefore, 23 ¼ 8 different gametes are formed from each parent,
33 ¼ 27 different genotypes are produced in this cross and it gives rise to 23 ¼ 8
different phenotypes. These are the numbers that we get from our calculations with
the forked line method as well as Punnett square method (Fig. 2.11).

2.4.3 The Probability Method

Probability is a mathematical tool that predicts the likelihood of occurrence of an

event.
We can easily use probability to predict the outcome of a genetic cross. Probabil-
ity can be calculated as the number of times an event occurs divided by the total
number of events. For example, the probability of picking a queen of hearts from a
deck of cards would be 1/52. This is because there is only one queen of hearts (one
2 Mendelian Principle of Inheritance 69

Fig. 2.11 Forked line method for obtaining genotypic and phenotypic ratios in a trihybrid cross:
For a monohybrid cross, genotypic ratios of 1:2:1 are obtained. Then for each of the genotypes, the
ratio for the next set of genes is calculated and further on and the proportions multiplied at the end.
Similarly, phenotypic ratios can be calculated by multiplying 3:1 ratios for each trait (Klug et al.
2012)

event) in a deck of 52 playing cards (all possible events). If, however, we were to
calculate the probability that a card picked would be any queen, this probability
would be 4/52 as there are four queen cards in a deck. There are two rules to be
followed for the calculation of slightly more complicated probabilities.

1. Multiplication rule: The probabilities of co-occurrence of two or more events

can be calculated by the product of their individual probabilities. This rule can
only be used if the events are occurring independent of each other. If the
occurrence of one event affects the probability of occurrence of the next event,
then their combined occurrence cannot be a simple product of their independent
probabilities. For example, the probability that two consecutive rolls of a dice are
a three and a six in that order is 1/6
1/6 ¼ 1/36. The individual probabilities of a
three and a six in a roll of dice are 1/6 and 1/6, respectively. Their product will
give the probabilities of their co-occurrence. We have already used the multipli-
cation rule while calculating the dihybrid and trihybrid cross ratio in the branch
diagram or forked line method. Multiplication rule is applied when the word and
is used. In the above example, we wanted to find the probability of ‘a three and a
six in a roll of dice’.
2. Addition rule: We can calculate the probability of either of two or more mutually
exclusive events occurring together by the addition rule. For example, if we just
wanted to obtain the probability of occurrence of a three or a six in a roll of the
dice, we would find the sum of individual probabilities. Thus, the probability is
1/6 + 1/6 ¼ 2/6 ¼ 1/3.
70 D. Patwardhan

The multiplication and addition rules can be used in predicting the outcome of
genetic crosses instead of Punnett square or forked line method. Let us consider
the cross between two plants having round seeds with genotypes Rr and Rr. The
probability of wrinkled seeds can be calculated using multiplication rule. The
probability of obtaining r allele from one parent is 1/2 and from the other parent is
also 1/2. For a wrinkled seed, the genotype needs to be rr and its corresponding
probability is 1/2
1/2 ¼ 1/4. If we were to calculate probability of round seeds,
both multiplication and addition rules need to be used. Round seeds can occur
because of three genotypes: RR, Rr and rR. Their individual probabilities are as
follows:

1=2 R
1=2 R ¼ 1=4 RR

1=2 R
1=2 r ¼ 1=4 Rr

1=2 r
1=2 R ¼ 1=4 rR

Their combined probabilities would therefore be 1/4 + 1/4 + 1/4 ¼ 3/4 round
seeds. It is easier to use probability method for calculation of complex crosses with
multiple traits as compared to Punnett square or forked line method.

2.4.3.1 Binomial Theorem

Binomial theory can be utilised when we want to calculate the occurrence of a
specific set of outcomes among a large number of potential events. Let us consider
the case of galactosemia. In this disorder, mutation in one of the galactose
metabolising genes prevents the individual from converting galactose to glucose.
The affected individual may show symptoms like lethargy, failure to gain weight and
liver damage. This disorder can only occur if both copies of the gene are mutated.
Thus, the parents of a child may each be carrying one mutated allele and not express
the disorder. Only if the child inherits both mutated copies will he/she be affected.
Let us assume that the gene affected here is a and its wild-type/normal allele is A. If
we now want to find the probability that both kids of heterozygous parents are
affected. The probability of one child being affected (aa) is 1/4 (Aa X Aa ¼ 1/4 aa,
3/4 A-) and that of two children being affected will be 1/4
1/4 ¼ 1/16.
Now, if we suppose the couple has three children and we want to find the
probability that two children are unaffected and one child is affected. There are
three scenarios in which this is possible:

1=4
3=4
3=4 ¼ 9=64 ðchild 1 affected, other 2 unaffectedÞ

3=4
1=4
3=4 ¼ 9=64 ðchild 1 and 3 unaffected, child 2 affectedÞ

3=4
3=4
1=4 ¼ 9=64 ðchild 1 and 2 unaffected, child 3 affectedÞ

Total probability ¼ 9=64 þ 9=64 þ 9=64 ¼ 27=64

2 Mendelian Principle of Inheritance 71

This calculation becomes more complex for situations with more number of
children and multiple different combinations. If we want to find the probability of
this couple having five children, three of whom are affected and the remaining two
are not, we can use the binomial expression. The binomial expression is of the form
(a + b)n where a and b are probabilities of two alternate events and n is the number of
times the event occurs. In the above case, we can define a as the probability that the
child suffers from galactosemia (1/4), while b is the probability that the child remains
unaffected (3/4). n here is the number of children which will be 5. The binomial can
be expanded as follows:

ða þ bÞ5 ¼ a5 þ 5a4 b þ 10a3 b2 þ 10a2 b3 þ 5ab4 þ b5

It follows the rule:

ða þ bÞn ¼ an þ an1 b þ an2 b2 þ an3 b3 þ . . . :bn

The expansion of (a + b)n consists of n + 1 terms. Each of these terms has a

numerical coefficient. The coefficient of the first term is always 1. The second term
has the coefficient same as the power to which the binomial is raised. So, in this case,
it is 5. For the next coefficient, multiply the coefficient of the previous term with the
exponent of a in that term. Divide this by the number of the term in the equation.
Thus, to calculate the coefficient for a3b2 in the above expansion, (5
4)/2 ¼ 10
where coefficient of earlier term is 5 and exponent of a in that term is 4 and it is the
second term in that equation. Similarly for the coefficient of a2b3, it can be calculated
as (10
3)/3 ¼ 10. We can calculate the coefficients for the rest of the terms and
expand the binomial.
Another method to calculate the coefficients in the equation is to use Pascal’s
triangle. We can determine the coefficients for each term in the binomial expression
from the terms in front of the corresponding n. Notice that all terms other than 1 in
Pascal’s triangle are the sum of terms directly above them (Fig. 2.12).
Once we have the equation, we can obtain the probability of any combination of
events by simply inserting the values of a and b. For example, to obtain the
probability of three out of five children having galactosemia, the term we use is
10a3b2:

10a3 b2 ¼ 10
ð1=4Þ3
ð3=4Þ2 ¼ 90=1024 ¼ 0:087

In this manner, we can easily calculate the probability of any combination of

events. There is another method to do the above calculation. It uses the formula:

n!
P¼
as bt
s!t!
P is the overall probability of co-occurrence of two events X and Y. Event X has a
probability of a occurring s times, while event Y has probability b occurring t times.
72 D. Patwardhan

Fig. 2.12 Pascal’s triangle: Pascal’s triangle can be used to obtain coefficients for terms in the
binomial expansion for any n. The terms other than 1 in Pascal’s triangle are a sum of the terms
directly above them

In the above case, X is the probability that the child is affected. Therefore, a is 1/4
and s is 3. Event Y is the probability that the child is unaffected. Here, b is 3/4 and t is
2. N is the total number of events which is 5 in this case. The symbol ! is for a
factorial which is the product of all positive integers from 1 to n. For example, 5! ¼ 5

4
3
2
1.
The calculation therefore is:
5!
P¼
ð ¼Þ 3 ð ¾Þ 2
3!2!
5
4
3
2
1
¼
ð¼Þ3 ð¾Þ2
ð2
1Þð3
2
1Þ
¼ 0:087

This value is the same as that obtained from binomial theorem.

2.5 Test of Genetic Hypothesis

Crosses between two individuals of known genotypes yield a certain genotypic and
phenotypic ratio. Based on Mendel’s laws, we can predict a certain ratio. However,
the experimental ratios may not match the expected values. Other than technical
difficulties (like death of plants before the phenotype can be observed), chance plays
a very important role in this deviation. This is easily illustrated with the example of a
coin flip. We know that the probability of getting a heads or tails in a coin flip is 1:1.
2 Mendelian Principle of Inheritance 73

If we do the coin toss for a large number of times, say 1000, we can expect that we
will get a number close to 1:1. However, if we toss the coin only ten times, we might
get seven heads and three tails or two heads and eight tails. This deviation from
expected ratio is just a chance event.
Genetic ratios however can also be different, if there is some linkage between the
traits being studied or if the gene is following some non-Mendelian pattern of
inheritance. An experimenter needs to know if the deviation from expected ratios
is just a matter of chance or it is of some biological significance. In such cases, we
can make use of a chi-square test.

2.5.1 The Chi-Square Test

Chi-square test, also written as χ 2 test, is used to evaluate how well the observations
support the null hypothesis. It is calculated from the sum of squared errors or sample
variance. A chi-square test can only tell us if the resulting ratio of genetic crosses is
deviating from the expected ratio merely due to chance. It cannot tell us if there is a
mistake during crossing or during calculation of expected ratios or there are some
complex inheritance patterns involved. In other words, it gives us a probability that
the difference in observed and expected ratio can be due to chance alone.
Let us take an example to understand how to use the chi-square test. A monohy-
brid cross between two tall plants resulted in a progeny of 100 tall plants and 40 short
plants. If we were to assume that the genes involved followed a Mendelian inheri-
tance pattern, we would expect a ratio of 3 tall:1 dwarf plants. For a total of
140 plants, 3/4
140 ¼ 105 plants should be tall and 1/4
140 ¼ 35 plants should
be short. We see that the observed ratio differs slightly from the expected ratio. Is
this merely an effect of chance?
We start by establishing a null hypothesis (Ho). The null hypothesis is called so
because it assumes that there is no real difference between our expected and
observed outcomes and any deviation is a result of chance events. Through the
probability derived from the chi-square test, we can then accept or reject the null
hypothesis. Our null hypothesis for this example will be that the inheritance follows
a ratio of 3:1. The formula for chi-square test is:

ðO  E Þ2
χ2 ¼ Σ
E
where E ¼ expected value for that category
O ¼ observed value for that category
Σ ¼ sum of calculated values for all categories
Plugging in the above values in this equation:
74 D. Patwardhan

ð100  105Þ2 ð40  35Þ2

χ2 ¼ þ
105 35
¼ 25=105 þ 25=35
¼ 0:238 þ 0:714 ¼ 0:952

After this calculation, we have to determine the degrees of freedom (df) which is
n  1 where n is the number of different categories the value may fall into. Here, the
plant can be tall or short. Thus, n ¼ 2 and df ¼ 1. Degrees of freedom are considered
because more categories introduce more deviation in the results. We now have to
interpret the χ 2 value in terms of its corresponding probability value. This calculation
is very complex, and we instead make use of a standard table which provides
probability values for different χ 2 values for each degree of freedom.
In the table below (Fig. 2.13), we can see that the calculated value of 0.952 for df
1 lies between p value of 0.5 and 0.1. We can interpret this as the probability that the
observed deviation from expected value is due to chance is between 10 and 50%.
Traditionally, scientists have accepted a p value cut-off of 0.05. That is to say that if
the p value is above 0.05, we can accept the null hypothesis. If the p value is less than
0.05, it means that the probability that the deviation is due to chance is less than 5%.
In this case, the null hypothesis is rejected. In our example, we can accept the null
hypothesis and conclude that the variation seen in the ratios is a product of chance
and that the inheritance indeed follows a 3:1 ratio.

Fig. 2.13 Probability values for χ 2 distribution: Figure giving probability values for estimated
χ 2 values at different degrees of freedom. The probability value keeps decreasing towards the right,
while the χ 2 values keep increasing
2 Mendelian Principle of Inheritance 75

Let us take another example. In a cross between plants having violet flowers and
white flowers, violet flowers were observed in F1. On self-fertilisation, it was seen
that 790 of the progeny had violet flowers and 210 had white flowers. Can we
ascertain if this follows the Mendelian pattern of inheritance?
Null hypothesis—The pattern of inheritance follows Mendelian genetics and does
not differ from a ratio of 3:1:

ðO  E Þ2
χ2 ¼ Σ
E
E: If the flower colour inheritance followed Mendelian genetics, we would see
that 3/4 of the total flowers would be violet and 1/4 would be white since violet is the
dominant character (because F1 flowers were violet). The expected numbers would
therefore be:
Violet 3/4
1000 ¼ 750, white 1/4
1000 ¼ 250

ð790  750Þ2 ð210  250Þ2

χ2 ¼ þ
750 250
¼ 2:133 þ 6:4 ¼ 8:533

Degree of freedom here too is 1, as only two characters are being observed.
P value for χ 2 8.533 at df 1 is less than 0.005
We can therefore reject the null hypothesis. The probability that deviation in ratio
is purely due to chance is very less and the gene is probably following some
non-Mendelian pattern of inheritance.

2.6 Application of Mendelian Principles in Human Genetics

Mendel’s work has shed light on the inheritance of genes and traits. We can use this
knowledge to analyse inheritance of various genetic diseases and traits in humans
too. We can do this by obtaining information regarding occurrence of the trait being
studied in the family of the affected individual.

2.6.1 Pedigree Analysis

Pedigree analysis is similar to a family tree for a specific trait. It is basically a chart
which illustrates which family members have the traits being studied. This aids in
understanding the method of inheritance of the trait. We can also predict the possible
genotype of individuals for that trait which can help in predicting probability of
inheritance of the trait in future generations.
A set of standardised symbols are used for illustrating a pedigree. Squares
represent males, and circles represent females. A shaded box denotes individuals
that expressed the phenotype being studied. A horizontal line between two
76 D. Patwardhan

individuals denotes mating. Their progeny are represented in the order of birth on a
horizontal line connected to the parental mating line. Different generations are
represented on descending levels. A double line connecting two individuals denotes
consanguineous marriage. A marriage between second cousins or even more closely
related individuals is referred to as a consanguineous marriage. Many studies have
shown that consanguinity is one of the major contributors of birth defects and
abnormalities. If an individual has a recessive gene, his progeny might inherit the
gene but not express the phenotype. Thus, individuals belonging to the same family
have a greater probability of carrying the recessive gene. A marriage between
members of a family increases the probability of a child from this union inheriting
two copies of a recessive gene and therefore suffering from a genetic disorder
inherited in an autosomal recessive manner. Although consanguineous marriages
have reduced over the years, they are still prevalent in the Middle East and parts of
Asia and Africa. More symbols and their meanings are given in Fig. 2.14.
Let us examine the pedigree shown in Fig. 2.15. Individual 4 from generation III
is the proband. This means that this individual was the first to be investigated for this

Fig. 2.14 Standard symbols

used while drawing a pedigree
chart: Proband denotes the
first individual studied in the
family for this trait.
Consanguineous marriage
means marriage between
individuals from the same
family, usually first
cousins (Klug et al. 2012)
2 Mendelian Principle of Inheritance 77

Fig. 2.15 Pedigree showing autosomal recessive mode of inheritance: The pedigree shown above
shows that the character has skipped a generation. Fewer members of the family are affected and the
trait is evenly distributed between males and females. Based on this, we can conclude that the mode
of inheritance of the gene being studied is autosomal recessive (Klug et al. 2012)

phenotype and prompted construction of the pedigree. We can see that one of the
siblings of individual 4 is also affected. None of the parental generation (Gen II) has
any affected members. Among the grandparental generation (Gen I), individual 1 is
affected. We can draw a few conclusions from this information. First is that the trait
being studied is recessive. Based on Mendel’s law of dominance, if the trait was
dominant, at least one parent of the affected individual would have expressed the
trait. Since none of the parents show the trait, they are most likely carriers of the
recessive allele. This skipping of generation in expression of traits is a characteristic
feature of recessive traits. Second, although there aren’t many affected individuals,
the trait seems to be passed equally between males and females (Gen III individuals).
We can therefore assume that the recessive trait is on an autosome. Of the 23 pairs of
chromosomes that are present in humans, 22 are autosomes and 1 pair is a sex
chromosome (X and Y chromosomes). This means that the 22 pairs are inherited
randomly between males and females. However, the sex chromosomes determine
the sex of the individual. In humans, XX determines a female and XY determines
males. Therefore, the inheritance of traits present on sex chromosomes will not
follow Mendelian patterns and instead show different ratios for males and females.
For example, genes on Y chromosomes will only be passed on to males and not to
the females. The probability of inheritance of a mutated gene between males and
females remains the same for an autosomal disorder irrespective of which parent
carries the mutated gene. In case of recessive disorders, however, the probability of
inheritance of mutated gene between the sons and daughters will differ based on
whether the father or the mother is carrying the mutated gene. We will discuss this
further in the next chapter. For the context of this discussion, it is enough to
78 D. Patwardhan

understand that any trait which seems to be passed equally between males and
females is most likely present on an autosome.

2.6.2 Mendelian Segregation

We can also deduce from this pedigree that either individual I-3 or I-4 was hetero-
zygous for the allele being studied. For individual III-4 to be affected, both his
parents need to be heterozygous for the allele in question. Based on Mendel’s law of
segregation, for individual III-4 to be homozygous recessive, he has to inherit one
recessive allele from each of his parents. Individual II-3 could have obtained the
recessive trait from individual I-1 since he was affected. For individual II-4 to be a
carrier, either individual I-3 or I-4 would have to be a carrier as they do not show the
phenotype. We can determine the pattern of inheritance and composition of the
genotype of an individual from a pedigree based on Mendel’s laws of dominance and
segregation.
Some examples of autosomal recessive disorders are cystic fibrosis, sickle cell
anaemia and Tay-Sachs disease. Cystic fibrosis is caused by a defect in both copies
of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The bodily
fluids become thick and sticky. Due to this, the individuals suffer from respiratory
and digestive problems. The abnormal mucous clogs airways and damages the
pancreas. Tay-Sachs disease is a progressive neuronal disorder that affects the
neurons in the brain and spinal cord. It is a rare disease in which infants start
showing symptoms after 3–6 months. Their development slows and they develop
muscle weakness. Progression of disease leads to loss of hearing, paralysis and
seizures. The disease is caused due to two defective copies of the HEXA gene. This
gene codes for the hexosaminidase A enzyme that plays a role in the breakdown of a
fatty substance called GM2 ganglioside. Build of GM2 ganglioside is toxic for the
neurons.
Let us now take the example of an autosomal dominant disorder. A typical
pedigree is shown in Fig. 2.16 for inheritance of an autosomal dominant trait. We
can immediately observe that this pedigree has at least one affected member in each
generation. This is a typical characteristic of inheritance of a dominant allele. We can
also see that the disorder has been passed on to both the males and the females. We
can therefore infer that the allele is present on the autosomes. An example of
autosomal dominant disorder is the Marfan syndrome. Marfan syndrome affects
the connective tissue due to which a number of abnormalities in the heart, bones,
joints, eyes and blood vessels can be observed. Marfan syndrome patients are tall
and slender with long narrow faces. Their arm span exceeds their body height and
they have elongated fingers and toes. Marfan syndrome is caused by a mutation in
the FBN1 gene which codes for the fibrillin 1 protein. Fibrillin 1 is instrumental in
the formation of microfibrils. Microfibrils are threadlike filaments that provide
strength and flexibility to the connective tissue. They also bind to growth factors
2 Mendelian Principle of Inheritance 79

Fig. 2.16 Pedigree showing autosomal dominant mode of inheritance: The pedigree shown above
has an affected member in each generation. A number of members of the family are affected and the
trait is evenly distributed between males and females. Due to this, we can assume that the mode of
inheritance of the trait being studied is autosomal dominant (Griffiths et al. 2011)

and control their release. Absence of functional fibrillin 1 reduces the amount of
microfibrils leading to lack of control in the availability of growth factors. An
excessive amount of available growth factors leads to overgrowth and abnormal
tissue formation. Being an autosomal dominant disorder, the presence of even one
mutated allele is sufficient for the manifestation of this disease.
Neurofibromatosis type 1 is also an autosomal dominant disorder. It is associated
with a range of symptoms. Individuals suffering from the disease show a pigmenta-
tion change with appearance of dark patches of the skin. Benign tumours
(non-cancerous) grow along the nerves in the brain and other parts of the body. In
some cases, these tumours may turn cancerous. Additionally, these individuals may
suffer from hypertension, macrocephaly, skeletal abnormalities and abnormal cur-
vature of the spine. Some affected individuals may develop learning disabilities or
attention deficit/hyperactivity disorder (ADHD). Neurofibromatosis type 1 is caused
due to mutations in the NF1 gene which codes for a protein called neurofibromin.
This protein is produced in the neurons as well as glial cells like oligodendrocytes
and Schwann cells. Neurofibromin acts as a brake for cell division and is known as a
tumour suppressing gene. Non-functional neurofibromin leads to lifting of this brake
and rampant and uncontrolled cell division leading to formation of tumours as seen
in the disease.
80 D. Patwardhan

Observations of the pedigree charts given above will make it clear that we do not
always see expected Mendelian ratios in these inheritances. This is mainly because
we do not have a large number of progeny which can be observed to reach the
expected ratio. The inheritance of gametes is dependent on chance, and as discussed
in chi-square analysis, we can see vastly different ratios than expected for a small
sample size. The second factor is that in a population, some alleles are more
commonly found than others. Most people are carriers of the rare allele and very
few are homozygous for the rare allele. Thus, mating usually happens between
individuals who are either heterozygous or homozygous for the most common allele,
making the appearance of individuals homozygous for the rarer allele very
uncommon.

2.6.3 Genetic Counselling

Pedigree analysis can also be used to predict the probability of the progeny inheriting
a certain trait or disease. Couples having certain disorders running in the family or
who themselves are affected may wish to know the probability of their children
inheriting the disease. Couples with one of their children affected with a genetic
disorder may seek to understand the possibility of their next child having the disease.
Genetic counselling may help in such situations. Genetic counsellors will obtain
information from the couples about affected family members and draw a pedigree.
From this, they can deduce the mode of inheritance and further calculate the
possibility of their unborn offspring inheriting the disorder. They can provide
information and educate as well as address concerns of the family members regard-
ing the disorders and provide support. They can also inform individuals about their
genetic predisposition to certain diseases and lifestyle changes if any that can prevent
or manage the disorder.
The Human Genome Project completed in 2003 was a 13-year-long study aimed
at sequencing the entire human genome. This sequencing was carried out at multiple
labs around the world and DNA was taken from a number of donors. The sequence is
therefore a mosaic and not from any one individual. This prompted the 100,000
genome project in the UK which aims at sequencing 100,000 individuals comprising
people with rare diseases, their families and cancer patients. With the mapping of
these genomes, we can hope to understand more and more about our genes and the
functions that they play in health and disease. We may be able to pinpoint the causes
of a number of genetic diseases which remain unknown till now. Genomic sequences
from patients will aid in developing diagnostics and therapeutics for individuals
suffering from Mendelian disorders. It may allow us to get closer to personalised
medicine where the analysis of an individual’s genome may provide clues as to what
treatment would be most effective for the individual.
2 Mendelian Principle of Inheritance 81

Box 2.1 Scientific Concept: Gregor Mendel’s Genetic Experiments:

A Statistical Analysis After 150 Years (Jan Kalina)
Gregor Mendel is not only regarded as the founder of genetics but also as one
of the pioneers of applying statistical principles in their experiments. Mendel’s
parents were peasants and bred fruit trees. After completing his secondary
education, Mendel joined the St Thomas Augustinian abbey where he was
ordained as a priest after some years. The abbey fostered an interest in science
and encouraged Mendel to carry out his plant breeding experiments for which
he was also given the use of one of the greenhouses in the abbey. In 1866,
Mendel published his landmark paper which defined basic laws of genetics.
He could not, however, enjoy the recognition of his work in his lifetime.
Mendel passed away in 1884, aged 61 years, due to chronic nephritis. His
work was rediscovered in 1900 independently by three scientists. In 1936,
Robert Fisher reanalysed Mendel’s data in light of the new statistical methods
available. After a detailed analysis of the data, he concluded that Mendel’s
data seemed to be too much in agreement with his theoretical expectations.
Given the variation caused by chance, his data seemed ‘too good to be true’.
He therefore concluded that Mendel must have falsified most, if not all, of his
data to fit his theoretical assumptions (Kalina 2016).
Since then, a large number of papers have come out analysing both sides of
the debate. In 2008, many scientists from different fields came together to
write a review on the subject hoping to put an end to the argument. The authors
were able to refute all arguments except the too good to be true claim for the
data. There were few Mendel supporters like Pilgrim, who found fault with the
way in which Fisher had performed his analysis (Pilgrim 1986). However, his
arguments were later refuted by Edward (Edwards 1986). Then, there was a
group of people who believed Fisher’s analysis but found it too stringent and
sought ways to analyse the data in an alternative manner. They too however
found that Mendel might have adjusted the data to suit his hypothesis. Then
there are those who believe that Fisher’s analysis holds true given the
assumptions. However, they try to provide suggestions for explaining the
high p values other than deliberate malpractice. Some of the explanations
provided are stopping experiments when the results seem to be good, error
carried out by some anonymous assistant and discarding plants due to suspi-
cion of some errors like pollen contamination and data selection for presenta-
tion (Novitski 2004).
In spite of the range of explanations provided, the bias towards expected
values seen in Mendel’s data has not been completely resolved. New statistical
models to explain the bias are being proposed to provide a satisfactory
explanation and end the controversy regarding falsification in Mendel’s data.
Based on the distribution of p values observed in Mendel’s data and different
simulation approaches, a plausible explanation of a two-stage model has been

(continued)
82 D. Patwardhan

Box 2.1 (continued)

put forth. It states that Mendel repeated some of his experiments, presumably
those which showed the largest deviation from expected values. He then
reported only the best of two or a combination of results of the two which
would result in values closer to those that were expected. This is called as a
two-stage model where experiments were performed and results evaluated in
second stage again for those experiments which did not meet the distribution.
This speaks of an unconscious bias that Mendel introduced in his experimental
approach but cannot be called as intentional scientific fraud (Pires and Branco
2010; Kalina 2016). Mendel’s laws have stood the test of time and are still
accepted today. His data might have been biased or adjusted but his
conclusions about the nature of genes and their inheritance are still relevant
today. We must therefore focus on Mendel’s contribution to the field of
genetics and quantitative biology and put an end to the debate as we seem to
have some possible explanations.

2.7 Summary

• Gregor Mendel’s painstaking decade long experiments and theories derived from
them have laid the foundation of genetics, and he is known as the father of
genetics. Mendel’s three laws of genetics provide a framework for understanding
the inheritance of genes. His work was rediscovered independently by three
botanists in 1900.
• In monohybrid crosses, plants which differed in only one trait were crossed. The
F1 hybrids carry the alleles for both parental traits but only express one of the
parental traits which was termed as the dominant trait. The parental trait that was
not expressed in the F1 hybrid was termed as the recessive trait. This was called as
the law of dominance.
• The F1 hybrid produces gametes possessing the dominant and recessive alleles
with equal probability. These can get paired randomly in F2 generation. This was
called the law of segregation of alleles.
• Mendel also carried out dihybrid crosses where he crossed plants differing in two
traits. He observed that the traits were inherited independently of each other. This
was referred to as the law of independent assortment.
• He developed the method of test cross to determine the genotype of plants. Plants
showing a dominant trait may either be heterozygous or homozygous for the
dominant allele. Test cross involved crossing the plant in question with a plant
showing the recessive trait. The ratio and phenotype of progeny from this cross
could indicate heterozygosity or homozygosity of the plant being studied.
• Phenotypic and genotypic ratios for a cross with multiple traits can be predicted
by using the methods of Punnett square, forked line method or probability
2 Mendelian Principle of Inheritance 83

method. Statistical methods like chi-square goodness of fit test can be used to

determine if the observed ratio from a cross vary from the expected ratios purely
due to chance.
• Application of Mendelian principles can contribute to understanding and
predicting genetic disorders in humans. Pedigrees can be constructed for a
particular trait to understand mode of inheritance of a gene. Genetic counselling
may help in counselling parents suffering from genetic disorders who want to
understand the chances of their children inheriting the disease.

References
Edwards AWF (1986) Are Mendel’s results really too close? Biol Rev 61:295–312
Griffiths AJ, Wessler SR, Lewontin RC, Gelbart WM, Suzuki DT, Miller JH (2011) An introduc-
tion to genetic analysis, 10th edn. Macmillan
Kalina J (2016) Gregor Mendel’s genetic experiments a statistical analysis after 150 years. EJBI 12:
20–26
Klug WS, Cummings MR, Spencer CA, Palladino MA (2012) Concepts of genetics, 10th edn.
Pearson Education, Inc.
Novitski E (2004) On Fisher’s criticism of Mendel’s results with the garden pea. Genetics 166:
1133–1136
Pierce BA (2010) Genetics: a conceptual approach. Macmillan
Pilgrim I (1986) A solution to the too-good-to-be-true paradox and Gregor Mendel. J Hered 77:
218–220
Pires AM, Branco JA (2010) A statistical model to explain the Mendel–Fisher controversy. Stat Sci
25:545–565
Reece JB, Meyers N, Urry LA, Cain ML, Wasserman SA, Minorsky PV, Jackson RB, Cooke BJ,
Campbell NA (2011) Campbell biology. Pearson, Frenchs Forest, NSW
Reid JB, Ross JJ (2011) Mendel’s genes: toward a full molecular characterization. Genetics 189:
3–10
The Punnett Square Approach for a Monohybrid Cross (2020) August 15. https://bio.libretexts.org/
@go/page/13264
Extension of Mendelism
3
Rohini Keshava

3.1 Multiple Alleles

Mendelian principles of heredity dealt with the segregation of genes that had only
two alternative forms (two alleles) at a gene locus. However, many genes exist in
several alternative forms. In other words, any given gene can have several alternative
variants/alleles, which occupy the same locus on the chromosome. When a gene
exists in more than two alternative forms, it is called multiple allelism, and the allelic
forms are called multiple alleles. Such alleles are said to constitute multiple allelic
series. However, any given individual can possess only two of such alleles, on a pair
of homologous chromosomes. Both homologous chromosomes can carry the same
allele (homozygous) or carry different alleles (heterozygous).

3.1.1 ABO Blood Groups

Several genes in humans consist of multiple alleles. One of the best examples of the
multiple allelic series in humans are those that determine the ABO blood grouping
systems, i.e., multiple alleles of the ABO gene locus determine the ABO blood
groups. Presently, as per the International Society of Blood Transfusion, there are
about 33 blood group systems. However, of these 33 systems, one of the most
significant is the ABO blood group system. It is of great clinical importance,
particularly in transfusion medicine. The ABO system comprises of four different
blood groups, viz., A, B, AB, and O. Worldwide distribution studies of these blood
groups have shown that O group is the most common, and is followed by B, A and
AB groups in the descending order of their abundance.

R. Keshava (*)
Ramaiah University of Applied Sciences, Bangalore, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 85

Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_3
86 R. Keshava

3.1.2 The A and B Antigens

Characterization of the blood groups depends on the specific antigens located on the
red blood cells (RBCs), i.e., erythrocytes. Currently known 33 blood group systems
are represented by more than 300 antigens. The multiple allelic series of the ABO
system determine the type of antigens on the RBCs, which in turn determines an
individual’s blood group. In an individual, the allelic pair occupying the ABO locus
determines his/her blood group. Any given individual will belong to any one of the
four blood groups. The two main antigens of the ABO blood system are called as
“A” and “B” antigens. The presence or absence of these determines the type of blood
group.
The multiple allelic series of the ABO blood group locus consists of three alleles,
IA, IB, and i. Alleles IA and IB code for antigens A and B. Allele i does not code for
any antigen. The dominance relationship among the ABO alleles can be represented
as follows: IA > i, IB > i, IA ¼ IB. This is an example of both dominance and
codominance. When present in an allelic pair, both IA and IB are dominant, i.e., both
phenotypes are equally expressed, and hence called codominant (refer to Sect.
3.2.4), whereas allele i is recessive to both these alleles. Hence, within this multiple
allele series, the dominance relationship can be depicted as follows: IA ¼ IB > i.
Presence of only IA or IB, in homozygous condition (IAIA or IBIB), or in hetero-
zygous condition (IAi or IBi), results in the expression of A or B antigen, and hence
the individual will be of either A or B blood group, respectively. If both IA and IB
alleles are present in an individual, both A and B antigens are expressed and the
individual is said to be of AB blood group. The genotype ii produces neither A nor B
antigen on the RBCs and is said to be of O blood group. The possible genotypes and
their corresponding blood types are shown in Table 3.1.
An antigen is a substance identified as foreign by the body and hence generates an
immune response. In response to antigens, the immune system produces proteins
called antibodies as a defense mechanism against the antigens. These antibodies
possess the ability to specifically bind the antigens against which they are produced.
The specific binding of the antibodies to the antigens results in large molecular
aggregates that may precipitate. Such an antigen-antibody reaction is called as
agglutination. However, an organism does not produce antibodies against its own
antigens due to a mechanism called tolerance. For example, an individual whose
blood group is A does not produce antibodies against antigen A, i.e., does not
produce anti-A antibodies. Similarly, B group person will not produce anti-B
antibodies and an AB group person will produce neither anti-A nor anti-B antibody.
This also implies that an A group person will produce anti-B antibodies, an B group

Table 3.1 ABO blood Genotype Blood type

typing system—genotypes
IAIA or IAi, A
and corresponding
phenotypes IBIB or IBi B
IAIB AB
ii O
3 Extension of Mendelism 87

Fig. 3.1 ABO blood groups and transfusion compatibilities. (Left to right) Phenotype/blood group,
and their corresponding genotypes, blood antigens, and serum antibodies of the respective blood
groups, and their respective transfusion compatibilities are given

person will produce anti-A antibodies, and an O group person will produce both anti-
A and anti-B antibodies.
Although antibodies generally are not produced without prior exposure to that
particular antigen, the ABO system is an exception. In this case, the serum of
individuals with A and B blood groups will possess anti-B and anti-A antibodies,
respectively, even without prior exposure to these antigens. Likewise, both
antibodies (anti-A and anti-B) will be present in the serum of an O group person.
However, it has been suggested that the production of these antibodies in unexposed
individuals is triggered by similar antigens present on the surfaces of commonly
encountered bacteria. Therefore, if an individual is exposed to a non-self, blood
antigen, then he/she is bound to generate an immune response, resulting in an
agglutination or clumping of erythrocytes. For example, when an individual receives
blood of incompatible type, i.e., whose antigens could stimulate an antibody pro-
duction, the blood cells will agglutinate within the blood vessels and several blood
vessels may become blocked. Recipient of such transfusion would go into a shock.
Such a reaction may also be fatal to the recipient of such a transfusion. Therefore,
matching the correct blood type is of great importance in blood transfusion, and
hence the ABO blood grouping system is of high clinical significance (Fig. 3.1).
From Fig. 3.1, it can be seen that a person with blood group A can donate blood to
A or AB group person; likewise, a person with B blood group can donate blood to a
B or AB group person. However, as far as receiving the blood is concerned, A group
can receive from both A and O; likewise, B group can receive from both B and O,
because O group does not possess any antigens and hence does not cause
88 R. Keshava

agglutination in the recipient. It is important to note that although the blood serum of
the O contains both anti-A and anti-B antibodies, it gets rapidly diluted during the
process of transfusion and, therefore, will not cause an agglutination reaction. Due to
these reasons, O blood group is considered as a universal donor, i.e., the O individual
can donate blood to any other ABO type. However, an O group person can receive
blood from only another O individual. An individual with AB blood group is another
exception and is called universal acceptor, because an AB individual can receive
blood from any of the ABO blood types. This is due to the fact that the AB
individuals consist of both A and B antigen on their RBCs and hence do not produce
anti-A or anti-B antibodies.
These agglutination reactions also form the basis for identification of blood
groups in vitro. Such identification is of primary importance and a prerequisite to
choose the compatible blood types during transfusions. In such a reaction, anti-A
antibodies will agglutinate RBCs of blood type A and blood type AB, because both
carry “A” antigen. Similarly, RBCs of both blood type B and AB will be
agglutinated by anti-B antibody. Agglutination with only anti-A antibody indicates
A blood group, and with only anti-B antibody indicates B blood group. However,
agglutination with both indicates AB blood group. If there is no agglutination
reaction with either antibodies, it indicates O blood group (Fig. 3.2).

3.1.2.1 Biochemistry of ABO Blood Antigens

Biochemically, ABO blood antigens are polysaccharide molecules, attached with
proteins, forming glycoproteins. Antigens A and B, of the ABO blood grouping
system, are both formed from a precursor substance called as the H substance or H
antigen that is present in the RBC. The “H” substance is a mucopolysaccharide
which is modified by enzymes encoded by IA or IB or both alleles, depending on
whether the individual is of A, B, or AB blood type. The genes IA and IB produce
enzymes called glycosyltransferases. Each of the glycosyltransferase catalyzes dif-
ferent modification of the H antigen (refer to Sect. 3.1.2.1), which forms the building
block for the A and B antigens. The glycosyltransferases (encoded by IA or IB) attach
either of the two sugar units (N-acetylgalactosamine or galactose) to the terminal
sugars of the H substance, thus producing A or B antigen, respectively (Fig. 3.3).
Hence, people with IAIA and IAi genotypes produce RBCs consisting of “A”
polysaccharide, while those with genotypes IBIB and IBi produce RBCs with “B”
polysaccharide. Likewise, individuals with genotype IAIB produce RBCs with both
“A” and “B” polysaccharides, while those with ii genotype do not produce either of
the polysaccharides on their RBC.
As explained earlier, the IAIB genotype shows codominance. This can be
explained as follows: enzymes coded by both IA and IB alleles are about equally
expressed resulting in the production of both A and B polysaccharides. The H
substance will be modified by whichever enzyme it first interacts with. Once a
particular enzyme modifies the H substance, it will no more be available for any
modification with the other enzyme. This mechanism makes it possible that both A
and B antigens are produced, in about equal proportions. The third allele, i, which is
recessive to both IA and IB alleles, encodes a defective enzyme that is unable to
3 Extension of Mendelism 89

Fig. 3.2 Agglutination reaction in ABO blood groups. Blog group testing by addition of anti-A
and anti-B antibodies is shown. Blood type AB shows agglutination with both antibodies. Blood
type A and B show agglutination with anti-A and anti-B antibodies, respectively. Blood type O does
not show agglutination with either of the antibodies

modify the H substance; hence, in homozygous (ii) condition, RBCs consist of only
H antigen (Fig. 3.3).
Note: Currently, efforts are being made to enzymatically convert blood types A
and B to O, thus increasing availability of universal donor blood. Blood transfusion
is a vital part of health care and it requires cautious blood type matching in order to
avoid adverse effects. Among the ABO blood types, only O is a universal donor.
Therefore, for emergency situations, the O type is critical, but the resources or time
involved in blood typing procedures is limited. Often there is a shortage of supply.
90 R. Keshava

Fig. 3.3 Antigens of the ABO blood grouping system. The ABO antigens are located on the
surface of RBCs and chemically these are carbohydrates. They are formed by glycosyltransferase-
mediated modification of a precursor carbohydrate. Glycosyltransferases are encoded by alleles IA
and IB. The enzyme coded by the i allele is inactive and the precursor carbohydrate remains
unmodified, and is called the H substance. The enzyme coded by the IA allele adds N-
acetylgalactosamine and that of IB allele adds galactose to the precursor H. The precursor H consists
of fucose, galactose and N-acetylglucosamine

As already discussed above, the presence of an added sugar antigen differentiates A

and B from O. A consists of GalNAc and B consists of Gal, additionally on the
H-antigen characteristic of O type RBC. Therefore, removal of additional sugar
antigen using an appropriate glycosidase has been attempted to convert A, B, or AB
RBCs to O type and has met with significant success. The B-to-O was the first
successful conversion and was demonstrated by Goldstein in 1982. Although enor-
mous quantities of enzyme were required, proof of principle was successfully
established and safe transfusions could be carried out in humans. By screening of
bacterial libraries, novel α-galactosidases and α-N-acetylgalactosaminidases have
been identified. This has allowed improvisations in B conversion and also led to
successful A conversion. Screening of metagenomic libraries generated by feces
3 Extension of Mendelism 91

analysis of an AB individual has led to detection of more efficient enzyme systems

for the conversion of A.

3.1.3 The Bombay Phenotype

Bombay blood group and its variant para-Bombay are rare blood phenotype. In
India, the frequency of both phenotypes combined is 1 in 10,000. It is slightly more
common in Taiwan, affecting 1 out of 8000 people. A relatively large number of
individuals with this phenotype were reported on a small French island 800 km east
of Madagascar in the Indian Ocean, called Reunion Island. In Europe, one per
million people possesses this phenotype.
The Bombay phenotype was named after the city Bombay, now known as
Mumbai, in India, where it was first discovered in 1952 by Dr. Y. M. Bhende. An
individual who had a peculiar blood type that reacted to other blood types in a way
that was not observed before was discovered. When the serum from this individual
was tested, it was found to contain antibodies that reacted with all RBCs of ABO
phenotypes (i.e., blood groups O, A, B, and AB). The individual’s RBCs apparently
seemed to lack all antigens of the ABO blood group and an additional antigen that
was earlier unknown. Based on these observations, a research paper was published in
1952, and the paper reported the presence of another antigen, related to the ABO
blood grouping system, in addition to the known A and B antigens. This new blood
antigen was called as the H substance or H antigen, and was found to be the building
block for the A and B antigens of the ABO blood group system (refer to
Sect. 3.1.2.1).
The “Bombay phenotype” refers to individuals whose RBCs lack the H antigen.
Since the formation of A and B antigens is dependent on the presence of the
precursor H substance, the RBCs of individuals with this phenotype also lack A
and/or B antigens. Hence, these individuals produce anti-H, anti-A, and anti-B
antibodies, and therefore during blood transfusion, they can receive blood only
from another Bombay phenotype individual who also lacks the H, A, and B antigens.
The H antigen gene locus is represented by two alternate alleles, H and h. H
antigen is coded by a dominant H allele, whereas the recessive form of this allele is
an amorph, i.e., it does not code for any known product. The HH/Hh genotype codes
for the presence of H antigen, which practically is found on all RBCs and is the
building block for the production of the antigens within the ABO blood group. The
deficiency of H antigen is known as the “Bombay phenotype.” It is also known as hh
blood group or Oh blood group. Being deficient in H by itself does not have any ill
effects, but in case a blood transfusion is required for the individual with such a
blood group, the donor also should be H deficient. If in case a transfusion is
performed with even an O blood group donor, there can be a severe transfusion
reaction. Since H antigen is required for the formation of ABO blood group antigens,
its absence blocks the formation of the ABO blood group antigens. This can be
misleading in paternity cases.
92 R. Keshava

M (mother) F (father)
Blood group ‘A’ Blood group ‘AB’
A A B
Genotype - I i Genotype - I I

Child
Blood group ‘O’
Genotype - ii

Fig. 3.4 Predicted ABO phenotypes and the genotypes of “M,” “F” and child. F’s ABO genotype
does not consist of i allele. Paternity test would reveal that F is not the father of the child considering
inheritance at the ABO locus

For example, consider the following hypothetical situation: “M” is the mother of
a child, whose father “F” is in doubt of his paternity. In this case M’s blood type was
A (genotype IAi), her child’s blood type was O (genotype ii), and F’s blood type was
AB (genotype IAIB). In this case, as the child’s blood group is O, it must inherit an
i allele from the father. Therefore, the possible genotypes of the father are IAi, IBi, or
ii, i.e., the blood group of the child’s father can be A or B or O. As per this situation,
“F” cannot be the father of the child (Fig. 3.4). This could have been the final
conclusion if only the ABO gene locus was considered. However, another gene
locus also plays a role in the expression of the ABO blood phenotypes, i.e., the H
antigen gene locus.
With respect to the H antigen gene locus, both “M” and “F” are carriers of
incomplete H deficiency. In other words, both “M” and “F” are heterozygous, Hh
at the H antigen gene locus. Therefore, their child is homozygous for the recessive
h allele. Due to this, the child will not be expressing the H antigen and hence is
unable to produce any ABO blood group antigens. Hence, despite inheriting the A or
B allele from “F,” the child’s RBC lacks the A and B antigens similar to blood type
O; therefore, it can be concluded that “F” is the father of the child (Fig. 3.5).

3.1.3.1 Biochemistry of H Antigen

As explained earlier (refer to Sect. 3.1.2.1), the biosynthesis of A and B antigens
involves a series of enzymes, called the glycosyltransferases that transfer
monosaccharides, resulting in antigens that are oligosaccharide chains. Similarly,
the biosynthesis of the H antigen also involves such glycosyltransferases. Production
of the H antigen involves a specific fucosyltransferase. Depending upon what alleles
of the ABO system are present in an individual, the H antigen gets converted into
either A, B, or both antigens (refer to Sect. 3.1.2.1). In a person of blood group O, the
H antigen stays unmodified. Hence, the H antigen is characteristically present in
individuals with O blood group.
3 Extension of Mendelism 93

M (mother) F (father)
Blood group ‘A’ Blood group ‘AB’
A B
Genotype - Ii Hh Genotype - I I Hh

Child
Blood group ‘O’
Genotype - hh

Fig. 3.5 Actual ABO and Hh genotypes of “M,” “F,” and child. Both “M” and “F” are heterozy-
gous, Hh. Hence, their child is homozygous hh and lacks H antigen and is unable to produce any
ABO antigens. Considering the H locus in addition to the ABO locus resolves the paternity issue
and determines “F” is the father of the child

Table 3.2 Common and uncommon H phenotypes

Common H phenotypes Uncommon H phenotypes
Secretor (common) Bombay phenotype
• H antigen present on RBC • H antigen absent in RBC
• H antigen secreted in saliva • H antigen absent in saliva
• Anti-H is absent • Anti-H present in serum
• Genotype: H/H or H/h, Se/Se, or Se/se • Genotype: h/h se/se
Nonsecretor (common) Para-Bombay phenotype
• Presence of H antigen on RBC • H antigen weakly expressed on RBC
• H antigen not secreted in saliva • Saliva may or may not contain H antigen
• Anti-H not produced • Anti-H is present in the saliva
• Genotype: H/H or H/h, se/se • Genotype: (H ), Se/Se or Se/se or se/se

Two different loci of the genome encode two fucosyltransferases with similar
substrate specificities, the H locus, consisting of FUT1 gene and the Se locus
consisting of the FUT2 gene. The FUT1 gene is expressed in RBCs. At least one
functional copy (i.e., at least one dominant H allele) of FUT1 gene is required for H
antigen synthesis. The HH or Hh genotypes enable expression of active
fucosyltransferase. The hh genotype makes inactive copies of FUT1 and results in
Bombay phenotype. The Se locus consists of the FUT2 gene, and it is expressed in
secretory glands. Individuals who possess at least one copy of the functional enzyme
(Se/Se or Se/se) are “secretors.” They produce soluble form of H antigen, and are
found in bodily fluids such as saliva. “Nonsecretors” (se/se) cannot produce soluble
H antigen. The FUT2 gene encoded enzyme is also involved in synthesis of Lewis
blood group antigens. The two commonly found H phenotypes are “secretor” and
“nonsecretor,” whereas Bombay and the para-Bombay are less common. The com-
parison of the two is given in Table 3.2.
94 R. Keshava

3.1.3.2 Clinical Significance of Anti-H Antibodies

3.1.3.2.1 Transfusion Reactions

If individuals with anti-H antibodies in their serum receive transfusions of blood that
contains the H antigen (e.g., blood group O), they are at risk of an acute hemolytic
transfusion reaction.

3.1.3.2.2 Hemolytic Disease of the Newborn (HDN)

Theoretically, the maternal production of anti-H antibodies during pregnancy could
cause hemolytic disease in a fetus who has not inherited the Bombay phenotype from
the mother. In practice, such cases of HDN have not been described, possibly due to
the rarity of the Bombay phenotype.

3.1.4 Drosophila Eye Color

Drosophila consists of several multiple allelic genes. Genes that code for eye color
of Drosophila melanogaster are one such example. A series called the “white-eye” is
a good example. It is named so because it consists of alleles of the white locus
(white-eye is one of the first discovered mutations of Drosophila). The series
consists of several alleles, which in homozygous condition produce a series of eye
colors of increasing intensity. The eye colors range from white through yellowish to
red. Heterozygotes of these alleles produce eye colors that are intermediate to that of
the parental homozygotes for those alleles. In this series, the wild type, i.e., the allele
for red eye color is dominant to all the other alleles of the series, whereas the allele
for white-eye color is recessive to all. Some of the alleles of this series and their
symbols are given in Table 3.3. Note that the names of the alleles correspond to the
eye color they produce. The dominance relationship/hierarchy of the series can be
depicted as follows:
W > wco > wbl > we > wch > wa > wh > wbf > wt > w p > wi > w.

Table 3.3 Allelic series of Allele Symbol

“white-eye” locus in
White (recessive to all) w
Drosophila melanogaster
Ivory wi
Pearl wp
Tinged wt
Buff wbf
Honey wh
Apricot wa
Cherry wch
Eosin we
Blood wbl
Coral wco
Red (wild type—dominant to all) W
3 Extension of Mendelism 95

3.2 Dominance Relationship

3.2.1 Dominance

The dominance concept states that among the pair of alleles in a genotype, only the
dominant allele expresses itself in the phenotype and recessive allele gets suppressed
or hidden. In other words, for a given pair of alleles, dominance is a condition in
which a trait is expressed both in the homozygous and heterozygous conditions.
Such an allele that expresses itself in the heterozygous condition is termed as
dominant. The allele which is not expressed in the heterozygous condition is called
recessive. A recessive allele can be expressed only in a homozygous state.
For example, in F1 offspring of a monohybrid cross between round seed (R) and
wrinkled seed (r) plants, plants inherit an R allele from round-seeded parent and an
r allele from wrinkled-seeded parent (Fig. 3.6). But only the round trait encoded by
R allele is observed in all the F1 progeny, i.e., the heterozygote’s phenotype is same
as one of homozygous parent’s phenotypes. Similar results were obtained in a cross
between a tall (T) and short (t) plant (Fig. 3.7), where all the F1 plants were tall. In
these examples, the alleles for round (R) seed and tallness (T) are dominant, whereas
the alleles for wrinkled seeds (r) and shortness (t) were recessive. The dominance
concept is one of the most important conclusions derived by Mendel from the results
of the monohybrid crosses.

3.2.2 Incomplete Dominance

All seven characters of pea plants that were chosen by Mendel clearly exhibited
complete dominance, i.e., there was a clear dominance-recessive relationship among
the pair of alleles. In other words, the dominant and the recessive traits of a character
were clearly distinct, and there were no intermediate phenotypes. But as Mendel
performed further studies, he observed that variations occurred in the dominance
relationships of alleles of a gene. One such cross that he performed was related to the
length of time taken by the pea plants for flowering. He performed a cross with two
homozygous varieties with different flowering time. The flowering time between the
two varieties differed by an average of 20 days. Interestingly, he observed that the F1
offspring of this cross showed a flowering time that was intermediate of the two
parents.
Such a phenomenon where the heterozygote has an intermediate phenotype
compared to its homozygous parents is termed as incomplete dominance. The
phenotype expressed by the heterozygous genotype will be in range between
phenotypes expressed by homozygous genotypes. Incomplete dominance is also
known by alternative terms such as semidominance and partial dominance.

3.2.2.1 Incomplete Dominance in Fruit Color of Eggplants

When a true breeding purple fruit (PP) producing plant is crossed with a true
breeding white fruit (pp) producing plant, all F1 (Pp) heterozygotes produce violet
96 R. Keshava

Fig. 3.6 Mendel’s monohybrid cross depicting the law of segregation and dominance phenome-
non. This is a monohybrid cross between purebred round seed (R) and wrinkled seed (r) pea plants.
All plants of the F1 generation expressed round seeds indicating that round shape of seeds is
dominant over wrinkled shape. The F2 generation produces both round and wrinkled shape plants in
the ratio of 3:1, indicating the segregation of the two alleles
3 Extension of Mendelism 97

Fig. 3.6 (continued)

colored fruit (Fig. 3.8). After selfing F1 plants, the F2 obtained consists of 1/4 purple
(PP), 1/2 violet (Pp), and 1/4 white (pp) (Fig. 3.8). This 1:2:1 phenotypic ratio is
different than the usual 3:1 ratio obtained when alleles with complete dominance-
recessive relationship are involved.

3.2.2.2 Incomplete Dominance in Feather Color in Chicken

Feather color in chicken provides another example of incomplete dominance. When
a homozygous black and homozygous white chicken are crossed, they produce gray
F1 chickens. If these gray F1 are further selfed, they produce F2 in a phenotypic ratio
equal to 1 black:2 gray:1 white.

3.2.2.3 Incomplete Dominance in Skin Pattern of Horses

Leopard spotting trait in horses exhibits incomplete dominance over unspotted trait.
Horses with LL genotype (homozygous dominant) are white with numerous dark
98 R. Keshava

Fig. 3.7 Mendel’s monohybrid cross depicting the law of segregation and dominance phenome-
non. This is a monohybrid cross between purebred tall (T) and short (t) pea plants. All plants of the
F1 generation expressed tallness indicating that tall is dominant over short. The F2 generation
produces both tall and short plants in the ratio of 3:1, indicating the segregation of the two alleles

spots and those with ll genotype (homozygous recessive) are unspotted, whereas the
heterozygote, with Ll genotype, has fewer spots (Fig. 3.9).

3.2.2.4 Mechanism of Incomplete Dominance

Most genes code for enzymes. For example, among a pair of dominant alleles coding
for a particular enzyme, each allele often individually contributes to the total quantity
of the enzyme in a cell or organism. A heterozygote will therefore produce an
intermediate quantity of the enzyme, whereas a homozygote recessive will not
produce the enzyme (Fig. 3.10). This example gives an insight into the mechanism
3 Extension of Mendelism 99

Fig. 3.8 Incomplete dominance in eggplant. The fruit color of the heterozygote is violet, which is
intermediate to the purple and white fruit color of the parents
100 R. Keshava

Fig. 3.9 Incomplete

dominance in horses. The
leopard spotting trait observed
in horses exhibits incomplete
dominance

Fig. 3.10 Mechanism of incomplete dominance. The figure depicts the level of phenotypic
expression (e.g., enzyme production) in a state of dominance and incomplete dominance

underlying incomplete dominance. Therefore, in incomplete dominance, the

heterozygote’s phenotype is intermediate to phenotypes of homozygous parents.
A typical example of incomplete dominance with such an underlying mechanism
is related to the inheritance of flower color in Antirrhinum (snapdragon). Flowers of
the wild-type plant produce a red colored anthocyanin pigment. The pigment is the
end product of a sequence of enzymatic reactions. The dominant allele I encodes the
wild-type enzyme, and is limiting to the rate of complete biosynthetic reaction.
Therefore, the quantity of red pigment produced is proportional to the amount of
enzyme expressed by I allele. An inactive enzyme is coded by the recessive i allele,
3 Extension of Mendelism 101

and homozygous recessive (ii) flowers are ivory in color. The heterozygous (Ii)
condition leads to a reduction in the quantity of the critical enzyme due to the
presence of a single I allele, as compared with the amount of enzyme in the flowers
of the wild-type homozygous for the dominant allele (II). Due to the reduction in the
amount of the enzyme, the amount of red pigment is also reduced, compared to the
wild-type. As a result, the flower color of the heterozygote is diluted and appears
pink, which is intermediate of red and white (Fig. 3.11). When snapdragon plants
with different flower color, such as a pure-breeding red-flowered variety and a pure-
breeding ivory-flowered variety are crossed, F1 plants produce pink flowers. The F2
progeny produced red, pink, and white flowers in the ratio of 1:2:1, respectively.
Further, among the F2, the red-flowered plants produced only red-flowered plants,
and similarly the ivory-flowered plants produced only ivory, whereas the pink-
flowered plants produced again 1/4 red:1/2 pink:1/4 ivory.
Similar to snapdragons, Mirabilis jalapa (four o’clock plant) also shows another
example of incomplete dominance. In this plant also, a cross between red flower
producing and white flower producing plants results in pink-flowered offspring. If
these pink-flowered F1 are further selfed, an F2 with the ratio of 1 red:2 pink:1 white
is obtained. Similar to that observed in snapdragons, in this case also, pink flower is
heterozygous and is intermediate of red and white homozygotes.
Incomplete dominance is frequently observed in quantitative phenotypes rather
than in discrete phenotypes. A quantitative trait is a trait where it is possible to
measure the phenotype on a continuous scale. In quantitative traits, the phenotype of
heterozygous offspring is a measured value, usually falling in the range between
phenotypes of homozygotes. Traits such as number of eggs laid, flowering time,
amount of enzyme, height, weight, etc. are examples of quantitative traits. On the
contrary, a discrete trait is either fully expressed or not expressed, and there is no
intermediate phenotype. Round/wrinkled and yellow/green with respect to seeds in
pea plants are examples of discrete traits.

3.2.2.5 Examples of Incomplete Dominance in Humans

A rare autosomal recessive genetic disorder, called Tay-Sachs disease, is
characterized by a progressive neurodegeneration in the brain and spinal cord.
Usually, infants get affected by the common form of this disease. In homozygous
recessive condition, it causes death before the age of 3 in children. However,
interestingly, the heterozygous individuals do not express the disease phenotype
and appear normal.
The manifestation of the disease is due to a defective enzyme called
hexosaminidase A, which is required for proper lipid metabolism. Enzyme activity
is completely lacking in homozygotes for this recessive mutation, whereas about half
the normal activity is observed in the heterozygotes. The individuals who are
homozygous for the normal allele show full level of the enzyme. Since the hetero-
zygote expresses an intermediate of the phenotypes of the homozygotes, this is a
case of incomplete dominance.
102 R. Keshava

Fig. 3.11 Incomplete dominance in snapdragons. Flower color in snapdragons shows incomplete
dominance, where the flower color of the F1 heterozygote (pink) is intermediate to the red and white
flower colors of homozygous parents

3.2.3 Complexities Involved in the Concept of Dominance

Although all traits described by Mendel had clear dominant-recessive patterns,

examples of departures from strict dominance are also commonly observed, for
example, even for a classical trait such as round versus wrinkled seeds in pea plants.
3 Extension of Mendelism 103

It is observed that to just consider round as dominant is an oversimplification of the

phenomenon.
Owing to the fact that one gene controls several functions, which is often the case,
the alleles that are responsible for the seed shape in peas not only determine the seed
shape but also determine several other attributes. In the visible phenotype, round
appears dominant over the wrinkled, and it only suggests that visually the effect of
WW and Ww genotypes cannot be distinguished and they both appear round. But at
the molecular level, they exhibit differences.
The shape of the wrinkled seeds is due to a genetic defect. The defect results in the
deficiency of an active form of an enzyme called starch-branching enzyme I (SBEI).
This enzyme is required for the synthesis of a branched-chain form of starch called as
amylopectin. Compared with homozygous WW seeds, the heterozygous Ww seeds
have only half the amount of SBEI. The seeds that are homozygous for the recessive
allele, i.e., ww, have nearly no SBEI (Fig. 3.12).
Homozygous WW pea seeds contain large, well-rounded starch grains, and hence
the seeds efficiently retain adequate water and shrink uniformly as they ripen, and do
not become wrinkled. In homozygous ww seeds, the starch grains lack amylopectin
and are irregular in shape. When these seeds ripen, they lose water very rapidly and
shrink unevenly, resulting in the wrinkled phenotype (Fig. 3.12). The w allele also
affects the shape of the starch grains in Ww heterozygotes. In heterozygotes, the
seeds contain starch grains of intermediate shape (Fig. 3.12), and these seeds have
high enough amylopectin content to enable uniform shrinking and thus prevent
wrinkling (Fig. 3.12).
From these observations, it is clear that same pair of alleles can show complete
dominance for one trait and show incomplete dominance for another trait. In this
particular example, if only the overall shape of the seeds is considered, round is
dominant over wrinkled and there are only two phenotypes. But if the shape of the
starch grains as observed with a microscope is also considered, all three genotypes
can be distinguished from each other, i.e., WW, with large, rounded starch grains;
Ww, with large, irregular grains; and ww, with small, irregular grains. If we also
consider the amount of the SBEI enzyme, the Ww genotype has an amount interme-
diate between the amounts in WW and ww genotypes (Fig. 3.12).
This particular example makes it clear that “dominance” is not a simple interac-
tion between a particular pair of alleles irrespective of how the resulting phenotypes
are observed. When a gene affects multiple traits, then a particular pair of alleles
might show simple dominance for some traits but may differ in others. A given
phenotype consists of many different physical and biochemical attributes, and
dominance may be observed for only some of these attributes and not for others.
Thus, dominance is a property of a pair of alleles in relation to a particular attribute of
a given phenotype (Table 3.4).
104 R. Keshava

Fig. 3.12 Complexities involved in dominance where alleles affect multiple traits. Alleles W
(round seed) and w (wrinkled seed) affect seed shape phenotype in pea plants. (a) Level of SBEI in
the heterozygous plant is intermediate to that of homozygous parents—incomplete dominance. (b)
Size and shape of the microscopic starch grains are intermediate in the heterozygote, compared to
the homozygous parents—incomplete dominance. (c) The seed shape trait depicts complete domi-
nance, with W coding for round and w coding for wrinkled seeds

Table 3.4 Comparison between the various forms of dominance relationships

Type of dominance Definition
Dominance Heterozygote’s phenotype is same as one of homozygous parent’s
phenotypes
Incomplete Heterozygote’s phenotype is intermediate to phenotypes of homozygous
dominance parents
Codominance Heterozygote’s phenotype expresses characteristics of both parental
alleles
3 Extension of Mendelism 105

3.2.4 Codominance

Codominance refers to a condition in which the heterozygote’s phenotype expresses

characteristics of both parental alleles. The heterozygous genotype thus results in an
equal mixture of the phenotypes of both of the corresponding homozygous
genotypes. Unlike incomplete dominance here, the heterozygous phenotype is not
intermediate between the homozygous genotypes (like pink colored flowers in
snapdragons) but rather has equal representation of the characteristics of both
homozygous genotypes. Some of the important examples of codominance are
observed in the alleles coding for the antigens determining human blood groups.

3.2.4.1 Codominance in Human Blood Groups

The ABO human blood grouping system consists of multiple allelic series where
alleles exhibit both simple dominance and codominance. IA and IB alleles of this
multiple allelic series are codominant (refer to Sect. 3.1.2).
Another example of codominance in human blood grouping systems is observed in
the MN blood group. The antigens M and N are determined by two alleles of a gene,
one coding for M and the other coding for N. Homozygotes for M allele produce M
antigen, and homozygotes for N allele produce N antigen, whereas both M and N
antigens are produced in the heterozygote, because of their codominant nature.
Accordingly, there are three possible blood types, i.e., M, N, and MN. The alleles
for M and N antigen are represented as LM and LN. Hence, the genotypes for the M, N,
and MN blood groups are LMLM, LNLN, and LMLN, respectively (Fig. 3.13). Here the
letter L is used as a tribute to Karl Landsteiner, the discoverer of blood typing.

Fig. 3.13 Agglutination reactions to detect M and N antigens. The M and N antigens on the RBC
are detected using specific anti-M and anti-N sera. The identification happens based on the
agglutination caused by the antisera on binding to the specific antigen. Three blood types, M, N,
and MN, can be identified
106 R. Keshava

3.2.5 Lethal Alleles

An allele that usually causes death, at an early developmental stage, and often before
birth is called as a lethal allele. Such an allele causes genotypes to be lost from the
progeny of a particular cross. The ratio of progeny from a specific cross can be
altered by lethal alleles. A peculiar pattern of inheritance was reported in mice in
1905, by Lucien Cuenot. A cross between two yellow mice yielded approximately
2/3 yellow and 1/3 nonyellow mice. A test cross of the yellow mice yielded all
heterozygous yellow mice. Cuenot was unable to obtain true breeding yellow mice.
Several discussions on these observations led to the realization that the lack of true
breeding yellow mice in the progeny could be due to the lethality of the yellow allele
when in homozygous condition (Fig. 3.14). In his experiment, Cuenot had originally
crossed two heterozygous mice (Yy  Yy). As per Mendelian segregation, this cross
would have resulted in 1/4 YY, 1/2 Yy, and 1/4 yy (Fig. 3.14). But in this particular
mating, the homozygous YY mice were conceived but did not last until complete

Fig. 3.14 Segregation of lethal alleles. Cross between two heterozygous yellow mice, resulting in
approximately 2/3 yellow and 1/3 nonyellow offspring. Although the segregation is in the typical
Mendelian pattern, presence of a lethal allele results in the given ratio of yellow and nonyellow
mice. Y allele in homozygous condition causes lethality in the early development
3 Extension of Mendelism 107

development, resulting in progeny, with a Yy (yellow): yy (nonyellow), a ratio of 2:1

where all yellow mice were heterozygotes (Yy).
Another case of a lethal allele is found in snapdragons. It was originally described
in 1907 by Erwin Baur, in a strain of snapdragons called aurea, which consisted of
yellow leaves. Two snapdragons with yellow leaves when crossed produced 2/3
progeny with yellow leaves and 1/3 progeny with green leaves. When two green-
leaved plants were crossed, all progeny had green leaves. But when yellow was
crossed with green, 1/2 progeny were green and 1/2 were yellow, indicating all
yellow-leaved snapdragons to be heterozygous. As typically observed, a ratio of 2:1,
among progeny of a cross between individuals of the same phenotype, indicated the
presence of a recessive lethal allele.
In both cases, it was observed that one allele affected both survival and color.
With respect to survival, the lethal alleles were recessive in nature, as they caused
death in homozygous condition. But the same allele expressed dominance with
respect to the color. However, lethal alleles can also be dominant in nature. In
such a case, both homozygotes and heterozygotes do not survive. It is not possible
for true dominant lethal alleles to be passed on from one generation to another unless
they affect the individual after the onset of reproductive age, as observed in
Huntington disease.

3.2.6 Sex-Limited Traits

Traits which are expressed particularly in one sex are called sex-limited traits.
Although the genes coding for these traits are present in both the sexes, expression
occurs in only one. In humans, formations of the breast and ovary are sex-limited
traits in women. Likewise, facial hair and sperm production are sex-limited traits in
men. In birds, the plumage patterns are sex-limited traits, where in many species,
only the male possesses brightly colored plumage patterns. Horns in only males of
certain sheep species and milk production only in mammalian females are other
examples of sex-limited traits.

3.2.7 Sex-Influenced Traits

Sex-influenced traits are also called as sex-conditioned traits. They appear in both
sexes but occur more frequently in one sex than the other. Premature pattern
baldness, for example, is a sex-influenced trait in human beings. It is due to an allele
that is differently expressed in the two sexes. In males, individuals develop bald
patches, both in homozygous and heterozygous conditions for the allele. However,
in females, only the homozygotes show a tendency for baldness, and that is usually
limited to general thinning of the hair rather than balding. The difference has been
suggested to be due to the requirement of the male hormone testosterone, for the full
expression of the allele. Females produce much less of testosterone and are therefore
rarely at risk of developing bald patches. The sex-influenced nature of this trait
108 R. Keshava

shows that intrinsic biological factors can play an important role in the regulation
gene expression.

3.3 X-Linkage Feature Genes

In organisms with XY chromosomal sex determination, the sex chromosomes are

heteromorphic unlike the autosomes which are homomorphic. Therefore, the
patterns of inheritance of genes located on heteromorphic sex chromosomes are
different when compared with autosomal inheritance. This happens because inheri-
tance of alleles located on the sex chromosomes is linked with sex of the offspring.
In XY sex determination, the sex of the offspring is dependent on the sperm that
fertilizes the egg. The Y chromosome containing sperms determine male sex, while
the X chromosome containing sperms determine the female sex. Therefore, alleles
located on the X chromosome of a male parent are always inherited by his daughters
but not his sons. Likewise, all sons receive the X chromosome from the mother. In
such a crisscross inheritance, traits carried on the X chromosomes are transmitted to
the sons by the mother and to the daughters by the father. For example, by the end of
the eighteenth century, the blood-clotting disorder hemophilia was known to be
associated with the X chromosome, because it was inherited mostly by men, and
women could transmit to next generations without themselves being affected.

3.3.1 X-Linkage in Drosophila

X-linked pattern of inheritance was first demonstrated in Drosophila by T. H.

Morgan in 1910. He observed white-eyed male in a culture of wild-type (red-eyed)
flies (Fig. 3.15). He crossed a wild-type female with a white-eyed male. All offspring

Fig. 3.15 Drosophila (fruit fly) eye color phenotype. (a) Wild-type red eye. (b) Mutant white-eye
3 Extension of Mendelism 109

Fig. 3.16 Inheritance pattern

of the white-eye mutant trait
in Drosophila

Fig. 3.17 X-linked inheritance of white-eye trait in Drosophila. The crosses depicted indicate
nonreciprocity, an important feature of sex-linked inheritance

born out of that cross were wild-type. However, selfing of these F1 flies produced
offspring of two different types (Fig. 3.16). All the female offspring were wild-type,
whereas one half of the males were white-eyed and the other half were wild-type.
Morgan and other researchers interpreted these observations by hypothesizing that
the gene for white-eye was positioned on the X chromosome (Fig. 3.17). X
chromosomes carrying the white-eye allele and wild-type allele are denoted as Xw
and X+, respectively. The Y chromosome does not contain this gene locus and is
denoted as Y.
Another characteristic with respect to X-linkage is as follows: since females
possess two X chromosomes, they can either be homozygous or heterozygous for
the given allele. But males have a single copy of X (Fig. 3.17) and a single copy of Y
110 R. Keshava

that are heteromorphic. Therefore, X-linked alleles can neither be homozygous nor
heterozygous in males. An X-linked allele, not having a homolog on the Y, is said to
be in a hemizygous condition. Hemizygosity causes a recessive allele to be
expressed, even if present in single copy. Therefore, a male Drosophila, although
with only one w allele, expresses white-eyed phenotype. Such a phenomenon is
called pseudodominance because it resembles the way in which a single dominant
autosomal allele determines the phenotype in a diploid heterozygous individual.
Nonreciprocity is another important feature of sex-linked inheritance. A cross is
said to be reciprocal if for a given trait, the results of the cross are equivalent, with
equal distribution of the trait in both sexes, irrespective of the sex of the parent that is
possessing the trait. A cross is said to be nonreciprocal if parental sex alters the
distribution of the trait in the following generations. Therefore, nonreciprocity
indicates the difference in the outcome of such a cross based on the association of
the trait with the sex of the organism. The inheritance pattern of sex linkage is not
reciprocal and it is observed when a white-eyed female is crossed with wild-type
male (Fig. 3.17). In this cross, the F1 males are white-eyed, and F1 females are wild-
type. Further in the F2, 50% of each sex are white-eyed. Such differences in the ratio
of distribution of a trait among the two sexes and nonreciprocity of a cross indicate
sex linkage, which are further confirmed by the crisscross pattern.

3.3.2 X-Linkage in Humans

Several X-linked traits are also observed in humans. The X-linked recessive traits are
more easily identified than recessive autosomal traits. In case of humans also, a male
needs to inherit only one recessive allele to show an X-linked trait; however, a
female needs to inherit two such alleles, one from each of her parents. Therefore, the
majority of people who show X-linked traits are male.

3.3.2.1 X-Linked Blood-Clotting Disorder: Hemophilia

Hemophilia is a disorder wherein the affected individuals lack a blood-clotting
factor. In case of bleeding due to injuries, the hemophiliacs continue to bleed and,
if it is not stopped by transfusion with clotting factor, it can cause death. In humans,
the major type of hemophilia is caused by an X-linked recessive mutation, and
therefore, almost all affected individuals are male. Males inherit the mutation from
their carrier mothers (heterozygous). If they reproduce, they will further transmit the
mutation to their daughters, who usually inherit a wild-type allele from their mother
and hence do not develop hemophilia, but remain as carriers that will transmit the
mutation to their sons. This results in more number of males than females being
affected by the disease. On the contrary, other blood-clotting disorders which occur
due to mutations in autosomal genes are equally distributed in both males and
females.
The most famous case of X-linked hemophilia occurred in the early twentieth
century, in the Russian imperial family (Fig. 3.18), where Czar Nicholas and Czarina
Alexandra had four daughters and one son, and the son, Alexis, was afflicted with
3 Extension of Mendelism 111

Fig. 3.18 Human X-linked disorder—hemophilia, the royal disease. (a) Russian imperial family of
Czar Nicholas II. (b) Hemophilia in the royal families of Europe. Due to intermarriage, the
hemophilia allele was transmitted from British royal family to Russian, German, and Spanish
families
112 R. Keshava

hemophilia. The X-linked mutation that caused the disorder in Alexis was transmit-
ted to him by his mother, who herself was a heterozygous carrier. Czarina Alexandra
happens to be the granddaughter of Queen Victoria of Great Britain, who was also a
carrier. Through pedigree records, it is known that Queen Victoria transmitted the
hemophilia allele to three of her nine children: Alice, Alexandra’s mother; Beatrice,
who had two sons with the disorder; and Leopold, who was also affected. The allele
that was carried by Queen Victoria evidently arose as a new mutation in her germ
cells, or her mother, father, or any other distant maternal ancestor. Throughout
history, hemophilia has been a fatal disorder and most of the affected people
deceased before the age of 20. Today, since more effective and relatively inexpen-
sive treatments are available, hemophiliacs can live longer, healthy lives.

3.3.2.2 X-Linked Vision Disorder: Color Blindness

Color perception in humans is mediated by light-absorbing proteins present in the
specialized cone cells of the retina. Three such light receptor proteins for absorption
of blue, green, and red light have been identified. Abnormality in any of these
receptor proteins can be the cause of color blindness. The classic type of color
blindness involves faulty perception of red and green light. It follows an X-linked
pattern of inheritance. Approximately 5–10% of human males are red-green color
blind; however, a much lesser fraction of females, i.e., less than 1%, has this
disability, suggesting that the alleles for this disorder are recessive. Studies at the
molecular level have shown that there are two separate genes on the X chromosome
for color perception. One of them encodes the receptor for green light, and the other
encodes the receptor for red light. Detailed analyses of these two receptors have
shown that they are structurally very similar.

3.4 Genotype to Phenotype

3.4.1 Penetrance

Phenotypic appearance of the genotypically determined traits is called as penetrance.

Not all genotypes are able to “penetrate” the phenotype. When individuals
possessing an appropriate genotype do not show the corresponding trait, such a
trait is said to show incomplete penetrance. Polydactyly, the presence of extra fingers
and toes in humans, is one such example (Fig. 3.19). A dominant mutant allele, P,
causes this condition, but is not expressed in all of its carriers. In the pedigree shown
in Fig. 3.19, the individual, symbolized III-2, must be a carrier even though he does
not possess extra fingers or toes, because both his mother and his three children are
polydactylous, which is an indication of the mutation being transmitted through
III-2. Incomplete penetrance can be a grave problem in pedigree analysis as it can
cause erroneous understanding of genotypes.
Another example of incomplete penetrance is as follows: a person could possess a
genotype that can cause vitamin D-resistant rickets (a bone disease), but may not
suffer from the disease. The allele which causes this disease is dominant and
3 Extension of Mendelism 113

Fig. 3.19 Incomplete penetrance. (a) Polydactyly in humans is a phenotype showing extra fingers.
(b) Pedigree showing inheritance pattern and the incomplete penetrance of the dominant trait
polydactyly. It can be seen that a male offspring (III-2) in generation III is not expressing
polydactyly although he is carrying the allele. The fact that one of his offspring expresses
polydactyly confirms that III-2 carried the allele

sex-linked. Vitamin D-resistant rickets is different from vitamin D deficiency,

because it cannot be treated by administering low levels of vitamin D. However,
the disease condition can be treated with very high levels of vitamin D. Similar to
polydactyly, in some family trees, unaffected parents give birth to children with
vitamin D-resistant rickets. This is unexpected with respect to inheritance of domi-
nant traits, because an affected offspring should have at least one affected parent. In
this case, the parent however must be carrying the disease allele because the children
are affected. Presence of low phosphorus levels in the parent’s blood, which is a
pleiotropic effect of the disease allele, proves the presence of the allele in the parent.
Interestingly, the low-phosphorus attribute of the phenotype shows complete pene-
trance, whereas vitamin D-resistant rickets shows incomplete penetrance.
However, most genotypes show complete penetrance. For example, albinism
(lack of melanin synthesis) is an autosomal recessive condition. Every individual
who has the homozygous recessive genotype for the albinism allele will certainly
express the disorder. There is no known example of a case where an individual with
the genotype did not express the condition. Therefore, albinism is completely
penetrant. However, certain genotypes, especially those that code for developmental
traits, frequently exhibit incomplete penetrance.

3.4.2 Expressivity

The term expressivity is particularly used when a trait is not uniformly expressed
among individuals that show a particular trait. A dominant mutation in Drosophila,
called the Lobe eye mutation (Fig. 3.20), is one such example. The phenotypic
expression of this mutation is extremely variable, in that it ranges from tiny com-
pound eyes in some heterozygous flies to large, lobulated eyes in others. In between
the two extremes, there exists an entire range of various phenotypes and hence the
lobe mutation is said to show variable expressivity.
114 R. Keshava

Fig. 3.20 Expressivity of Lobe eye mutation in Drosophila. The Lobe eye mutation of Drosophila
shows variable expressivity. Although each of the flies is heterozygous for the mutation, the
phenotypic expression varies from complete absence of an eye to nearly wild-type eye

Several developmental traits in addition to being incompletely penetrant also

exhibit variable expressivity, ranging from mild to extreme. One such example is the
cleft palate trait, which shows both variable penetrance and variable expressivity.
Once penetrance is attained by the genotype, severity of the condition varies
considerably, ranging from severe clefting of hard and soft palates to a mild external
cleft. Failure to penetrate and variable expressivity are characteristics of the devel-
opmental traits in various organisms. Incomplete penetrance and variable expressiv-
ity both indicate that the pathway between a genotype and its expression as a
phenotype goes through considerable modulation. Some of these modulations are
suggested to be due to environmental factors, but some are also due to genetic
factors. Breeding experiments have provided clear evidence that two or more genes
can affect a particular trait.

3.4.3 Environmental Effects on Gene Expression

Phenotypes depend both on environmental and genetic factors, i.e., the genotype.
The analyses of phenotypes have shown that genes do not act in isolation. Instead,
they act in the background of an environment and in coordination with other genes.
These analyses have also shown that a particular gene can influence various different
traits.
3 Extension of Mendelism 115

A gene needs to function in the framework of both biological and physical

environments. The physical environment factors are easier to study, because partic-
ular genotypes can be reared under controlled conditions in the laboratory, thus
allowing an assessment of the effects of temperature, light, nutrition, and humidity.

3.4.3.1 Position Effect

The function and expression of a gene is very much dependent on its location or
position in the genome/chromosome, to an extent that a change of location or
position can alter its function. Such repositioning of genes usually occurs when
chromosomal aberrations lead to rearrangement of chromosomal segments. When
chromosomal rearrangements occur, genes that exist near the breakpoints become
repositioned in the genome and they become flanked by novel neighboring genes
and chromosomal segments. In several cases, such a repositioning of the gene affects
its expression level or, in certain cases, it alters its ability to function; this is called
position effect.
Examples of such effects have been extensively studied in organisms such as
Drosophila and yeast. Position effect was first observed in the Bar eye mutation of
Drosophila. The Bar eye mutation is a duplication of 16A region on the X chromo-
some of Drosophila, which reduces the eye to a bar shape, due to reduction in the
number of facets. A Bar homozygote (B/B) and a Doublebar/wild-type heterozygote
(BB/B_) both have four copies of the 16A region. Hence, it would be expected that
both genotypes produce the same phenotype, i.e., with respect to the number of
facets in the Bar eye. But interestingly, the number of facets in a Bar homozygote is
70 and in a heterozygote it is 45. Therefore, from this example, it is clear that in
addition to the amount of genetic material, its arrangement or position where it is
placed in the genome also plays a significant role in determining the extent of the
phenotype.
The most common position effect observed in Drosophila results in a mottled or
mosaic eye phenotype. The eye of such Drosophila is structured with interspersed
patch cells expressing wild-type phenotype, and mutant cells, with an inactivated
wild-type gene. This mosaic phenotype is called as a variegation, and hence this
effect is called position-effect variegation (PEV).
Usually, PEV results from a chromosomal aberration that alters the position of a
wild-type gene in euchromatin and relocates it to a new position near or within a
heterochromatin (Fig. 3.21). In Drosophila, an inversion mutation in the X chromo-
some that changed the position of wild-type w+ allele into heterochromatin was
identified. This change of position resulted in the variegation phenotype, where
patterns of red (wild-type) and white (mutant) facets appeared in the eyes of flies
(Fig. 3.22).
Inactivation of the euchromatic region is known to occur through a length ranging
from 1 to 50 bands in the polytene chromosome of Drosophila. The length depends
on the particular chromosomal abnormality that has occurred. At the molecular level,
this range approximately includes 20–1000 kb. Similar inactivation events are also
reported to take place in cells of female mammals. It has been reported that when
116 R. Keshava

Fig. 3.21 Position-effect variegation (PEV). Commonly observed due to an inversion mutation,
where chromosome rearrangement changes the location of a gene in euchromatin, to a location in or
near heterochromatin. This is an inversion within X chromosome of Drosophila melanogaster
involving wild-type allele of the white gene locus, causing its relocation near heterochromatin. The
effect of PEV is observed as spotted red and white eyes. This phenomenon is called “position-effect
variegation,” because here the change occurs only in the gene position but not in the gene

Fig. 3.22 Patterns of spotted eyes due to PEV in Drosophila. Each group of cells of the same color
is a product of a single cell during the developmental process. Commonly, several small patches of
red or a mixture of small and large patches is observed

euchromatic genes get translocated to the X chromosome, they become heterochro-

matic and inactive.
Note: PEV occurs when a gene usually located in euchromatin gets relocated near
a heterochromatin due to transposition or chromosomal rearrangement. Intensive
3 Extension of Mendelism 117

studies on PEV have been performed using white gene in Drosophila. Analysis of
PEV in Drosophila at the biochemical, molecular, and genetic levels has provided
useful insights into this process. The variegating phenotype shown by genes that
abnormally get placed in heterochromatin is a result of gene silencing. A spread of
the heterochromatin packaging across the heterochromatin/euchromatin border is
suggested to cause the transcriptional silencing of the juxtaposed/repositioned gene.
Several approaches have been used to identify the key contributors to this process.
This has led to the characterization of several modifying enzymes and structural
proteins that play important roles in establishment and maintenance of heterochro-
matin and the associated gene silencing. Heterochromatin formation is shown to
critically depend on histone H3 methylation at lysine 9, with simultaneous interac-
tion with other proteins and enzymes such as methyltransferases. It is shown that the
spreading and maintenance of heterochromatin is dependent on multiple interactions
between these proteins.

3.4.3.2 Temperature Effect

Environmental conditions such as temperature also affect gene expression. One such
example is found in a Drosophila mutation called as shibire. Shibire is a Japanese
word meaning “paralysis.” At 25  C, the normal culturing temperature, shibire flies
are viable and fertile. But these flies are highly sensitive to sudden shock, such that
when a shibire culture vessel is shaken, the flies get temporarily paralyzed and fall to
the bottom of the culture vessel. If the same culture of shibire flies is kept at a slightly
higher temperature of 29  C, all the flies fall to the bottom and die, even without
exposure to a shock. Hence, the phenotypic expression of the shibire mutation is
temperature-sensitive, i.e., it is dependent on the ambient temperature. It is a
mutation that is viable at 25  C, but is lethal at 29  C. A probable explanation to
this temperature sensitivity is that at 25  C, the mutant gene produces a protein that is
partially functional, whereas at 29  C, the protein is completely nonfunctional.

3.4.3.3 Nutritional Effect

Phenylketonuria (PKU) is an example of a recessive disorder with faulty amino acid
metabolism. Children homozygous for the mutant allele accumulate toxic
metabolites in their brains which affect brain development and impair mental ability.
The harmful aspects of PKU are correlated to a particular amino acid, phenylalanine,
which is present in the diet of the individual suffering from this disorder. Although
phenylalanine itself is not toxic, it gets metabolized into other toxic intermediates.
Infants affected with PKU if fed with normal diets ingest enough amounts of
phenylalanine that can bring about severe manifestations of the disorder. However,
if such affected infants are fed with diets low in phenylalanine, they usually develop
without serious mental impairment. Because PKU can be well diagnosed in
newborns, the clinical impact of this disease can be significantly reduced by placing
infants that are PKU homozygotes on a low-phenylalanine diet soon after birth.
Thus, PKU clearly indicates the effect of nutrition on the gene expression. In this
case, the presence of phenylalanine in the diet triggers the expression of the disease,
and hence the same can be controlled by altering the diet of the individual.
118 R. Keshava

3.4.3.4 Pleiotropy
It is now known that any given phenotype can be influenced by several genes. The
converse of this is also true, i.e., one gene can influence many phenotypes. When a
mutant gene affects many aspects of the phenotype, it is said to be pleiotropic (Greek
for “to take many turns”). Such a phenomenon is called pleiotropy, and the effects
caused are called pleiotropic effects.
PKU gene in humans is an example for pleiotropy. The primary effect of the
recessive mutation in this gene is that it causes accumulation of toxic substances in
the brain, leading to mental impairment. However, these mutations also interfere
with the synthesis of the pigment melanin, leading to lightening hair color; and
hence, individuals with PKU frequently possess light brown or blond hair. Biochem-
ical tests also reveal the presence of rare compounds in the blood and urine of PKU
patients, which is otherwise not found in normal individuals. This range of pheno-
typic effects is typical of most genes and results from interconnections between the
biochemical and cellular pathways regulated by these genes.
Another example of pleiotropy is the mutation causing sickle cell anemia. Sickle
cell mutation primarily affects the oxygen-carrying protein hemoglobin, present in
the RBC. In addition, this mutation is also associated with several pleiotropic effects.
In addition to its effect on RBC, it also affects several major organs and organ
systems (Fig. 3.23). The sickle cell mutation is present in the β-globin gene that
codes for a β polypeptide chain of hemoglobin. The molecular basis of the disease is
illustrated in Fig. 3.24. The β-globin polypeptide chain is 146 amino acids long. The
part of the β-globin gene coding for amino acids in positions 5 through 8 is shown in
Fig. 3.24. The sickle cell mutation involves a base pair change indicated with an
arrow. The mutation replaces the normal A-T base pair with a T-A base pair. Due to
this mutation, the mRNA codon is replaced with a GUG instead of a GAG codon.
GUG codes for valine (Val), while GAG codes for glutamic acid (Glu). Therefore,
normal glutamic acid is replaced with valine in the β-globin polypeptide chain at
position number 6 (Fig. 3.24). Thus, formed abnormal β polypeptide chain attributes
long, needlelike polymer forming ability to hemoglobin. Due to the polymerization
of hemoglobin, RBCs become deformed into crescent-like sickle-shaped cells. Some
amount of these abnormal RBCs are immediately destroyed, reducing the blood
oxygen-carrying capacity resulting in anemia. The remaining sickle-shaped RBC
may clump and clog the capillaries, thus interrupting blood circulation.
The Glu ! Val replacement results in profound pleiotropic effects. All of these
effects are due to the breakdown of RBCs, or the reduced oxygen-carrying capacity
of the blood, or the physiological alterations the body makes in an effort to
compensate for the disease. Patients with sickle cell anemia suffer spells of severe
pain. Anemia causes impaired growth, weakness, and jaundice. Affected people are
also generally immunologically weakened, and they become susceptible to bacterial
infections, which are the most frequent cause of death in children with the disease.
Hence, sickle cell anemia is a severe genetic disease which can cause premature
death.
In spite of its severity, it is quite prevalent in areas of Africa and Middle East.
These regions also have an extensive incidence of malaria caused by Plasmodium
3 Extension of Mendelism 119

Fig. 3.23 Pleiotropy in sickle cell anemia. The sickle cell mutation affects several major organ
systems in the human body, in addition to causing abnormal hemoglobin in RBC

falciparum. There is a significant association between the incidence of malaria and

sickle cell anemia in these regions. The association is because of the mutant form of
β-hemoglobin, which to a certain extent provides resistance against malarial infec-
tion. This is due to the fact that the malarial parasite requires the RBCs as part of its
life cycle. The proliferation of the parasite in the RBCs is checked due to the sickling
of the cells, and hence the severity of the malarial infection is reduced.
120 R. Keshava

Fig. 3.24 Molecular basis of sickle cell anemia. (a) Shows a segment of the normal β-globin gene,
and corresponding mRNA, coding for the amino acid sequence Pro-Glu-Glu-Lys. The GUG codon
at the sixth position in the mRNA codes for glutamic acid (Glu). (b) Shows mutant form of the
β-globin gene, showing an A-T transversion mutation. Likewise, the codon in mRNA alters from
GUG to GAG, which in turn alters the amino acid at the sixth position from glutamic acid to valine
(Val)

Another example of pleiotropy is the mutations that affect the formation of

bristles in Drosophila. Wild-type flies possess smoothly curved, long bristles on
the head and thorax. Flies which are homozygous for the singed bristle mutation
have short, twisted bristles on the head and thorax. Thus, the wild-type form of the
singed gene product is required for proper formation of bristles. In addition, it is also
required for the production of healthy, fertile eggs. This fact is known because
females homozygous for certain singed mutations are sterile; they lay fragile,
ill-formed eggs that fail to hatch. However, these mutations do not affect male
fertility. Thus, the singed gene pleiotropically regulates the formation of both bristles
and eggs in females, whereas in males it regulated the formation of bristles.

3.4.4 Gene Interaction

In the dihybrid crosses performed by Mendel, each gene locus independently

controlled the phenotype. In other words, one gene locus did not have any influence
3 Extension of Mendelism 121

Table 3.5 Proportions of 9/16 R_Y_ Round, yellow

F2 progeny in Mendel’s
3/16 R_yy Round, green
dihybrid cross
3/16 rrY_ Wrinkled, yellow
1/16 rryy Wrinkled, green

on the functioning of the other gene locus. For example, when Mendel self-fertilized
the F1 plants obtained from a dihybrid cross between homozygous round yellow and
wrinkled green plants, the phenotypic proportions in the F2 progeny were as shown
in Table 3.5.
This was a typical example of independent assortment. Here the independence
observed is at two levels. Firstly, the genes at each locus were independently
assorted during meiosis, and hence produced a 9:3:3:1 phenotypic ratio in the
progeny. Secondly, each gene independently controlled particular phenotypes, i.e.,
R and r allele determined round and wrinkled seed shape, respectively. Similarly, the
Y and y alleles determined yellow and green color, respectively. Alleles that con-
trolled seed shape did not affect those that controlled color and vice versa.
Although genes often show independent assortment, they are not independent in
their phenotypic expression. In several cases, a gene at one locus influences the
expression of a gene at another locus. This kind of interaction among genes at
different loci (nonallelic genes), which affects the phenotypic outcome, is termed
gene interaction. Due to such gene interactions, products from different genes
interact with one another to produce novel phenotypes, which cannot otherwise be
formed by the effects of single loci. When there is involvement of two genes in the
outcome of a single characteristic, the dihybrid phenotypic ratios observed in such
crosses can be quite a deviation from the typical 9:3:3:1. Although gene interactions
among three, four, or more loci are common, the examples discussed in this chapter
are primarily focused on interaction of genes at two loci.

3.4.4.1 Production of Novel Phenotypes by Gene Interaction

Fruit color trait in a pepper, Capsicum annuum, is controlled by interactions between
genes at two separate loci. These peppers come in four colors: red, brown, yellow, or
green. In a plant homozygous for red peppers (RRCC) when crossed with a plant
homozygous for green peppers (rrcc), all F1 (RrCc) produce red peppers (Fig. 3.25).
When the F1 are self-pollinated (RrCc  RrCc), red, brown, yellow, and green
peppers are obtained in the ratio of 9:3:3:1, respectively, in the F2 (Fig. 3.25).
The red pigment in peppers is produced by a dominant allele R at one gene locus
(locus 1); recessive allele r at the same locus does not produce red pigment. At
another gene locus (locus 2), a dominant allele C causes green pigment chlorophyll
to degrade, whereas the recessive allele c allows retention of chlorophyll. Therefore,
genes at locus 1 and 2 interact with each other to produce the various colors observed
in fruits of pepper Capsicum annuum. The genotypes and the phenotypes of the F2
plants are given in Table 3.6.
This can be explained as follows: in case of R_C_ genotype, dominant R produces
red pigment, dominant C results in decomposition of chlorophyll, and together the
122 R. Keshava

Fig. 3.25 Gene interaction in pepper Capsicum annuum resulting in novel phenotype. The fruit
color in pepper is determined by the interaction between two gene loci, an example of a single
character being influenced by two different genes
3 Extension of Mendelism 123

Table 3.6 Production of Genotype Phenotype

novel characters by gene
R_C_ Red
interaction—F2 genotypes
and phenotypes for fruit R_cc Brown
color in Capsicum annuum rrC_ Yellow
rrcc Green

Fig. 3.26 Gene interaction resulting in chicken a novel phenotype. The combs of chicken are
determined by the interaction between two gene loci, an example of a single character being
influenced by two different genes

action of both nonallelic genes results in a red color. In the genotype R_cc, dominant
R produces red pigment, recessive c retains chlorophyll, and the resultant is a brown
color. In the genotype rrC_, recessive allele r does not produce red pigment,
dominant allele C leads to the decomposition of chlorophyll, and as a result, the
fruit color is yellow. In the genotype rrcc, recessive allele r does not produce red
pigment and the recessive allele c retains chlorophyll and hence the fruit color is
green.
Chicken combs provide another example of a novel phenotype produced due to
gene interaction. A comb is a fleshy structure found on the heads of chicken. In this
example, two loci (R, r and P, p) interact with each other and produce four types of
combs (Fig. 3.26). The presence of at least one dominant R at locus 1, and at least
one dominant P at locus 2 (genotype R_P_), produces a walnut comb in the chicken.
Chicken with one dominant allele (R) at locus 1 when the alleles at the second locus
are homozygous recessive (pp), i.e., a genotype R_pp, produces a rose comb. If the
locus 1 is homozygous recessive (rr), and locus 2 consists of at least one dominant
allele (P_), the chicken (rrP_) produces pea comb. When both loci are homozygous
recessive (rrpp), the chicken produces a single comb.

3.4.4.2 Epistasis
Classically, epistasis is defined as a gene interaction where one gene overrides the
influence of another gene at a different locus in such a way that its phenotype is
suppressed. This is similar to phenomenon of dominance, but unlike dominance
which involves alleles of the same gene, epistasis involves alleles of two different
genes. The overriding gene is called epistatic, and the suppressed gene is called
hypostatic. Genes that are epistatic genes may be either dominant or recessive.
124 R. Keshava

Table 3.7 Recessive epis- Genotype Phenotype

tasis in coat color of
B_E_ Black
Labrador retrievers—F2
genotypes and phenotypes bbE_ Brown (usually called chocolate)
B_ee Yellow
bbee Yellow

Fig. 3.27 Dihybrid cross

depicting recessive epistasis
in coat color of Labrador
retrievers. The phenotypic
ratio of recessive epistasis is
9:3:4

3.4.4.3 Recessive Epistasis

Coat color in Labrador retrievers is an example of recessive epistasis. These dogs
have different coat colors such as black, brown, or yellow. Coat color is determined
by two different loci, where one locus determines pigment type to be produced by
the skin cells—dominant allele B codes for a black pigment; recessive allele b codes
for a brown pigment. The second gene locus controls pigment deposition into the
hair shaft—dominant allele E allows deposition of pigments (black or brown) into
the hair shaft; recessive allele e prevents deposition of pigments (absence of
pigments causes hair to appear yellow). If the second locus consists of genotype
ee, the expression of the first locus (black or brown pigments) gets concealed.
Therefore, genotypes with dominant alleles at both loci result in black coat color,
homozygous recessive at locus 1, and at least one dominant allele at locus 2 results in
brown coat color. But if the second locus is homozygous recessive, no pigments will
be expressed irrespective of the genotype at locus 1. In the absence of any of the dark
pigments, the coat color appears yellow. The possible genotypes and their
corresponding phenotypes (coat color) are given in Table 3.7.
A cross between a homozygous dominant black Labrador and a homozygous
recessive yellow Labrador produces black F1 hybrids. An intercross among the F2
results in the phenotypic ratio of 9 black:3 brown:4 yellow Labradors, as shown in
Fig. 3.27.
3 Extension of Mendelism 125

From Fig. 3.27, it can be noted that genotypes B_ee and bbee are both yellow in
color, although they possess alleles for black and brown coat color, respectively.
Here the recessive allele e, at the second locus, is epistatic to both B and b alleles of
the first locus, because e masks both their expression; likewise, B and b alleles are
said to be hypostatic to the allele e. In this example, e is a recessive epistatic allele,
because it can exert the epistatic effect only in the homozygous condition.

3.4.4.4 Dominant Epistasis

The phenomenon where a dominant allele of one gene locus masks the expression of
alleles at another gene locus is called dominant epistasis. Dominant epistasis is
observed in summer squash with respect to the inheritance of fruit color. Commonly,
summer squashes come in yellow, white, or green color. In homozygous plant
(WWYY) for white squash when crossed with a homozygous plant for green squash,
all F1 plants are white. When the F1 are intercrossed, the following results are
obtained in F2 generation:
The proportions of phenotypes observed in the F2 generation are 3/4 plants
(12 out of 16) produced white squash, and 1/4 of the plants produced colored squash,
i.e., 3 out of 16 produced yellow and 1 out of 16 produced green (3/16 + 1/16 ¼ 4/
16). These observations suggest that production of pigment is inhibited by a domi-
nant allele (W ) at one locus, resulting in white progeny, i.e., W_ genotype inhibits
pigment production and white squash is produced, and ww enables production of
pigment producing colored squash. F2 plants with homozygous recessive (ww)
genotype at the W locus produced pigmented fruits. The fruits were either yellow
or green in color, and were produced in the ratio of 3:1 as seen in Fig. 3.28. The type
of pigment (green or yellow) is dependent on a second locus. The dominant Y allele
(Y_) produces yellow pigment, and the recessive allele (yy) determines green. This
second locus is expressed only in plants homozygous recessive (ww) for the first
locus, i.e., in the absence of the dominant inhibitory W allele. Therefore, the plants
producing yellow squash have the genotype wwY_, and those with genotype wwyy

Fig. 3.28 Dihybrid cross

depicting dominant epistasis
in fruit color of summer
squash. The phenotypic ratio
of dominant epistasis is 12:3:1
126 R. Keshava

Table 3.8 Dominant epis- W_Y_ White squash

tasis in summer squash—F2
W_yy White squash
genotypes and phenotypes
wwY_ Yellow squash
wwyy Green squash

Fig. 3.29 Mechanism of dominant epistasis in summer squash. The yellow pigment in summer
squash is synthesized by a two-step biosynthetic pathway. A colorless compound A is converted to
a green compound B by enzyme I. Enzyme II converts compound B to a yellow colored compound
C. The dominant allele W inhibits conversion from A to B. Plants with Y allele produce enzyme II
which converts compound B to C. The yy genotype does not code for active enzyme II and hence
compound B to C conversion does not occur

produce green squash. The possible genotypes and their respective phenotypes are
given in Table 3.8.
Dominant allele W is epistatic and both Y and y are hypostatic, because presence
of even one copy dominant W allele suppresses production of both pigments: yellow
and green. This is in contrast to allele e in Labrador retriever where two copies of the
recessive allele are required to bring about epistasis. Therefore, W is a dominant
epistatic allele.
Summer squash is a good example to understand how epistasis occurs. This is a
typical example of genes which take part in a series of reactions in a biochemical
pathway. The mechanism of epistasis in summer squash can be explained as follows:
production of yellow pigment is suggested to follow a two-step biochemical path-
way (Fig. 3.29). The pathway starts with a colorless (white) precursor compound A
which is converted to a green colored compound B, by enzyme I. In the second step,
the green colored B is further converted by enzyme II to C which is yellow in color.
Plants with genotype ww produce enzyme I, and the squash produced by these plants
may be yellow or green, depending on presence or absence of enzyme II, respec-
tively. Presence of allele Y at the second locus produces enzyme II and hence yellow
fruits are obtained.
Presence of homozygous recessive y does not encode a functional enzyme II, and
green squash are produced. Conversion of A to B is inhibited in the presence of
dominant W at the first locus; plants with W_ genotype cannot synthesize B and their
fruits remain white, irrespective of the alleles at the second locus. Several examples
of epistasis which involve such a mechanism, where a gene (such as W ) is involved
3 Extension of Mendelism 127

in an earlier step of a biochemical pathway, will be epistatic to genes (such as Y and

y) that are involved in the later steps.

3.4.4.5 Duplicate Gene Interaction

Epistatic interaction between genes is not always in opposition to one another.
Sometimes genes can have the same role in a particular process and substitute for
one another. This kind of a mechanism is called duplicate gene interaction.

3.4.4.5.1 Duplicate Recessive Epistasis

When expression of dominant alleles is masked by recessive alleles at two gene loci,
it is called duplicate recessive epistasis. Earlier this was called as complementary
gene interaction because both genes (dominant alleles of both genes) are required to
express the particular phenotype.

Flower Color in Peas

Bateson and Punnett, for the first time, explained epistasis based on their work in pea
plants. They performed a cross with two pure-breeding varieties of pea bearing white
flowers. All F1 progeny had purple flowers. When these purple-flowered F1 plants
were selfed, the F2 generation produced both purple and white flowers in the ratio
9.4:6.6, or approximately 9:7. Bateson and Punnett counted a total of 382 purple-
flowered plants and 269 white-flowered plants in the F2 generation. In a report
published in 1909, Bateson first proposed an explanation and he called it the ability
of one “allelomorphic pair” (pair of gene alleles) to mask the effect of alleles for
another gene. Later he used the word “epistasis,” (meaning, standing upon) to define
the masking action of one gene by another.
The mechanism involved can be explained as follows: the alleles of genes
controlling flower color in pea were designated C and P. The genes C and P are
related to biosynthetic pathway involved in the synthesis of a pigment called
anthocyanins. This biosynthetic pathway in peas happens in two steps. The first
step requires gene C and the second step requires gene P (Fig. 3.30). If either of the
gene is nonfunctional, the respective step fails and purple pigment is not produced.
In such a case, the affected pea plants will bear only white flowers. Dominant alleles
C and P code for functional enzymes involved in anthocyanin production, while the
recessive alleles c and p code for nonfunctional enzymes. Therefore, if any one of the

Fig. 3.30 Two-step anthocyanin biosynthesis process. Presence of genes C and P is required for
anthocyanin synthesis. Anthocyanin pigment production involves biochemical conversion of a
precursor compound. In the first step, precursor is converted into an intermediate by C. In the
second step, intermediate is converted into anthocyanin by P
128 R. Keshava

Table 3.9 Dihybrid cross between two purple-flowered (CcPp) pea plants

Female Gametes
CP Cp cP cp
CP CCPP CCPp CcPP CcPp

Male Cp CCPp CCpp CcPp Ccpp

Gametes cP CcPP CcPp ccPP ccPp
cp CcPp Ccpp ccPp ccpp

loci is homozygous recessive, anthocyanin pigment will not be synthesized

(Table 3.9).

Albinism in Freshwater Snail Physa heterostropha

Albinism is a common genetic trait which is characterized by absence of a pigment,
and is observed in both plants and animals. Production of pigments in most cases
takes place through a multistep biosynthetic pathway, which is also applicable to
albinism and hence may involve gene interaction. Researchers Robert T. Dillon and
Amy R. Wethington studied albinism in Physa heterostropha, a common freshwater
snail. They showed that the causal factor for albinism is the presence of homozygous
recessive alleles, at either of two gene loci.
For their study, they collected inseminated snails from natural populations and
placed them in cups of water. The snails laid their eggs in the cups. The progeny was
studied after the eggs hatched. Some of the snails that hatched were albinos. When
two such albino snails were mated, all of the F1 were pigmented. The F2 obtained on
intercrossing the F1 progeny consisted of 9/16 pigmented snails and 7/16 albino
snails, giving rise to a phenotypic ratio of 9:7, which is obtained when dominant
alleles present at both loci (A_B_) are responsible for the production of pigment, and
all other genotypes produce albinos as shown in Fig. 3.31.
The production of pigments in the snails is suggested to be due to a two-step
biosynthetic pathway (Fig. 3.32), where pigment (compound C) is produced only
after enzyme I converts compound A into compound B and enzyme II converts
compound B into compound C. At least one dominant allele A is required at the first
locus to produce enzyme I, and at least one dominant allele B is required at the
second locus to produce enzyme II. Absence of compound C causes albinism, and it
can happen in three ways. Firstly, production of enzyme I can be prevented due to
recessive alleles in homozygous condition at the first locus (genotype aaB_), and
thus compound B is never produced. Secondly, production of enzyme II may be
prevented because of homozygous recessive alleles at the second locus (genotype
A_bb), and thus compound B will not be converted into C. Thirdly, both loci may be
occupied by two recessive alleles (aabb), resulting in the absence of enzymes I and
3 Extension of Mendelism 129

Fig. 3.31 Dihybrid cross

depicting duplicate recessive
epistasis in snail
pigmentation. The phenotypic
ratio of duplicate recessive
epistasis is 9:7

Fig. 3.32 Mechanism of duplicate recessive epistasis. Pigment synthesis in snails is by a two-step
biosynthetic pathway. Compound A is converted to compound B by enzyme I. Enzyme II converts
compound B to compound C (pigment). The dominant allele A is required to express enzyme I and
dominant B is required for enzyme II expression. For pigmentation both enzymes are required
(A_B_). Lack of either of them results in albinism

II. In this example, a is epistatic to B, and b is epistatic to A; both are recessive

epistatic alleles because two copies of either allele a or b are needed to suppress
pigment production. Epistasis in snails differs from the epistasis of coat color in
Labrador retrievers in that suppression of pigment production in snails is caused by
recessive alleles at either of the two loci, whereas in Labradors, recessive alleles at a
single locus suppress pigment production.

3.4.4.5.2 Duplicate Dominant Epistasis

When expressions of recessive alleles present at two gene loci are masked by a
dominant allele, it is known as duplicate gene action or duplicate dominant epistasis.
130 R. Keshava

Fig. 3.33 Color production

in wheat kernel. Kernel color
in wheat is determined by two
genes, A and B. Colored
kernel will be produced if
either gene is functional.
Kernel will lack color (white)
only if both genes are
nonfunctional

Table 3.10 Dihybrid cross between wheat plants of genotype AaBb

Female Gametes

AB Ab aB ab

AB AABB AABb AaBB AaBb

Male Ab AABb AAbb AaBb Aabb

Gametes aB AaBB AaBb aaBB aaBb

ab AaBb Aabb aaBb aabb

Wheat Kernel Color

Kernel color in wheat is dependent on a biochemical reaction which converts a
precursor molecule into a pigment. This biosynthetic reaction can be performed by
either of the products of genes A or B (Fig. 3.33). Therefore, presence of either
dominant A or B allele produces kernel color. But white kernel (lacking pigment)
will be produced when neither dominant allele is present, i.e., when the genotype is
double homozygous recessive. The outcome of such an interaction is a phenotypic
ratio of 15:1 (15 color:1 noncolor) (Table 3.10).
Some of the possible gene interactions and their modified dihybrid ratios are
given in Table 3.11.
The term “epistasis” was given by William Bateson to a particular type of genetic
interaction, wherein one gene masks the effects of another gene. Earlier, different
terms, such as complementary (duplicate recessive epistasis), supplementary (reces-
sive epistasis), etc., were used to distinguish different forms of gene interaction.
Ronald Fisher generalized the term epistasis to include several other forms of gene
interactions. He included any multigene interactions that lead to a phenotype differ-
ent than that of the expected phenotypes associated with combination of
corresponding individual genes. Eventually, the term epistasis has evolved and
presently it includes all gene interactions. It is now defined as any phenomenon
3 Extension of Mendelism 131

Table 3.11 Types of gene interactions

Ratio Description Name of interaction
9: No gene interaction; each allele produces Independent assortment without gene
3:3: its own phenotype interaction
1
9: Both gene pairs show complete dominance; Not named, ratio similar to independent
3:3:1 dominant alleles and also both homozygous assortment, but phenotypes are formed
recessives interact to produce new by gene interactions
phenotypes
9:4:3 Both gene pairs show complete dominance; Recessive epistasis
But, one gene when in homozygous
recessive state, suppresses other gene’s
phenotype
9:7 Both gene pairs show complete dominance; Duplicate recessive epistasis
either of the genes can suppress the other
gene when in homozygous recessive state
12: Both gene pairs show complete dominance; Dominant epistasis
3:1 when one gene is dominant, the phenotype
of the other gene is suppressed
15:1 Both gene pairs show complete dominance; Duplicate dominant epistasis
when either of the gene is dominant, it
suppresses the other gene’s effect
13:3 Both gene pairs show complete dominance; Dominant and recessive epistasis
but when either of the gene is dominant, it
suppresses the other gene’s effect
9:6:1 Both gene pairs show complete dominance; Duplicate interaction
when either of the gene is dominant, it
suppresses the other gene’s effect
7:6:3 One gene pair shows complete dominance No name
and the other shows partial dominance;
when in homozygous recessive state, the
first gene is epistatic to the second gene
3: One gene pair shows complete dominance No name
6:3:4 and the other shows partial dominance; in
homozygous recessive state, either gene
hides other gene’s effect; when both genes
are in homozygous recessive state, the
effects of the first gene are suppressed by the
second
11:5 Both gene pairs show complete dominance, No name
only when both types of dominant alleles are
present; if not, the recessive phenotype
appears

that involves different genes contributing to a single phenotype such that their effects
are not just additive. Such genes are said to be epistatic. Therefore, epistasis is not
restricted to the interactions of only two genes. Rather, epistasis can occur in all of
the following scenarios: (1) whenever two or more loci interact to create novel
phenotypes, (2) whenever an allele at one locus masks the effects of alleles at one or
132 R. Keshava

more other loci, and (3) whenever an allele at one locus modifies the effects of alleles
at one or more other loci.

Box 3.1 Scientific Concept: Epistasis and Disease

When factors contributing to a disease phenotype are analyzed, epistasis forms
a very significant factor along with gene mutations or environmental factors.
Understanding epistatic gene interactions is believed to be crucial to fathom
complex diseases, which include diabetes, cardiovascular disease, cancer, and
Alzheimer’s disease. Understanding the genetic basis and the causal factors of
these diseases has been elusive when single genes are studied. But the growing
focus on epistatic interactions is expected to contribute toward greater under-
standing of these complex human diseases. Researchers have attempted to
understand epistatic interactions that could be involved in Alzheimer’s dis-
ease. More than 100 probable epistatic interactions have been evaluated in
sporadic cases of Alzheimer’s disease. Sporadic cases are those cases of
disease incidence which have no known hereditary background. Evaluation
of epistatic interactions between associated pairs of genes involved measure-
ment of magnitude and its statistical significance. Overall, 27 significant
epistatic interactions have been shown to play a role in Alzheimer’s disease
process. They have been divided into five groups, which include (1) beta-
amyloid production, (2) oxidative stress, (3) inflammation, (4) cholesterol
metabolism, and (5) others. Certain epistatic interactions appeared to be
synergistic, i.e., increased disease risk, while others appeared to be antagonis-
tic, i.e., have a protective relationship.
Diabetes is another example of a complex disease wherein epistasis plays a
role. In case of diabetes, both epistatic interactions and the environment play
an important role. Interactions between numerous chromosomal loci have
been discovered in type II diabetes patients. Experimental evidences have
also shown involvement of epistasis in several other complex diseases such
as autism, hypertension, cardiovascular disease, neurological disorders, and
several types of cancer. Presently, it has become possible to detect
relationships between epistatic interactions, genes, and networks at a systems
level. With the currently available high-throughput experimental tools, it is
possible to measure biochemical and molecular data. Examples of such high-
throughput tools include DNA microarrays, bioinformatics, and computational
methods. These tools can be applied to identify and understand epistatic
relationships on a large scale, and the knowledge obtained can be applied for
better diagnosis and treatment of complex diseases.
3 Extension of Mendelism 133

Box 3.2 Scientific Concept: Diseases with Complex Inheritance Patterns

Typically, monogenic disorders follow Mendelian patterns of inheritance.
However, there are several disorders that exhibit complex patterns of inheri-
tance. A common progressive neurodegenerative disorder such as Parkinson’s
disease (PD) is one such example. Earlier, PD was considered to be a sporadic
disease without a genetic basis. However, research spanning over a decade
have established the underlying role of genetic factors in PD pathogenesis.
Interestingly, the causal factors include highly penetrant singular genes,
unique variants with incomplete penetrance, and also the more common
idiopathic forms of the disease. Therefore, it involves monogenic forms with
Mendelian inheritance and also forms with non-Mendelian inheritance. How-
ever, the monogenic Mendelian forms are rare, but have provided insight into
the genetic architecture underlying this disease. Monogenic mutations
associated with both autosomal dominant and autosomal recessive PD have
been characterized. In addition, a few gene variants exhibiting incomplete
penetrance are shown to be strong risk factors for the disease incidence in
some populations.

3.5 Extranuclear Inheritance

Extranuclear inheritance indicates transmission of characters through factors that

reside outside of the nucleus. It is also called as non-Mendelian inheritance because
Mendel’s laws of heredity are not applicable to their inheritance patterns. Some of
the other terminologies used include extrachromosomal, cytoplasmic, or nonchro-
mosomal inheritance. Cytoplasmic inheritance involves several factors such as
cellular organelles (e.g., chloroplast and mitochondria) containing their own DNA
and parasitic or symbiotic particles (infective particles) that reside in the cytoplasm
and possess their own genetic material. Organelles such as chloroplasts and
mitochondria and infectious agents such as bacteria, viruses, and DNA molecules
such as plasmids are all mediators of cytoplasmic inheritance. The genetic materials
in these entities are also susceptible to mutation and their inheritance does not follow
Mendelian rules and ratios.
Another type of extranuclear inheritance, called maternal effect, includes those
traits and characters that are influenced by the mother’s genotype or phenotype.
When gametes are formed, the egg cell of the female gamete receives a greater
volume of cytoplasm than the sperms or male gametes. This difference in the
cytoplasmic content of the gametes is responsible for the maternal effect. During
fertilization, the cytoplasmic contribution of the female gamete to the zygote is
greater than that of the sperm. This results in an unequal contribution of the female
parent toward the development of zygotes. Although chromosomal genes are
contributed equally to the zygote by both parents, sex chromosomes being an
134 R. Keshava

exception, the sperm rarely contributes any additional material toward zygotic
development. The role of the female parent in this regard is very significant. In
addition to the chromosomal genes, the female parent often contributes to the initial
cytoplasm and organelles to the zygote. Hence, zygotic development is facilitated by
the maternal environment provided by the cytoplasm of the egg cell. Coiling in snails
and moth pigments are examples of maternal effect.
Mitochondria and chloroplasts are cytoplasmic organelles with specialized
functions. The unique feature of both these organelles is the presence of small
circular organellar DNA, which make them semiautonomous. A specific subset of
the total cellular genome is carried by these small circular organellar chromosomes.
Genes concerned with energy production are located on the mitochondrial DNA,
whereas genes important for photosynthesis are present in chloroplast DNA. How-
ever, neither of the organelle is completely independent, because they both depend
on the nuclear genome to a certain extent for their functions and hence called
semiautonomous. Another distinct feature of organellar genomes is their copy
numbers, i.e., they are present in large number of copies in the cell. In addition,
the organelles themselves are also present in multiple copies per cell. Collectively,
any given cell consists of hundreds to thousands of organellar chromosomes. These
organelle genes follow their own pattern of inheritance and are called uniparental
inheritance, indicating that the progeny inherit organelle genes exclusively from one
parent, which is in most cases the mother, and hence also called maternal inheritance.
Cytoplasmic inheritance can be deduced by performing crosses and studying the
patterns of inheritance for several generations. It is specifically the results of the
reciprocal crosses that reveal the cytoplasmic inheritance pattern. In any given cross,
the variant phenotype will be transmitted to the progeny particularly by the female
parent, but not the male parent. Hence, cytoplasmic inheritance pattern can be
generally represented as follows:
Mutant ♀  wild-type ♂ ! all mutant progeny
Wild-type ♀  mutant ♂ ! all wild-type progeny

3.5.1 Extranuclear Genomes: Mitochondria

Several mitochondrial DNA (mtDNA) including that of humans have been

sequenced. The genome/DNA of mitochondria (mtDNA) is compact, circular
(Fig. 3.35), and double stranded. It consists of 16,569 base pairs. The genome
includes 37 genes, of which 13 codes for polypeptides, 22 codes for transfer RNA
(tRNA), and 2 codes for ribosomal RNA (rRNA) (Fig. 3.34). Majority of proteins
required by the mitochondria for its functions are encoded by the nuclear genome.
Each strand of the mtDNA duplex is transcribed into a single RNA which is further
cleaved into smaller segments, releasing 22 tRNAs, along with 16S and 12S rRNA.
The mitochondria are fairly self-sufficient with respect to availability of mRNAs
required for protein synthesis. The oxidative phosphorylation process occurs within
the mitochondrion, and it requires the participation of about 69 polypeptides. Human
mtDNA carries 13 of these which include cytochrome b, two subunits of ATPase,
3 Extension of Mendelism 135

Fig. 3.34 Human mitochondrial DNA. The gene map of the human mitochondrial DNA shows the
presence of a heavy (H) strand and light (L) strand. Except nine gene loci, all are located on the H
strand (labeled on the outside). The remaining nine loci are located on the L strand (labeled on the
inside). The origin of replication and the direction of transcription of the H and L strand are shown

three subunits of cytochrome-c oxidase, and seven subunits of NADH dehydroge-

nase. The synthesis of the rest of the polypeptides required for oxidative phosphory-
lation is regulated by the nuclear genes and takes place in the cytoplasm and then it
gets transported into the mitochondrion.
The mitochondrial rRNA shares a great similarity to prokaryotic rRNA than to
eukaryotic rRNA. This is demonstrated by sensitivity of mitochondrial ribosome to
prokaryotic antibiotics such as chloramphenicol and streptomycin, which inhibit its
function. The similarity observed between prokaryotes and mitochondria strongly
supports the theory of symbiotic origin of mitochondria. This theory has been
proposed by L. Margulis for both organelles, i.e., mitochondria and chloroplasts.
The theory hypothesizes that initially cyanobacteria and free-living bacteria have
been transformed into chloroplast and mitochondria, respectively. This is a theory
that has been accepted. It is suggested that the prokaryotes invaded or were eaten up
by primitive cells, which eventually evolved within these primitive cells to become
the respective organelles.
Mitochondrial inheritance shows two general patterns in animals. Firstly,
mitochondria are inherited in a maternal fashion where it is typically transmitted
136 R. Keshava

through the egg cell but not the male gamete. However, exceptions to the general
rule of maternal inheritance of mitochondria do exist. A certain amount of “leaki-
ness” however occurs in this process, as it has been recently shown in mice that
nearly one out of thousand mitochondria is of paternal origin. Species, such as
mussels, show biparental inheritance of mitochondria. In such species, the mito-
chondrial population of an offspring are obtained almost equally from both parents.
Certain gymnospermous plants, e.g., coastal redwoods, show paternal inheritance of
mitochondria, i.e., zygote receives only paternal mitochondria.
The second general pattern observed in mitochondrial inheritance is
homoplasmy, which means existence of uniform populations of mitochondria within
a cell or organism. In general, all mitochondria of an individual are genetically
identical. However, phenomena such as biparental inheritance and leakiness of
paternal mitochondria cause heteroplasmy, leading to mitochondrial heterogeneity
within a cell or organism. When organelle populations are a mixture of two geneti-
cally distinct chromosomes, then they often show segregation of the two types into
the daughter cells during cell division, a phenomenon called cytoplasmic
segregation.

3.5.2 Extranuclear Genomes: Chloroplast

Chloroplasts are characterized by the presence of the photosynthetic pigment chlo-

rophyll. The precursors of chloroplasts are called plastids, and they become
chloroplasts after they develop chlorophyll. However, in certain conditions, plastids
can fail to develop into chloroplasts, but still persist with reduced size and complex-
ity. Such undeveloped plastids are referred to as proplastids, and each is approxi-
mately the size and shape of a mitochondrion. Similar to mitochondria, both DNA
and ribosomes present in the chloroplast show prokaryotic affinities. The DNA of
chloroplasts (cpDNA) is circular in shape and its size ranges from 85 kilobases
(kb) in Codium, a green alga, to 2000 kilobases in Acetabularia, also a green alga.
The size of cpDNA is nearly five times that of an animal mtDNA. Like mtDNA, the
cpDNA also regulates synthesis of tRNA, ribosomal RNA, and certain proteins that
constitute the organelle. Data obtained by sequencing several cpDNA has shown that
about 100 genes are located in the chloroplast genome. Nearly 30 of these genes are
known to code subunits of five protein complexes involved in photosynthesis:
photosystem I, photosystem II, ribulose 1,5-bisphosphate carboxylase-oxygenase,
cytochrome b6-f complex, and ATP synthase. Nearly 60 genes code for machinery
involved in protein synthesis within the chloroplast (Fig. 3.36).
The prokaryotic affinities shown by the DNA and ribosomes of the chloroplast
led to the hypothesis that chloroplast originated from symbiotic cyanobacteria (blue-
green algae). These cyanobacteria have been shown to possess many similarities
with the chloroplast, and the rRNAs of cyanobacteria have been shown to hybridize
with the cpDNA. Similarities between mitochondria and chloroplasts have made it
possible to predict the inheritance patterns of chloroplast mutations on the basis of
knowledge derived from the study of mitochondrial genetics. However, in these
3 Extension of Mendelism 137

Fig. 3.35 Electron

micrograph of circular mouse
mitochondrial DNA.
Magnification 48,000

inheritances, it is important to discover and distinguish whether the mutation affect-

ing chloroplast function has occurred in chromosomal DNA or cpDNA. If mutations
undergo simple segregation, they will be chromosomal, whereas cpDNA mutations
show cytoplasmic patterns of inheritance. Also, adding to the complexity, particu-
larly in plants, is the presence of both mitochondria and chloroplasts, which makes it
difficult to determine whether a given trait is due to a mutation in cpDNA or mtDNA.
Similar to mitochondria, chloroplasts also exhibit homoplasmy and heteroplasmy. It
is important to note that cytoplasmic inheritance in plants happens in two
dimensions, i.e., via both chloroplast and mitochondria. In many cases, it may be
hard to distinguish between the two. Note that heteroplasmic cells are also called
cytohets or heteroplasmons.

3.5.3 Experimental Methods to Deduce Extranuclear Inheritance

Identification of extranuclear inheritance typically happened due to the observation

of peculiar results from reciprocal crosses. The progeny of a reciprocal cross has to
be followed for several generations; to deduct the patterns of extranuclear inheri-
tance, any premature interpretation can become misleading. Nuclear transplantation
has been utilized as powerful technique to identify extranuclear inheritance. The
138 R. Keshava

Fig. 3.36 Chloroplast DNA of Marchantia polymorpha. Circular DNA, 121,024 base pairs
(bp) long, consisting of 128 genes. Majority of chloroplast proteins are encoded by nuclear genome
and the rest are encoded by cpDNA. Consists of large inverted repeats A and B (IRA and IRB), small
single-copy region (SSC), and a large single-copy region (LSC). The 128 possible genes include
4 rRNAs, 32 tRNAs, and 55 proteins

technique involves removal of a nucleus from a cell such as an amoeba or a frog egg
by microsurgery or destroying using irradiation, and then substituted with nucleus
from another source. A heterokaryon test is another experiment that can be
performed with fungi such as Neurospora and Aspergillus, to determine extranuclear
inheritance. In this technique, the ability of mycelia to fuse and form a heterokaryon,
i.e., a cell containing nuclei from different strains, is utilized. The cytoplasm of a
heterokaryon consists of nuclei of both strains, which subsequently produce spores
(conidia) containing either of the two nuclei, and hence can be isolated. Isolated
3 Extension of Mendelism 139

conidia can be cultured to form colonies, whose phenotype demonstrates whether the
trait under study is determined by the cytoplasm or the nucleus.
It is also possible to isolate chromosomal genes in a particular cytoplasm by
repeated backcrossing of offspring with the male parent. Each such cross reduces the
quantity of the female chromosomal genes to half, but the cytoplasm remains similar
to the female line. After several generations, female cytoplasm will consist of male
genes, and the phenotypes resulting from this final cross will show whether the
inheritance of a particular trait is chromosomal or extrachromosomal.

3.5.4 Extranuclear Inheritance: Examples

3.5.4.1 Maternal Effects

A phenomenon in which the phenotype of an offspring is influenced by maternal
genotype or phenotype is called as maternal effect.

3.5.4.1.1 Snail Coiling

Shells of snails are coiled either dextrally (to the right) or sinistrally (to the left).
The direction of coiling is observed by holding the snail and looking at the opening
of the shell, top down from the apex. The snail is said to be coiled dextrally when the
opening comes from the right-hand side. It is said to be coiled sinistrally if the
opening comes from the left-hand side (Fig. 3.37).
The left panel of Fig. 3.37 shows fertilization between eggs of a dextral snail and
sperms of a sinistral snail. All offspring are dextral, which could indicate that dextral

Fig. 3.37 Maternal effects—shell coiling in snails. Genotype of the mother and not phenotype
determines coiling in snails. Figure depicts reciprocal crosses, where D, dominant, causes dextral
and d, recessive, causes sinistral coiling. In both crosses shown, DD was crossed with dd. The F1 in
both crosses have Dd genotype, but express the mother’s coiling phenotype. Offspring of DD
mothers show dextral coiling, whereas offspring of dd mothers show sinistral coiling. F2 in both
crosses are identical because of identical genotypes (Dd) of F1 mothers
140 R. Keshava

coiling is dominant. When the F1 snails were self-fertilized (snails are

hermaphrodites), all offspring were dextrally coiled. If dextral coiling was a domi-
nant trait, these results are unexpected. However, when F2 were self-fertilized,
one-fourth of offspring were sinistral, and three-fourths were dextral. If shell coiling
is considered as a dominant trait, the continued self-fertilization should reveal the 3:1
phenotypic ratio obtained to be 1:2:1 in the following generations, but observations
made did not correspond to it.
Results from a reciprocal cross (Fig. 3.37, right) showed that all F1 have the same
genotype similar to what was observed in the previous cross, but showed sinistral
coiling, similar to the female parent. But further results were the same for both
crosses. For both cases, it was observed that F1 had a phenotype similar to the female
parent although offspring of both crosses had the same genotype (Dd). The
explanations for these observations are as follows: the genotype of the maternal
parent determines phenotype of the offspring, with dextral dominant. Thus, the DD
mother in Fig. 3.37 produces dextral F1 with Dd genotype, whereas the dd mother
produced sinistral progeny, which also had the same Dd genotype.
The spiral cleavage of zygotes of molluscs during development is involved in the
determination of coiling phenotype. During such cleavage, the way the spindle is
tipped with respect to the egg axis determines the coiling. Tipping of the spindle in
one way produces sinistral and in another produces dextral coiling. It is the maternal
cytoplasm that controls the direction of tipping, which is in turn under the regulation
of the maternal genotype.

3.5.4.1.2 Moth Pigmentation

Pigmentation in the moth flour moth, Ephestia kuehniella, is another example of
maternal effect. In flour moth, the cytoplasm of the egg cell influences the pigmen-
tation phenotype of the offspring, under regulation of chromosomal genes.
Kynurenine, which is a precursor molecule for pigment synthesis, accumulates in
the eggs. When the recessive allele, a, is in homozygous condition (aa), it results in
absence of precursor. The results of reciprocal crosses are different for larvae and
adults. When a cross is performed with a nonpigmented female and a pigmented
male, the results obtained adhere to Mendelian principles. But when a reciprocal
cross is performed with a pigmented female (a+a), all larvae produced are pigmented
irrespective of their genotype, but will retain (a+a) or lose (aa) pigments at adult
stage based on their genotype (Fig. 3.38). The pigmentation observed in the larval
stage is due to the residual kynurenine that was present in the egg’s cytoplasm,
which eventually got diluted out, and the adults showed pigmentation as per their
own genotype.

3.5.4.2 Mitochondria-Mediated Inheritance

3.5.4.2.1 Poky Strain in Neurospora

In the haploid fungi Neurospora, studies on certain mutants can clearly establish
maternal inheritance. For example, a mutant of Neurospora called poky (Fig. 3.39)
shows a slow growth phenotype. Crosses in Neurospora can be performed in such
3 Extension of Mendelism 141

Fig. 3.38 Maternal effects—inheritance of pigmentation in larval and adult flour moth Ephestia
kuehniella. Single locus controls presence (a+) or absence (a) of kynurenine. Nonpigmented (aa)
mother produces aa offspring that are also nonpigmented in both larval and adult stages (left). In a
reciprocal cross (right), the pigmented mother with a+a genotype produces aa offspring that are
nonpigmented in the adult stage, but pigmented in the larval stage. This is due to the residual
kynurenine present in the egg from a pigmented mother

way that cytoplasm is contributed by one of the parents and hence behaves as the
maternal parent. Results of reciprocal crosses suggested that the mutation existed in
the mitochondrial gene(s). From the reciprocal crosses, it was evident that the female
parent or cytoplasmic parent’s phenotype determines the phenotype of all offspring.
The progeny of the reciprocal crosses are as follows:
Poky ♀  wild-type ♂ ! all progeny poky
Wild-type ♀  poky ♂ ! all progeny wild-type
Neurospora, being a fungus, does not possess chloroplasts, and hence the pheno-
type can be attributed to mitochondria based on its inheritance in reciprocal crosses.
Hence, the poky mutation is now known to be in mitochondrial DNA.

3.5.4.2.2 Petites in Yeast

Yeast, a facultative anaerobe, can grow in both aerobic and anaerobic conditions. In
aerobic conditions, the growth is characterized by distinctive colony morphology. In
anaerobic conditions, the yeast forms smaller colonies, and also mitochondria are
observed to be structurally reduced. Small anaerobic-like colonies can appear
sometimes, even if the yeast is grown in aerobic conditions. However, in such
small colonies, the mitochondrial structure appears perfectly normal. Such colonies
are a result of mutations called as petite. A cross between wild-type and petites reveal
three modes of inheritance (Fig. 3.40).
A petite caused by a chromosomal mutation is called a segregational petite, and
follows Mendelian inheritance. Neutral petite is lost instantly when crossed with a
wild-type. The suppressive petite shows variability in expression from one strain to
another but is able to convert the wild-type mitochondria into the petite form. All
142 R. Keshava

Fig. 3.39 Poky strain of Neurospora. Parent which contributes most of the cytoplasm to the
progeny is called the female. Brown shading indicates mitochondria containing poky mutation.
Green indicates normal mitochondrion. (a) All progeny is poky; (b) all progeny is normal. Both
crosses indicate maternal inheritance pattern. The ad+ (black) and ad (red) are representatives of
nuclear genes shown to indicate 1:1 segregation

types of petites are formed due to a failure in mitochondrial function. Whether the
regulation of the defective mitochondrial function resides in the mitochondria or
within the cell’s nucleus, they are usually deficient in one or the other cytochromes.
The composition of a DNA molecule can be measured by a technique called
density gradient centrifugation. Here the term buoyant density is used to describe the
equilibration position DNA attains when subjected to density gradient
3 Extension of Mendelism 143

Fig. 3.40 Categorization of petite yeasts based on their segregation patterns. There are three
categories of petites—segregational, neutral, and suppressive—depending on their meiotic segre-
gation pattern of a cross between petite and wild-type diploids. In spores of segregational petite
heterozygotes, a 1:1 ratio segregation is observed; heterozygous neutrals are lost; and suppressive
petites behave as dominant under the same circumstances

centrifugation. Therefore, buoyant density is a measure used to indicate any changes

in the composition of a DNA molecule when compared with the normal. When DNA
from the petites is subjected to such an analysis, some petites do not show any
change, while others show changes ranging from very small to complete absence of
DNA. From these results, it can be inferred that petites can occur due to a point
mutation, with no measurable change in the buoyant density, or can occur due to
marked changes in DNA, or even total absence of DNA. Most petites are
characterized by a mitochondrion, defective protein synthesis. Mitochondria that
completely lack DNA are known to occur in neutral petites. When wild-type are
mated with neutral petites, diploid cells are formed, in which normal mitochondria
dominate. Almost every spore during meiosis obtains numerous normal
mitochondria; therefore, all progeny is normal, whereas suppressive petites are
known to influence normal mitochondria in the following ways: suppressive
mitochondria may outnumber normal mitochondria by reproducing faster within a
cell, or crossing over between the DNAs of wild-type and suppressive petite may
affect normal DNA if the petite’s DNA harbors severe damage.
Several crosses involving a wild-type and a suppressive petite, with mtDNA of
known buoyant densities, have been performed. The mtDNA buoyant densities of
petite offspring were variable. For example, if a normal strain with mtDNA buoyant
144 R. Keshava

density of 1.684 g/cm3 is crossed with a suppressive petite of 1.677 g/cm3 buoyant
density, the mtDNA of the offspring had buoyant densities of 1.671, 1.674, and
1.683 g/cm3. These results suggested that the suppressive character took over the
colony by way of mtDNA recombination.

3.5.4.2.3 Human Mitochondrial Inheritance

In humans, root of certain diseases can be traced to mitochondrial pathologies.
Human pedigrees showing transmission of rare phenotypes particularly through
females but never through males have been identified. This pattern where a trait is
transmitted only through females suggests cytoplasmic inheritance. Cytoplasmic
inheritance in humans is mediated via the mtDNA and these rare phenotypes are
caused by a mutation in mtDNA.
The first mitochondrial disease was reported in 1962 and was called as Luft
disease, characterized by symptoms such as excessive sweating and general weak-
ness. Douglas Wallace and colleagues in 1988 showed cytoplasmic mode of inheri-
tance of another disease called Leber optic atrophy. As the name suggests, the
disease affects the optical nerve. A large number of mitochondria are usually present
in nerve cells, as their functioning requires high energy, and therefore defects in
mitochondria are not tolerated by nerve cells. In addition to damaging the optic
nerve, the disease also damages the heart. Maternal mode of inheritance of Leber
optic atrophy has been shown in the pedigrees. Point mutations associated with the
disease have been identified after sequencing mtDNA of individuals from affected
families. The point mutations involve a change in nucleotide 11,778, in the NADH
dehydrogenase subunit 4 gene, which is responsible for the disease phenotype. The
point mutation replaces the nucleotide guanine with adenine at codon 340, which in
turn substitutes amino acid arginine with histidine. Therefore, Leber optic atrophy is
the first human disease to be specifically linked to a particular mutation in mtDNA.
Presently, more than 260 different pathogenic mutations involved in a spectrum
of mitochondrial diseases have been reported, and the number is continuing to rise.
Three types of mtDNA mutations are as follows: point mutations in genes coding for
proteins and genes related to protein synthesis (rRNA or tRNA genes), and
rearrangements in mtDNA, including deletions and insertions.
MERRF (myoclonic epilepsy with ragged red fiber) is another phenotype caused
because of a point mutation mtDNA. The disease not only affects the muscle but also
causes hearing disorders and affects the eye. Kearns-Sayre syndrome is another
condition which exhibits an array of symptoms affecting the eyes, heart, muscles and
brain, and is caused by a deletion in the mtDNA. Currently, it is known that large
proportion of human diseases are associated with mitochondrial dysfunction. A
spectrum of disorders such as neurodegenerative disorders, cardiovascular disorders,
neurometabolic diseases, cancer, and obesity are associated with mtDNA
mutations (Table 3.12). Inability of the mitochondria to produce adequate amounts
of ATP is considered as one of the primary basis for most mitochondrial pathologies,
and hence resulting in multisystemic disorders. Extremely severe clinical
presentations are observed in high-energy demanding tissues, such as skeletal
muscle, central nervous system, and heart muscles. However, it is also important
3 Extension of Mendelism 145

Table 3.12 A list of some mitochondrial diseases and their clinical features
Disorder Primary features
CPEO (chronic progressive external External ophthalmoplegia, bilateral ptosis
ophthalmoplegia)
KSS (Kearns-Sayre syndrome) Progressive external ophthalmoplegia,
pigmentary retinopathy
Pearson syndrome Sideroblastic anemia of childhood,
pancytopenia, exocrine pancreatic failure
Leigh syndrome Subacute relapsing encephalopathy
NARP (neurogenic weakness with ataxia and Late-childhood or adult-onset peripheral
retinitis pigmentosa) neuropathy, ataxia
MELAS (mitochondrial encephalomyopathy Stroke-like episodes, seizures and/or
with lactic acidosis and stroke-like episodes) dementia, ragged red fibers and/or lactic
acidosis
MERRF (myoclonic epilepsy with ragged red Myoclonus, seizures, cerebellar ataxia,
fibers) myopathy
LHON (Leber hereditary optic neuropathy) Subacute painless bilateral visual failure

to note that mitochondrial disorder can also occur due to mutations in the nuclear
genome.
Some individuals with a mitochondrial disorder may be homoplasmic for mtDNA
mutation, but heteroplasmic condition is more prevalent. In heteroplasmy, cytoplas-
mic segregation causes varying proportions of normal and mutant organelles to be
transmitted to the progeny (Fig. 3.41). Heteroplasmic individuals have both mutant
and wild-type mtDNA in various proportions. In such a case, a proportion of mutant
to wild-type mtDNA determines disease expression. Therefore, higher level of
mutant mtDNA is usually associated with increased severity of the clinical
symptoms. This is called as the threshold effect. Only when the level of mtDNA
mutation exceeds a critical threshold the associated defect can be detected. Even in
an individual, the proportions of mutant and normal organelles vary temporally and
based on the different tissue types. Accumulation of certain types of mitochondrial
mutations over time has been considered as one of the possible causes of aging.

3.5.4.2.4 Mitochondrial Inheritance in Yeast: Antibiotic Influences

Correlating with the prokaryotic origin of the mitochondria, the protein synthesis
machinery within the mitochondrion is prokaryotic in nature. Therefore, antibiotics
such as erythromycin and chloramphenicol can inhibit protein synthesis in the
mitochondrion. Exposure of yeast cells to these antibiotics causes a petite-type
growth pattern. Yeast strains resistant to antibiotics can be obtained by growing
them on a medium containing antibiotic. In such case, only mutant yeast cells,
resistant to antibiotics, will grow in the medium. The resistance phenotype has
been observed to be inherited through mtDNA, and not through cellular DNA.
Crosses performed between resistant and sensitive (wild-type) yeast have shown a
mitochondrial pattern of inheritance (Fig. 3.42). Diploid yeast colonies obtained
from these crosses segregate into both resistant and sensitive cells. These results are
146 R. Keshava

Fig. 3.41 Cytoplasmic segregation of mitochondria. In heteroplasmic cells, organelles randomly

divide into the progeny. The diagram shows replicative segregation of mitochondria during mitosis.
Similar process takes place in meiosis also
3 Extension of Mendelism 147

Fig. 3.42 Mitochondrial inheritance—chloramphenicol (antibiotic) resistance in yeast. Diploid

cells formed from a cross between resistant and sensitive haploids produce both resistant and
sensitive cells. Segregation of the resistance trait is non-Mendelian, and relies on random assort-
ment of the mitochondria. Sensitive yeast does not possess resistant mitochondria. Resistant cells
possess resistant mitochondria

unlike chromosomal gene inheritance, and random distribution of mitochondria

during cell division such that a wild-type cell consisting of only sensitive
mitochondria is formed is a possibility. Since only one to ten mitochondria are
present per cell in some yeast, such random distribution can occur relatively at a high
frequency resulting in yeast cells with only sensitive mitochondria.
148 R. Keshava

3.5.4.3 Chloroplast-Mediated Inheritance

3.5.4.3.1 Variegation in Zea mays

As explained earlier in Sect. 3.5.1, cytoplasmic inheritance involves heteroplasmons.
The heteroplasmons undergo cytoplasmic segregation in which the two types of
organelles are distributed into different daughter cells. This is a process that mostly
arises due to chance segregation at the time of cell division. A good example of such
segregation can be seen in the variegated plants. Plants consisting of green and white
patches are called variegated.
M. Rhoades studied variegation in corn (Zea mays) and the phenotype is con-
trolled by a chromosomal locus called iojap. If the locus is homozygous for iojap, it
inhibits development of proplastids into chloroplasts, thus causing variegation.
Plastids affected by iojap lack ribosomes or ribosomal RNA and therefore are
deficient in protein synthesis. Reciprocal crosses pertaining to this trait are illustrated
in Fig. 3.43. The first cross between green female (IjIj) and variegated male (ijij)
produces green F1 (Ijij). Selfing of F1 resulted in green (IjIj and Ijij) and variegated
(ijij) with a genotypic ratio of 1:2:1, a typical Mendelian inheritance, with

Fig. 3.43 Chloroplast inheritance—variegation in Zea mays. Reciprocal cross involving iojap
gene for variegation in Zea mays. Variegation is induced by homozygous recessive, ijij. Blotch
variegation shows irregular white areas instead of stripes seen in regular variegation. Heterozygous
Ijij will be variegated if the mothers are variegated, because of transmittance of chloroplast from the
mother. Pollen parents do not transmit chloroplast
3 Extension of Mendelism 149

variegation induced by the homozygous recessive genotype (ijij). However, a

reciprocal cross between a variegated female (ijij) and green male (IjIj) resulted in
blotch variegation in F1 and F2 plants carrying dominant Ij allele. These observations
can be explained as follows: in the first cross, the ovule from the maternal parent
carries normal chloroplast and pollen grains do not carry any chloroplasts; hence,
normal chloroplasts are passed on to F2 generation. However, in the F2 generation,
variegation is induced by the genotype ijij. In this case, chloroplasts belonging to the
pollen parent are insignificant because they are not transmitted to the offspring. But
in case of a reciprocal cross, where the female (ovule) parent is variegated, the ovule
consists of abnormal proplastids in the cytoplasm. F1 heterozygotes therefore inherit
the cytoplasmic proplastids from the female parent, which remain unchanged even in
the presence of normal dominant allele, Ij, leading to a blotchy variegation, with
white spots consisting of colorless cells. Chloroplasts that get induced by the ij allele,
to become proplastids, cannot revert back to normal type even if Ij allele is present.
Evidences have suggested that iojap may bring about its effect by suppressing the
chloroplast rather than causing a functional mutation.

3.5.4.3.2 Variegation in Four O’clocks

Maternal inheritance of variegation in Mirabilis jalapa (four o’clock plant) was
discovered by Carl Correns in 1909. On the basis of the plant region on which the
stigma parent (female parent) was located, he predicted phenotypic outcome with
respect to color and variegation in the offspring. Flowers borne on white plant parts,
irrespective of the pollen type that pollinated them, produced white colored plants.
Similarly, flowers borne on green or variegated parts, irrespective of the pollen type
that pollinated them, produced green or variegated plants, respectively. This clearly
indicated maternal inheritance of variegation in Mirabilis jalapa. Figure 3.44 shows
a frequently observed variegated leaf and branch phenotype in Mirabilis jalapa,
Variegation observed in this plant is because of a mutant chloroplast gene. Mutation
in cpDNA causes chloroplasts to be white in color, which in turn makes the branches
and leaves white colored. Variegated branches display a mosaic of green and white
patches. Therefore, these plants produce three types of branches—green, white, and
variegated, and each branch produces flowers which consist of those respective
compositions of chloroplasts. Whenever a cross is performed (Fig. 3.45), the mater-
nal gamete determines leaf and branch color of the progeny. For example, an egg cell
belonging to a flower on green branch consisting of normal chloroplast will produce
all green progeny, regardless of the source of the pollen. Similarly, a white branch
consisting of white chloroplasts produces all white progeny. But due to the lethality
of the condition, descendants of white do not last beyond seedling stage.
The ova from variegated branches produce variegated zygotes consisting of both
green and white chloroplasts (Fig. 3.45). Since these zygotes are heteroplasmic, they
undergo cytoplasmic segregation. Division of such a zygote segregates green and
white chloroplast into separate cells, which appear as distinct green and white sector
characteristics of variegation. In this example, cytoplasmic segregation of an organ-
elle population containing a mixture of genetically distinct chromosomes is clearly
demonstrated.
150 R. Keshava

Fig. 3.44 Variegation in

leaves of Mirabilis jalapa.
Note that the plant consists of
three types of branches, i.e.,
green, white, and variegated.
Each of these branches gives
rise to flowers that can be used
for crosses

3.5.4.3.3 Antibiotic Resistance in Chlamydomonas

Chlamydomonas reinhardtii is a green alga, consisting of a single cell, and has been
extensively studied to understand extrachromosomal inheritance. There are several
advantages for choosing Chlamydomonas for these studies: (1) it consists of a single
large chloroplast; (2) it can survive in culture techniques even if chloroplast is
nonfunctional; and (3) it shows interesting non-Mendelian patterns of inheritance,
associated with the mating type. Ruth Sager has extensively researched inheritance
of streptomycin resistance in Chlamydomonas, and cells specific for antibiotic
resistance can be selected by various means. Antibiotic sensitive normal cells get
killed in a medium containing the antibiotic. But if these cells were initially grown in
low antibiotic concentrations (100 g/mL), some cells exhibit resistance. Further,
when these resistant cells are crossed with normal (sensitive) cells, 1:1 segregation of
resistance phenotype is observed indicating the chromosomal inheritance of strepto-
mycin resistance. If the experiment is repeated with higher concentrations of the
antibiotic (500–1600 g/mL), resistant colonies will again be obtained, but a cross
with wild-type will not result in a 1:1 segregation. Chlamydomonas cannot be
distinguished into male or female sex, but are identified as mating types mt+ and
mt. Mating in Chlamydomonas occurs only between cells of opposite mating types.
The mating type is encoded by a single gene locus consisting of a pair of alleles. A
diploid zygote (product of fusion of two haploids of opposite mating types mt+ and
mt) undergoes meiosis to produce four haploid cells, of which two are mt+ and two
are mt. Resistance to higher concentration of antibiotics always segregates with mt+
parent (Fig. 3.46), which is similar to maternal plastid inheritance in plants. Here it
can be considered that mt+ parent contributes cytoplasm to the zygote, while the mt
3 Extension of Mendelism 151

Fig. 3.45 Chloroplast inheritance in Mirabilis jalapa. Two different types of chloroplasts are
shown in the figure, green and colorless/white. The chloroplast content of the female cells
determines the type of offspring. Based on the chloroplast content and composition of the female
branch, the offspring can be green, white, or variegated

parent is similar to the pollen parent. The mechanism underlying such extrachromo-
somal inheritance in Chlamydomonas involves preferential digestion of cpDNA of
the mt parent.
Since Sager’s discovery, several other mutations in Chlamydomonas have been
discovered. All these have shown uniparental inheritance. All these mutations have
linked with the chloroplast. It is important to note that in Chlamydomonas several
152 R. Keshava

Fig. 3.46 Antibiotic resistance in Chlamydomonas. Streptomycin resistance is determined by the

mt+ parent. If the mt+ parent is resistant (left), then all meiotic products and the heterozygous diploid
will show resistance. If the mt+ parent is sensitive (right), then all products of meiosis and the
heterozygous diploid are sensitive

antibiotic resistance phenotypes are also transmitted via the mtDNA. But in these
cases, it has been observed that it is always transmitted by the mt parent, which is
opposite of what happens in chloroplast inheritance.

3.5.4.4 Infective Particles

3.5.4.4.1 Paramecium
Tracy Sonneborn discovered the cytoplasmic inheritance of a killer trait in Parame-
cium, a ciliated protozoan. Paramecium consists of two types of nuclei, a larger
macronucleus and a smaller micronucleus. Micronuclei are two in number and are
mainly involved in reproductive function. They consist of one macronucleus, which
is polyploid in nature, and regulate the vegetative functions. Paramecia divide by
binary fission, during which the micronuclei divide mitotically and macronuclei
divide into halves by constriction.
Conjugation and autogamy are two processes wherein Paramecia undergo
nuclear rearrangements of two types. The conjugation process involves coming
together of two Paramecia belonging to different mating types, and the formation
of a connecting bridge between them. The nuclear events that take place in each of
the Paramecia after the formation of the connecting bridge are shown in Fig. 3.47.
3 Extension of Mendelism 153

Fig. 3.47 Conjugation process in Paramecium. K and k are alleles of a gene present in
micronuclei. Exconjugants of a conjugation between KK and kk Paramecia acquire Kk genotype

The process begins with temporary disintegration of the macronucleus in each cell,
whereas micronuclei divide by meiosis to produce eight haploid micronuclei per
cell. Out of the eight haploid micronuclei, seven disintegrate and the remaining one
micronucleus divides by mitosis to form two haploid nuclei. At this stage, both the
conjugant Paramecia consist of two haploid nuclei each. This is followed by a
reciprocal exchange of nuclei between the two Paramecia that take place through the
connecting bridge. After the reciprocal exchange, each Paramecium consists of one
haploid nucleus of its own and one received from the other conjugant Paramecium.
These two haploid nuclei undergo fusion to give rise to a diploid nucleus. After
reciprocal exchange, the diploid nuclei of both conjugating cells are genetically
identical. Two more mitoses occur in the diploid nuclei to give rise to four diploid
nuclei in each cell. Two of these remain as micronuclei, whereas the other two
become macronuclei. After the process is complete, the contact between the conju-
gant paramecia is released, and these separated cells are called exconjugants. These
paramecia next undergo cell division, where the macronuclei separate, and the two
micronuclei undergo one more mitosis before separation. At the completion of
conjugation, each exconjugant gives rise to two daughter Paramecia. Each daughter
Paramecium consists of one macronucleus and two micronuclei. Depending
154 R. Keshava

Fig. 3.48 Autogamy in Paramecium. K and k are alleles of a gene located in micronuclei.
Autogamy in a heterozygote results in homozygosity for either K (KK) or k (kk)

particularly on the duration of contact (conjugation bridge) between the conjugating

cells, cytoplasmic exchange may also occur in addition to exchange of nuclei. From
Fig. 3.47, it can be seen that for a given pair of alleles (K and k), conjugation between
two homozygous Paramecia (KK and kk) results in heterozygous (Kk) daughter
Paramecia.
The process of autogamy involves a single Paramecium (Fig. 3.48). The nuclear
events in autogamy are same as in conjugation, except for absence of nuclear
reciprocal exchange, instead haploid nuclei of the same cell fuse to form the diploid
product. If autogamy happens in a Paramecium heterozygous (Kk) for a given pair of
alleles (K and k), after the completion of the process, the daughter cells become
homozygous with respect to one of the alleles (KK or kk) with a 50% chance for each
(Fig. 3.48).
3 Extension of Mendelism 155

3.5.4.4.2 Kappa Particles and Killer Paramecium

Sonneborn and his fellow researchers made some interesting observations when
working with Paramecium. They observed that mixing of certain stocks of Parame-
cium resulted in killing of one of the stocks by the members of the other stock. Such
Paramecia, which killed Paramecia of other stock, were called “killers,” and those
that got killed were called “sensitives.” It was observed that sensitives could become
temporarily resistant to killers during conjugation. If conjugation lasted long enough
to allow cytoplasmic exchange, then both exconjugants became killers. This
indicated that sensitives could acquire killer property if exchange of cytoplasm
occurred. On the contrary, if such cytoplasmic exchange did not occur, then killers
remained killers and sensitives remained sensitive. These observations indicated that
transfer of certain cytoplasmic factors could be involved in conversion of sensitives
to killers. Further studies by Sonneborn led to the discovery of cytoplasmic particles
called kappa in the killer Paramecia (Fig. 3.49).
Initial studies on the killer trait could not establish any link between chromosomal
genes and expression of killer phenotype in Paramecium. Later Sonneborn reported
a case of conjugation in which the killer exconjugant of hybrid origin (originated
from conjugation between killer and sensitive; see Fig. 3.47) underwent autogamy.
Fifty percent of the Paramecia that were produced by autogamy had lost kappa
particles and had become sensitive. Based on these results, he concluded that a
particular nuclear gene (dominant allele K ) is required to enable retention of kappa
particles, and hence to maintain the killer status. This was subsequently verified by
performing several crosses in Paramecia. Figure 3.50 shows the sequence of events
that produce a killer Paramecium heterozygous for K allele (Kk) which, upon

Fig. 3.49 (a) Normal (sensitive) Paramecium lacking kappa particles. (b) Killer Paramecium,
containing kappa
156 R. Keshava

Fig. 3.50 Autogamy in

heterozygous (Kk) killer
Paramecium. There is a 50%
chance of the heterozygote
becoming either KK (killer) or
kk (sensitive). The
Paramecium with kk genotype
becomes sensitive as it
eventually loses the kappa
particles
3 Extension of Mendelism 157

Fig. 3.51 A sectioned kappa

particle (Caedobacter
taeniospiralis) visualized
under an electron microscope.
The dark inclusions observed
are kappa containing phage
particles. Plane of the
sectioning is through a rolled-
up R body. Magnification
61,200

autogamy, has a 50% chance to become either killer or sensitive. As seen in

Fig. 3.50, Paramecium with genotype kk lose the kappa particles in the successive
cell divisions and become sensitive. Kappa is presumed to be a bacterium as it
possesses several prokaryotic features. Kappa was further studied by J. Preer and his
colleagues, and was named Caedobacter taeniospiralis. Killer Paramecia execute
killer effect by releasing a toxin called paramecin into its surrounding environment
and the sensitives are killed.
Kappa are found in two visibly distinct forms, which can be distinguished when
observed using bright phase contrast microscopy. There is a larger kappa called
bright kappa, which consists of one or more discrete refractile regions. The other is a
smaller kappa, called nonbright kappa, which does not contain refractile bodies
(Fig. 3.51). These two forms are abbreviated as B kappa and N kappa, respectively.
The refractile region is named R body. N kappa divide to produce more N kappa and
are also highly infective. B kappa is associated with the killing activity. However, it
is suggested that only a small portion of B kappa possess killing activity, and such B
kappa are designated as killing or P kappa (P stands for paramecin).

3.5.4.4.3 Mate-Killer Infection and Mu Particles

Apart from kappa, Paramecium also harbors other infective agents. Another such
infective agent is called the mate-killer infection. The killer cells in this case also
possess visible, bacteria-like particles in the cytoplasm, and are called mu particles.
Preer and colleagues named the bacteria Caedobacter conjugatus. The mode of
action of the mu particles is different than that of the kappa, i.e., they do not produce
any toxin, but instead kill their conjugating partners during conjugation. Like the K,
allele for kappa, either M1 or M2 dominant alleles are shown to be required to
maintain mu particles in the cytoplasm of Paramecia. Mate-killer become homozy-
gous recessive (m1m1 or m2m2) by autogamy. In the homozygous recessive condi-
tion, all offspring eventually lose their mu particles, but are able to retain up to
almost the eighth generation. By about the 15th generation, mu particles are present
in only around 7% of the cells. The reduction in the quantity of mu over generations
has been attributed to progressive dilution of a factor called metagon, which is
158 R. Keshava

known to be essential for the maintenance of mu in the cell. In homozygous

recessive cells, metagon production is known to cease and hence results in loss of
mu. These metagons appeared to be mRNA as they could be degraded by the
enzyme RNase.

3.5.4.4.4 Sex-Ratio Phenotype in Drosophila

Another example of infective particles is found in Drosophila. The sex-ratio pheno-
type is found in Drosophila females, wherein such females mostly produce
daughters and fewer sons. Several forms of sex-ratio phenotypes are known. There
are forms that are inherited via chromosomal genes and also there are others which
are nonchromosomal. Females with the nonchromosomal form also typically pro-
duce more number of daughters and fewer sons. An interesting feature of these
nonchromosomal forms is that the sons do not transmit this trait to the next genera-
tion, whereas daughters transmit the sex-ratio trait to the next generations. This
pattern is typical of cytoplasmic inheritance. About 50% of eggs from the sex-ratio
females failed to develop, and cytoplasm from such eggs was collected and used to
infect other females. A thorough examination of the cytoplasm of sex-ratio females
has shown the presence of a spirochete (Fig. 3.52). Researchers could isolate the
spirochete and cause sex-ratio phenotype in other female through infection with the
isolated spirochetes. Therefore, it was established that the spirochete cytoplasmic
inheritance caused sex-ratio phenotype in female Drosophila.

Fig. 3.52 Spirochete that

transmits sex ratio, an
extrachromosomal trait in
Drosophila. Electron
micrograph at magnification
22,700
3 Extension of Mendelism 159

Box 3.3 Scientific Concept: Evolution of Approaches to Study Complex

Diseases and Mapping of Gene Interactions (GIs)
GIs are now known to underlie various aspects of biology, including complex
diseases, speciation, and evolution of sex. GIs are known to play a vital role in
gene regulation, signal transduction, biochemical networks, and several other
physiological and developmental pathways. These gene-gene interactions, put
together, produce a phenotype. Also, it is known that some genes alter
phenotype of other genes, the outcome of which is differences in penetrance
and expressivity of those genes.
Several diseases in humans are a result of multiple genetic alterations and
follow non-Mendelian inheritance patterns. In other words, these disease
phenotypes occur due to a collection of mutations in several genes that
individually control single phenotypic traits. Overall complex GIs make the
disease phenotypes more than simply additive and hence raise the difficulty to
point at the exact causative gene. In fact, GIs affect not only onset but also
susceptibility of several complex diseases. These challenges have paved the
way for new approaches to study these complex human diseases.
After completion of the Human Genome Project in 2003, a radical new idea
of health called personalized medicine emerged. Personalized medicine seeks
to assess the risk for a disease and determine appropriate treatment using one’s
genetic information obtained by sequencing. It was assumed that as more and
more people got their genomes sequenced, discovery of disease-related genes
would also happen alongside. Although this was true to an extent, it also
increased the complexity of genetic data. As a result, even after 16 years of
sequencing, where tens of thousands of genomes have been sequenced,
gathering meaningful inference from the large volume of available genomic
data is yet a major challenge.
The genome data also threw light on the vast genetic diversity prevalent in
the human populations. It has been observed that these diversities bring about
variability in the disease inheritance patterns of individuals. The situation is
further complicated by environmental factors such as diet, lifestyle, etc., which
also influence expression of a disease trait. Progressively, it has been realized
that susceptibility to complex diseases such as heart disease, schizophrenia,
etc. is an outcome of several combinations of subtle genetic changes scattered
across the genome and not because of alterations in a single gene. There are
also cases in which a single gene variant is potent enough to cause a disease,
e.g., cystic fibrosis, hemophilia, etc. Even in such cases, two people affected
by the same disease variant can experience vastly variable disease severity.
Even more surprisingly, sequencing studies have identified people who carry
damaging mutations, but are perfectly healthy. In such individuals, other
unknown genetic variants within their genomes could be preventing disease
expression. These observations clearly show that GIs play a crucial role in

(continued)
160 R. Keshava

Box 3.3 (continued)

disease susceptibility and expression. Therefore, in addition to sequence data,
it is also important to map the GIs so that the basis of a disease can be
deciphered.
One of the primary challenges of modern genetics is to decipher the
complexity involved in genotype to phenotype relationship, and to be able to
apply the understanding to predict trait heritability. Traditional methods used
to study heritable traits or diseases relied on pedigree construction, in which
traits are mapped to the family members, revealing inheritance patterns. With
the emergence of the molecular genetics, linkage mapping approaches were
used to identify stretches of DNA in the human genome that were co-inherited
with certain disease traits. This was done by using genomic landmarks such as
single nucleotide polymorphisms (SNPs), as points of reference. Hence,
linkage mapping has been a very useful strategy for studying genes involved
in complex diseases such as type I diabetes. In spite of being useful, linkage
mapping and other related approaches were limited with respect to their extent
of use. These methods could not be used to study traits influenced by environ-
mental factors, having low heritability, or affecting restricted groups of people.
To overcome these limitations, newer strategies were evolved, which include
whole-genome approaches, where whole-genome sequences of affected
individuals are compared with normal control population. The differences
between the two genomes are assumed to indicate possible “hot spots” for
identifying genes causing those disease traits. Initially, sequencing approaches
were not considered feasible due to excessive costs involved. But this scenario
changed with advances in DNA sequencing technology and creation of com-
prehensive databases that recorded genome information. This resulted in
application of whole-genome association studies for complex human disease
traits. An interesting outcome of these studies was that scientists continually
discovered new gene associations for complex disorders.
Currently, several tools are available to analyze the genotype-phenotype
relationships, especially with respect to human diseases. Next-generation
sequencing technologies have been applied to create a database of the millions
of genetic variations in humans. This data, when used in combination with
population-based registries and electronic health records, are producing
extraordinary genomic and phenomic resources for such analysis. Genome-
wide association studies (GWAS), scanning genomes of patient and healthy
controls, have discovered thousands of genetic variations or mutations that
are associated with diseases. As of 2018, the NHGRI (National Human
Genome Research Institute) EBI (European Bioinformatics Institute) Catalog
reported >50,000 associated loci for >3000 unique traits (https://www.ebi.ac.
uk/gwas/). Most of these genetic variations are associated with common
diseases that affect human populations globally.

(continued)
3 Extension of Mendelism 161

Box 3.3 (continued)

Although we currently understand the chief importance of GIs and also
possess sequencing technologies that can comprehensively genotype entire
genomes, mapping of vital GIs associated with naturally occurring variations
within genomes of individuals remains extremely difficult. In this regard,
many inbred model systems are available. These include organisms such as
yeasts and worms. Also, fruit fly and mammal cell cultures are used as model
systems. These systems provide an experimental setup for systematically
mapping GIs. Understanding of gene interaction and their impact on the
phenotype of the organism has been enabled by large-scale studies in such
model organisms.
Mapping of GIs in the inbred animal model systems will enable an under-
standing of the fundamental principles of genetic networks, which in turn may
make it possible to map GIs and networks in natural outbred populations
taking into account the existing natural variations in individuals. The budding
yeast, Saccharomyces cerevisiae, is the most well-studied model systems for
deciphering GIs. The most widely applied method for analysis of yeast cell
phenotypes involves independent gene mutations, and applies a mathematical
tool for evaluation of GIs. In this method, the expected double mutant pheno-
type is considered to correspond to the product of those respective single
mutant phenotypes. The scoring of GIs is done by distinguishing double
mutants which deviate from the expected values in their phenotypes.
Studies in yeast cells emphasized the need to look beyond effect of individ-
ual genes in order to effectively understand their function. Yeast has been a
suitable model due to its relatively smaller genome consisting of 6000 genes
and already existing extensive database. In addition, many of the genes found
in yeast cells are also found in humans. The first map showing the global
genetic interaction network was created for a yeast cell. The model explains
how thousands of genes synchronize with one another to perform life
functions. Just as human societies are organized hierarchically from local
communities up to countries, genes in cells form hierarchical networks to
become organized into a cell. Instead of probing for single genes that underlie
diseases, it would be more suitable to look for gene pairs. Studies to unearth
the rules involved in combinatorial gene function have been performed in
yeast cells. Using the yeast system, scientists have worked out how genes
work in pairs. Studies for mapping GIs in yeast initially involved removing all
possible gene pairs (18 million of them) and studying their effect. After
completely studying gene pairs, the next step was to study trigenic
combinations (36 billion possible combinations). It was found that similar to
interactions between gene pairs, trigenic interactions also primarily occurred
between functionally related genes. It was observed that such interacting genes
coded for parts of the same molecular machinery or same part of a cell.

(continued)
162 R. Keshava

Box 3.3 (continued)

Importantly with trigenic interactions, more unexpected partnerships between
genes having unrelated functions and involved in different bioprocesses were
observed.
If a similar study had to be performed with the human genome, it would
involve examining approximately 200 million possible gene pairs for their
association with a disease. But this has been relatively simplified, because of
the know-how obtained from the yeast map. This has enabled researchers to
start mapping genetic interactions in human cells. Recent advances in gene
editing have made it possible to map relationships between disease genes by
removing gene combinations from human cells.

Box 3.4 Scientific Concept: GI and Cancer

A characteristic feature of cancer is its heterogeneity. The disease consists of
hundreds of distinct subtypes and varying genetic backgrounds. Identifying
unique GIs specific to a particular cancer and its subtypes is of great signifi-
cance. The Achilles heel of cancer therapy is systemic damage; hence, targeted
therapy is the most pressing goal of current research. Genetic network analysis
standardized in yeast has been now extended to human cells. It has been used
to identify cancer cell line-specific GIs that can uncover unique functional
relationships specific for those cancer types. Such unique GIs represent pro-
spective therapeutic targets which can be inhibited either genetically or chem-
ically to kill only cancer cells, without harming identical normal cells because
they lack the specific alteration. For example, ovarian and breast cancer cells
harboring BRCA1 or BRCA2 gene mutations are highly susceptible to PARP1
(poly (ADP)ribose polymerase 1) inhibition.
Using the cutting-edge CRISPR-Cas9 genome editing, researchers have
developed a breakthrough technology, which allows faster creation of novel
cancer treatments. This new technology was applied to identify a new pro-
spective therapeutic target in a type of pancreatic cancer. This new treatment
destroys cancer cells, by exploiting genetic faults inherent and unique to the
type of pancreatic cancer. Function of every single gene expressed in pancre-
atic cancer was probed by the researchers to determine one of the receptors
called Frizzled-5 is particularly required by mutant pancreatic cancer cells for
their growth. Normally, Frizzled-5 activates a signaling pathway that controls
cell division, cell differentiation, and cell death. However, it initiates tumor
development when mutated or deregulated. After identifying the important
role Frizzled-5 receptor plays in promoting growth of pancreatic cancer, the
team of researchers quickly developed an antibody drug against the receptor to

(continued)
3 Extension of Mendelism 163

Box 3.4 (continued)

inhibit growth of these cancer cells. Thus, developed antibody has been shown
to effectively kill cancer cells derived from patient tumor samples, and it was
also shown to cause tumor shrinkage in mice. Most importantly, the antibody
caused tumor shrinkage in mice but without harming the surrounding healthy
cells. These studies hold promise of developing highly effective therapeutics
that target only cancer cells, without causing systemic damage.

3.6 Summary

• When a gene exists in more than two alternative forms, it is called multiple
allelism. One of the best examples of the multiple allelic series in humans are
those that determine the ABO blood grouping system, and it is of great clinical
importance, particularly in transfusion medicine. The multiple allelic series of the
ABO system determine the type of antigens on the RBCs, which in turn
determines an individual’s blood group. The multiple allelic series of the ABO
blood group locus consists of three alleles, IA, IB, and i. Bombay blood group and
its variant para-Bombay are rare blood phenotypes.
• The dominance concept states that among the pair of alleles in a genotype, only
the dominant allele expresses itself in the phenotype and recessive allele gets
suppressed or hidden. A phenomenon where the heterozygote has an intermediate
phenotype compared to its homozygous parents is termed as incomplete domi-
nance. Codominance refers to a condition in which the heterozygote expresses the
phenotype of both the alleles equally.
• An allele that usually causes death, at an early developmental stage, and often
before birth is called as a lethal allele. Such an allele causes genotypes to be lost
from the progeny of a particular cross.
• In organisms with XY chromosomal sex determination, the sex chromosomes are
heteromorphic unlike the autosomes which are homomorphic. Therefore, the
patterns of inheritance of genes located on heteromorphic sex chromosomes are
different when compared with autosomal inheritance. X-linked pattern of inheri-
tance was first demonstrated in Drosophila by T. H. Morgan in 1910.
Hemizygosity causes a recessive allele to be expressed, even if present in single
copy. Such a phenomenon is called pseudodominance. Nonreciprocity is another
important feature of sex-linked inheritance.
• Phenotypic appearance of the genotypically determined traits is called as pene-
trance. Not all genotypes are able to “penetrate” the phenotype. However, most
genotypes show complete penetrance. However, certain genotypes, especially
those that code for developmental traits, frequently exhibit incomplete
penetrance.
164 R. Keshava

• The term expressivity is particularly used when a trait is not uniformly expressed
among individuals that show a particular trait. Several developmental traits in
addition to being incompletely penetrant also exhibit variable expressivity, rang-
ing from mild to extreme.
• The function and expression of a gene is very much dependent on its location or
position in the genome/chromosome, to an extent that a change of location or
position can alter its function. In several cases, such a repositioning of the gene
affects its expression level or, in certain cases, it alters its ability to function; this
is called position effect.
• Any given phenotype can be influenced by several genes and conversely one gene
can influence many phenotypes. When a mutant gene affects many aspects of the
phenotype, it is said to be pleiotropic (Greek for “to take many turns”). Such a
phenomenon is called pleiotropy, and the various effects caused are called
pleiotropic effects.
• Although genes often show independent assortment, they are not independent in
their phenotypic expression. In several cases, a gene at one locus influences the
expression of a gene at another locus. This kind of interaction among genes at
different loci (nonallelic genes), which affects the phenotypic outcome, is termed
gene interaction. Classically, epistasis is defined as a gene interaction where one
gene overrides the influence of another gene at a different locus in such a way that
its phenotype is suppressed.
• Patterns of inheritance are typically of three types, and are categorized based on
the gene location: autosomal inheritance is the inheritance of a gene located on
the autosomes; sex-linked inheritance is the inheritance of a gene located on the
sex chromosomes; and cytoplasmic inheritance is the inheritance of a gene
located on organelle chromosomes such as chloroplast (cpDNA) and
mitochondria (mtDNA).
• Extranuclear inheritance indicates transmission of characters through factors that
reside outside of the nucleus. It is also called as non-Mendelian inheritance
because Mendel’s laws of heredity are not applicable to their inheritance patterns.
Some of the other terminologies used include extrachromosomal, cytoplasmic, or
nonchromosomal inheritance. Cytoplasmic inheritance involves several factors
such as cellular organelles (e.g., chloroplast and mitochondria) containing their
own DNA, and parasitic or symbiotic particles (infective particles) that reside in
the cytoplasm and possess their own genetic material. Any given phenotype in
cytoplasmic inheritance is transmitted to the progeny particularly by the female
parent, but not the male parent.
Chromosome Mapping in Eukaryotes
4
Rohini Keshava

Construction of a series of descriptions indicating position and spacing of

characteristics such as genes and other DNA sequence features along a chromosome
is called chromosome mapping. The proposition of the chromosomal theory of
inheritance by Sutton in 1903 led to the accumulation of evidence that genes are
located on chromosomes. Morgan, through his Drosophila breeding experiments,
determined location of white-eye gene locus on the X chromosome, and established
chromosomes as carriers of genetic information. Also, it soon became obvious that
the number of genes in any organism exceeded the number of chromosomes,
suggesting location of several gene loci on an individual chromosome. Further
studies on inheritances deviating from Mendelism, cell division (meiotic and
mitotic) processes, and linear structure of eukaryotic chromosomes led to the
concepts of genetic linkage, crossing over, and recombination.
All these together contributed toward the development of the chromosome
mapping techniques. Mapping of positions of the genes on a chromosome was key
to analyzing their functions. Although presently complete genomes are being
sequenced, chromosome maps are still relevant. This is so because the functions of
most genes identified through sequencing of genomes are unknown. Hence, it is
required that the genes thus identified be correlated with information derived by their
phenotypic analysis. In this regard, the chromosomal maps play a very vital role.

4.1 Linkage, Recombination, and Crossing over

The law of independent assortment was the second law of inheritance proposed by
Mendel. This law stated that pairs of alleles segregate independently, i.e., if two
hypothetical genes A and B are considered, inheritances of alleles of gene A and B

R. Keshava (*)
Ramaiah University of Applied Sciences, Bangalore, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 165
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_4
166 R. Keshava

Fig. 4.1 Mendel’s law of independent assortment. (Top) The second law of inheritance proposed
by Mendel states that allelic pairs segregate independently. The figure depicts a parent heterozygous
for a pair of genes A and B. It can be seen that among the F1 generation, each member has equal
chance of inheriting either allele of each of these genes from the parent. (Bottom) The independent
segregation of the allelic pairs result in predictable outcome of the genetic crosses as depicted by the
9:3:3:1 phenotypic ratio of the dihybrid cross

are mutually exclusive and do not influence one another. Going by this law, it is
possible to predict the outcomes of genetic crosses (Fig. 4.1).
Followed by the rediscovery of Mendel’s work in 1900, it was soon observed that
in meiotic cell division, pairing of homologous chromosomes takes place and that
individual chromosomes of each pair segregate into separate daughter cells. These
observations led to the assumption that homologous chromosomes behaved as whole
units. Therefore, it was expected that all genes located on one chromosome are
transmitted together (Fig. 4.2) without undergoing independent assortment. This
concept was called as complete linkage.
However, the observations made by various scientists indicated otherwise. It was
observed that pairs of genes either showed independent assortment, as it was
4 Chromosome Mapping in Eukaryotes 167

Fig. 4.2 Genes on the same chromosome were expected to show complete linkage. Since A and B
are located on the same chromosome, they were expected to be always inherited together and hence
show complete linkage. Therefore, the law of independent assortment was not applied to genes A
and B, but was applicable to A and C or B and C as they were located on separate chromosomes

expected for genes present on different chromosomes, or showed partial or incom-

plete linkage (i.e., they were inherited together only sometimes, while at other times,
they were apart) (Fig. 4.3).
Therefore, linkage or genetic linkage can be defined as the tendency of genes
closely located on a chromosome to be frequently inherited together. The linkage
phenomenon was first discovered in 1905, by William Bateson, Edith Rebecca
Saunders, and Reginald C. Punnett (Fig. 4.3). Studies on the sweet pea plant
indicated linkage between flower color and pollen shape. In 1911, studies conducted
on inheritance patterns of fruit flies, Drosophila melanogaster, by Thomas Hunt
Morgan, revealed that the eye color trait was linked with the sex of the fly. Morgan
examined two genes on the X chromosome of Drosophila. One was a mutant gene
for white-eye color, and the other was a mutant gene causing miniature wings.
Morgan observed that the linkage was incomplete, i.e., he found that in female
flies white and miniature alleles generally tended to remain together but however
some recombinant X chromosomes with new combinations were produced.
These observations resulted in the emergence of the genetic linkage concept,
which describes how two closely associated genes located on the same chromosome
are often inherited together. Incomplete linkage is the general rule for genes located
on the same chromosome. The reason for this incomplete linkage is the phenomenon
of crossing over during meiosis. Crossing over leads to genetic recombination.
Recombination during meiosis occurs due to the exchange of chromosomal
fragments between non-sister chromatids.

4.1.1 Determination of Linkage

Bateson and Punnett’s study in sweet pea (Lathyrus odoratus) included the gene
influencing flower color (P, purple, and p, red) and the gene affecting the pollen
grain shape (L, long, and l, round). Pure lines of the plants producing purple flowers
168 R. Keshava

Fig. 4.3 Incomplete or partial linkage. Studies on inheritance conducted in the early twentieth
century by Bateson, Punnett, and Saunders with sweet pea. The cross depicted in the figure shows
that a result typical of a dihybrid cross is obtained in the parental cross, wherein all F1 plants have
the dominant phenotypes, viz., flowers with purple color and pollen grains that are long. But selfing
of F1 neither yielded an independent assortment ratio of 9:3:3:1 nor the complete linkage ratio of 1:
1. The unusual ratios obtained indicated partial linkage

and long pollen (P/P|
|L/L) were crossed with pure lines producing red flowers and
round pollen grains ( p/p|
|l/l). The F1 heterozygotes (P/p|
|L/l ) thus obtained were
further selfed to obtain the F2 plants. Figure 4.3 and Table 4.1 show the phenotypes
and their proportions in the F2 generation.
Although it was a dihybrid cross, the phenotypic ratio of the F2 phenotypes
deviated extremely from the expected 9:3:3:1 ratio as observed in the Mendelian
4 Chromosome Mapping in Eukaryotes 169

Table 4.1 Bateson and Punnett’s breeding experiments in sweet pea by F2 generation phenotypes
Number of progeny
Phenotype (and genotype) Expected from independent assortment (9:3:3:1 ratio) Observed
Purple, long (P/–k
kL/–) 3911 4831
Purple, round (/–kP
kl/l ) 1303 390
Red, long ( p/pk
kL/–) 1303 393
Red, round ( p/pk
kl/l ) 435 1338
6952 6952

crosses. Also, the observed ratios could not be explained as modification of the
Mendelian ratio. It was observed that the number of two phenotypic classes, i.e., the
purple, long and the red, round, was larger than expected.

4.1.1.1 Coupling and Repulsion Hypothesis

In an attempt to explain the basis of the observations made in sweet pea, Bateson and
Punnett hypothesized that the F1 plants had produced more numbers of gametes with
P|
|L and p|
|l combination than would be otherwise produced by independent
assortment. Since these genotypes were the same as the gametes of the original
pure lines, it was suggested that physical coupling of the dominant alleles P and
L and the recessive alleles p and l could not have allowed their independent
assortment in the F1 generation. However, they could not explain the basis of this
coupling phenomenon.
The proof and understanding of the coupling hypothesis put forth by Bateson and
Punnett came with the application of Drosophila as a genetic tool. Thomas Hunt
Morgan performed experiments with Drosophila and found similar deviation from
Mendel’s law of independent assortment. Morgan studied two autosomal genes, the
genes affecting eye color (pr, purple, and pr+, red) and wing length (vg, vestigial,
and vg+, normal) in Drosophila. Wild-type alleles of both genes are dominant.
Morgan mated pr/pr|
|vg/vg flies with pr+/pr+|
|vg+/vg+ and then performed a test
cross of the F1 double heterozygote females with double recessive male: pr+/pr|
|vg+/
vgk♀kkpr/prk
kvg/vgk♂.
Test cross is an extremely important tool used to reveal the gametic composition
of a heterozygous offspring of the F1 generation. This is so, because in such a cross,
one parent called the tester (homozygous recessive for the concerned trait)
contributes gametes consisting of only recessive alleles, and hence the phenotypes
of the offspring reveal the gametic contribution of the other heterozygous parent.
Therefore, for analysis only the meiosis in one parent (heterozygote) needs to be
considered, thus simplifying the process. This is in contrast to analyzing progeny
from an F1 selfing experiment, where meiosis in both male and female parents has to
be considered. The results obtained by Morgan are as follows, and it can be observed
that the F2 classes are specified by the alleles contributed by the F1 female:
As seen in Table 4.2 the number of progeny deviated considerably from the
Mendelian test cross ratio for independent assortment 1:1:1:1, thus indicating the
coupling phenomenon among genes. The two largest groups were of pr+|
|vg+ and
170 R. Keshava

Table 4.2 Progeny from Genotype Number of progeny

the test cross between a F1
pr+k
kvg+ 1339
double heterozygote female
(pr+/pr|
|vg+/vg|) and a prk
kvg 1195
double recessive male (pr/ pr+k
kvg 151
prk
kvg/vg) prk
kvg+ 154
Total ¼ 2839

Table 4.3 Progeny from Genotype Number of progeny

the test cross between a F1
pr+k
kvg+ 157
double heterozygote female
(pr+/pr|
|vg+/vg) and a dou- prk
kvg 146
ble recessive male (pr/ pr+k
kvg 965
prk
kvg/vg) prk
kvg+ 1067
Total ¼ 2335

pr|
|vg. The test cross clearly indicates the combination of alleles within gametes of
one of the parents in F1. Thus, it enabled clear observation of the coupling phenom-
enon. The test cross also reveals a 1:1 ratio within the two parental and the two
nonparental types.
The crossing experiment was repeated by changing the allelic combinations of the
parents and hence the gametes. Further test cross of F1 females was carried out:

P ! pr þ =pr þ k
kvg=vgkpr=pr k
kvgþ =vgþ k

#
F 1 ! pr þ =pr j
jvgþ =vgk

The progeny obtained as a result of the test cross were as follows:

The results of this test cross also clearly deviated from the Mendelian dihybrid
test cross ratio of 1:1:1:1. In these results, the two largest groups were pr+k
kvg and
prk
kvg+ (Table 4.3).
Similar to the earlier cross, in this too, the allelic combinations originally
contributed to F1 generation by parents form the most frequent groups in test cross
progeny. The term repulsion was used by Bateson and Punnett to explain such
phenomenon, as it appeared that the nonallelic dominant alleles “repelled” each
other. This was in contrast to what was observed in case of coupling, where the
dominant alleles appeared to stay together.

4.1.1.2 Coupling and Repulsion Explained

Morgan proposed that the genes regulating both phenotypes are situated on the same
homologous pair of chromosomes. Hence, both pr and vg were suggested to be
physically present on the same homologous chromosome and passed on from one
parent; likewise, pr+ and vg+ were passed on from the other parent (Fig. 4.4). In case
of repulsion, pr and vg+ were suggested to be located on one parental chromosome
and pr+ and vg were located on the other. Therefore, repulsion could be visualized as
another case of coupling, but the coupling was between a dominant allele of one
4 Chromosome Mapping in Eukaryotes 171

Fig. 4.4 Inheritance pattern of two allelic pairs located on the same homologous chromosome pair

gene and recessive of another. Hence, the occurrence of large number of parental
allelic combinations in the progeny could be explained. However, this could not
explain the occurrence of nonparental allelic combinations.
The terms coupling and repulsion are presently used to denote two types of
linkage conformations in a double heterozygote. They are depicted as follows:

The linkage between two dominant alleles or between two recessive alleles is
termed as coupling. The term repulsion is used to indicate linkage between a
dominant allele and a recessive allele. A test cross has to be performed or the
genotypes of the parents have to be analyzed in order to determine the linkage
conformations of a given double heterozygote. Alternatively, the coupling and
repulsion phases are also termed respectively as the cis and trans (Fig. 4.5) arrange-
ment of alleles on chromosomes.

4.1.1.3 Mechanism of Crossing over and Recombination

The physical process that underlies the genetic recombination is crossing over of
non-sister chromatids of the homologous chromosomes. In an attempt to explain the
physical basis of coupling and repulsion, Morgan hypothesized that pairing of
homologous chromosomes occurred in meiosis, and occasionally chromosomes
exchanged segments through an event of physical crossing over between them
(Fig. 4.6). This phenomenon resulted in the new combinations, i.e., the nonparental
combinations, which are also called as crossover products.
172 R. Keshava

Fig. 4.5 The cis and trans

arrangement of alleles on
dihybrid chromosomes

Fig. 4.6 Meiotic crossing over. One homolog from each parent is contributed to an individual
offspring. The homologs undergo crossing-over and exchange parts of chromosomes, thereby
producing gametic recombinant chromosomes whose allelic combinations are different than that
of parental combinations

Fig. 4.7 Diagrammatic representation of crossing over and chiasma formation at meiosis.
Chromatids of a pair of synapsed homologous chromosomes are represented as lines. Crossing
over occurs between non-sister chromatids of homologous chromosomes

The physical phenomenon of crossing over was observed in meiosis, during

which pairing of replicated homologous chromosomes occurs. Cross-shaped
structures are often formed between the two non-sister chromatids of the paired
homologous chromosomes (Fig. 4.7), and are called chiasma (singular) or chiasmata
4 Chromosome Mapping in Eukaryotes 173

(plural). The discovery of the chiasmata corroborated with the concept of chromo-
somal crossing over proposed by Morgan. His success in linking this cytological
phenomenon with results of his breeding experiments emphasized the significance of
the chromosomal theory of inheritance. Such observations, of coupling and repul-
sion in F1 selfing and test crosses, are commonly come across in genetics and are a
clear deviation from independent assortment. In other words, it can be concluded
that independent assortment does not occur when two genes are located close to each
other on the same chromosome pair.
Crossing over occurs during prophase I of meiosis. There are two observable
outcomes of crossing over and they are:

1. Chiasmata formation in the late prophase of meiosis I

2. Recombination of genes located on the opposite chromatids at the point of
crossover

Although chiasmata can be visibly observed, the outcome of recombination can

only be observed in the next generation, because it is in the offspring that the
recombinant chromosomal genes are expressed.
The crossing over among homologous chromosomes results in production of
recombinant gametes. This process is characterized by a physical exchange between
the non-sister chromatids of homologous chromosomes (Fig. 4.8). As explained
earlier, this event occurs during the prophase I of meiosis, where pairing of
duplicated chromosomes occurs. At this stage, the paired chromosomes consist of
four homologous chromatids, and they are said to form a tetrad. Although there are
four homologous chromatids, only two of them take part in crossing over at any one
point. This is followed by breakage of chromatids at the crossover site. Thus,
detached fragments reattach with the opposite chromatid to produce recombinants.
The other two chromatids do not undergo crossover and remain nonrecombinant at
this site. Therefore, the outcome of each crossover event is two recombinant

Fig. 4.8 Crossing over forms the basis of genetic recombination. The figure depicts the formation
of recombinant chromosomes as a result of exchange of chromosomal segments between paired
homologous chromosomes during meiosis. The homologs have been differentiated by using
different colors. From each homolog, only one of the chromatids takes part in a specific event of
recombination, thus producing two each of recombinant and nonrecombinant chromosomes
174 R. Keshava

Fig. 4.9 Multiple meiotic crossovers. Multiple exchanges can take place between the chromatids
of paired homologs during the prophase I of meiosis. Double, triple, and quadruple crossovers
between non-sister chromatids are depicted in a, b, and c, respectively. Each crossover occurs at an
exclusive location on the chromosome. If a crossover occurs between sister chromatids as depicted
in d, it does not result in recombinants

chromatids out of four. It is important to note that only two chromatids involve in a
crossover and exchange at any given point. Nevertheless, the other two chromatids
may become involved in a crossover at a different point. Hence, in a given tetrad,
there is a chance for multiple crossovers, resulting in multiple exchanges (Fig. 4.9),
for example, two, three, or even four, which are called double, triple, or quadruple
crossovers, respectively. However, it is important to note that if an exchange occurs
between sister chromatids, genetic recombinants are not produced because they are
4 Chromosome Mapping in Eukaryotes 175

identical. At the molecular level, the breaks in the chromatids at the chiasmata are
caused by enzymes that act on the DNA constituting the chromatids. Further,
enzymes also mediate the rejoining of the broken chromatid fragments to the
opposite non-sister chromatid.

4.1.2 Proof That Crossing over Makes Recombination

The cytological evidence for the mechanism of crossing over was obtained in 1931
by two scientists: Harriet Creighton and Barbara McClintock. Through their experi-
ment, they obtained evidence to prove that the genetic recombination event is
associated with exchange of material between chromosomes. Morphologically dis-
tinguishable homologous chromosomes of maize were used by Creighton and
McClintock for their study. The goal of the study was to determine if recombination
between genes located on homologous chromosomes correlates with the physical
exchange between these homologs. For this study, two different forms of chromo-
some 9 were selected. One of the chromosome 9 had a cytological aberration on both
ends, whereas the other one was normal. On one of the ends, the abnormal chromo-
some 9 possessed a heterochromatic knob, and on the other end, a fragment of
another chromosome was attached (Fig. 4.10).
These chromosomes were also genetically characterized, which enabled to detect
recombination. One of the marker genes located on this chromosome coded for the
kernel color (C, colored; c, colorless), and the other regulated texture of the kernel
(Wx, starchy; wx, waxy). The following test cross (Fig. 4.11) was performed:
The progeny of this test cross was examined for recombination and the associated
exchange of chromosomal segments between the two distinguished forms of

Fig. 4.10 Creighton and

McClintock experiment—
cytological evidence of
crossing over. Two forms of
chromosome 9 of maize
which were genetically
characterized were used in the
experiment. Crossing over
could be visually observed by
the distinguishing
morphology of the
chromosome 9 and also the
progeny could be scored for
the characterized genetic
markers
176 R. Keshava

Fig. 4.11 Test cross between

a F1 heterozygote and
homozygous recessive plant

Fig. 4.12 Outcome of a test

cross between a F1
heterozygote and
homozygous recessive plant.
Recombinants C Wx and c wx
consisted of a chromosome
9 with only one of the two
cytological markers; exchange
with the normal homolog of
chromosome 9 via crossing
over had resulted in a loss of
the other marker

chromosome 9. The exchange of the chromosomal segments could be visualized due

to the abnormal chromosome 9, and the genetic recombination could be identified by
the expression of the marker genes. The results obtained were as follows: the
recombinants C Wx and c wx consisted of a chromosome 9 which retained only
one of the two cytological markers. The loss of the other marker could be clearly
attributed to a chromosomal exchange event that occurred through crossing over
with the normal chromosome 9 homolog in the previous generation (Fig. 4.12).
These findings strongly indicated that physical exchange of chromatin material
between paired homologous chromosomes formed the basis of recombination.
4 Chromosome Mapping in Eukaryotes 177

Fig. 4.13 Diplonema during meiosis in male grasshopper. Eight autosomal bivalents and X
chromosome univalent can be seen. Among the autosomal bivalents, one chiasma each is seen in
four of the smaller chromosomes, while the remaining have two to five chiasmata

4.1.3 Chiasmata and Crossing over

It is during the late prophase of meiosis I, chiasmata, the cytological evidence for
crossing over can be clearly visualized. It is at this stage that paired homologous
chromosomes slightly repel each other. Although the homologous chromosomes
repel each other, they maintain a close contact at the centromere and also at each
chiasma (Fig. 4.13). This slight repulsion between the paired homologs partly
separates them, thus enabling accurate counting of chiasmata. It can be expected
the large chromosomes form more chiasmata compared to small chromosomes.
Therefore, the number of chiasmata can be considered to be proportional to the
length of chromosomes. Although the chiasmata are visible in the late prophase of
meiosis I, experimental evidence has suggested that crossing over occurs at an earlier
stage. Heat shock experiments to alter the recombination frequency showed little
effect when heat shocks were administered in late prophase, but earlier administra-
tion changed the recombination frequency.
Therefore, the crossing over events that lead to recombination occur rather early
in the prophase of meiosis. Molecular studies on DNA synthesis revealed additional
evidence in this regard. Although most of the DNA synthesis is completed during the
interphase of meiosis, a small amount of synthesis is shown to occur in the prophase
178 R. Keshava

of meiosis I as well. This small amount of DNA synthesis has been attributed to the
repair of broken chromatids possibly associated with the crossing over. Experimen-
tally, it has been shown that this DNA synthesis occurred during early to
mid-prophase, but not later. From this evidence, the time of crossing over was
deduced to be during early to mid-prophase, i.e., much earlier than the appearance
of chiasmata.
Most geneticists consider chiasmata as mere vestiges of the actual genetic
exchange process. It is suggested that the chromatids, involved in the exchange
process, remain entangled through most of the prophase. Eventually, the resolution
of these entanglements occurs, resulting in separation of the chromatids to the
opposite poles of a cell by the meiotic spindle apparatus. Hence, the chiasma is
believed to represent an entanglement created by an earlier crossover event during
the prophase. Several geneticists consider these entanglements resulting from cross-
ing over as a means to hold the chromosomes of a bivalent together during prophase
I of meiosis. In some organisms, prophase I is prolonged, i.e., it can extend up to
40 years in human females. If the crossovers do not exist, then paired homologous
chromosomes may separate accidentally, particularly during extended periods. Also,
the homologs thus separated may fail to disjoin properly during the subsequent
anaphase. Defective chromosome disjunction during meiosis I ultimately yields
gametes that are aneuploid. Hence, crossing over can be considered as a mechanism
for holding paired homologous chromosomes together during cell division, thereby
ensuring their appropriate segregation to each of the daughter cells. The possibility
of nondisjunction can thus be minimized, thereby largely preventing the occurrence
of aneuploid gametes.

4.1.4 Sturtevant and Mapping

Alfred Henry Sturtevant was one of Morgan’s students. He worked in the “fly
room,” Morgan’s research laboratory where the study of Drosophila genetics was
born. As discussed, earlier Morgan contributed immensely to the concept of
chromosomes as carriers of genes and linkage. He also proposed that closely linked
genes are located close together on the same chromosome and rarely get shuffled by
recombination, whereas loosely linked genes lie farther apart and hence are more
frequently shuffled by recombination. Working along with Morgan on the recombi-
nation between genes, Sturtevant in 1911 proposed that variation in the strength of
linkage can indicate their relative position along a chromosome, and hence can be
used as a tool for mapping genes.
Sturtevant constructed a remarkably accurate and the first genetic map (Fig. 4.14)
and established the basic gene mapping methodology which is used until today.
Alfred Sturtevant furthered his research in gene mapping, Drosophila genetics, and
other areas of biology such as evolution and became a leading geneticist.
4 Chromosome Mapping in Eukaryotes 179

Fig. 4.14 Sturtevant’s first genetic map. The first genetic map worked out by Sturtevant involved
five genes on the Drosophila X chromosome. The genes were yellow body (y), white eyes (w),
vermilion eyes (v), miniature wings, (m) and rudimentary wings (r). Marked above are the symbols
initially used by Sturtevant; corresponding modern symbols along with their present X chromosome
locations are marked below

4.1.5 Linkage Analysis

Linkage analysis forms the foundation of genetic mapping. Consider two genes A
and B, each having two alleles A,a and B,b, respectively. Considering that these
genes are located on the same chromosome, their behavior during meiosis can be
analyzed as shown in Fig. 4.15. It is assumed that alleles A and B are located on one
homolog and alleles a and b on the other. Two alternative outcomes are depicted in
Fig. 4.15.
1. Meiosis without crossing over between genes A and B: Among the four products
of meiosis, two of the gametes consist of AB genotype and the other two consist of
ab genotype, i.e., 2 AB : 2 ab
2. Meiosis with crossing over between genes A and B: Results in four types of
gametes with all possible genotypes: 1AB : 1aB : 1Ab : 1ab.

If the results of meiosis like the first example are scored in a hundred identical
cells, wherein crossover never occurred, then the following genotypes will be
present in the resulting gametes: 200 AB and 200 ab.
Here both genes A and B participate in meiosis as a single entity, thus showing
complete linkage. But in the greater likelihood in at least some nuclei, crossovers
will occur between A and B. In such a case, the allele pairs will not behave as a single
entity and thus are not inherited together. Assuming crossovers occurred in 40 out of
100 meioses, the following outcome will be observed: 160 AB, 160 ab, 40 Ab, and
40 aB, thus displaying incomplete linkage. Here the gametes possess both the
parental genotypes (AB, ab) and the recombinant genotypes (Ab, aB).

4.1.5.1 Linkage Analysis with Different Types of Organisms Can Be

Categorized as Follows
• The first category includes linkage analysis in species wherein it is possible to
perform planned breeding experiments, e.g., fruit flies and mice.
This method is based on analyzing progeny derived from experimental crosses
between parents of known genotypes. This method can be applied to all
eukaryotes theoretically. However, this approach cannot be applied for human
beings due to ethical considerations. Also, certain practical problems such as
180 R. Keshava

Fig. 4.15 Outcome of

crossover between linked
genes. The figure depicts a
meiosis involving a
homologous chromosome pair
(the homologs are
distinguished by red and blue
color) consisting of linked
genes A and B, with alleles
A, a, B, and b. Two alternative
scenarios during meiosis have
been depicted. (Left) Meiosis
without crossing over between
the linked genes A and
B. Among the four gametes
formed in meiosis, two are AB
genotype, and two are ab
genotype; (Right) Crossover
between A and B produces
gametes of all four possible
genotypes: AB, aB, Ab, and ab

prolonged gestation periods and time duration required by the newborn to mature
and grow to a reproducible age limit the effectiveness of this approach to some
animals and plants as well. As explained in Fig. 4.15, the key to gene mapping
lies in the determination of genotypes of gametes resulting from meiosis.
4 Chromosome Mapping in Eukaryotes 181

In some cases, such as in Saccharomyces cerevisiae (yeast), a microbial

eukaryote, gametes can be directly examined. It is possible to grow haploid
colonies of these organisms and using biochemical tests, their genotypes can be
determined. Gametes of higher eukaryotes can also be directly genotyped, by
PCR-based analysis of DNA markers (such as RFLPs, SSLPs, and SNPs; refer to
Sect. 4.4.2). Usually, spermatozoa are used for this purpose, but the typing of the
sperms is a laborious process. Therefore, routine linkage analysis in higher
eukaryotes does not involve examining the gametes directly. Rather, genotypes
of the diploid progeny are determined. Diploid progeny is an outcome of gametic
fusion, i.e., a genetic cross.
Nevertheless, a certain complication is inherent within a genetic cross, i.e., the
resulting diploid progeny are a product of not one but two different meiosis (one
in each parent), and in most organisms, there is an equal likelihood of crossover
events during production of both male and female gametes. Therefore, it becomes
very important that the crossover events occurring in each of the two meioses
need to be deciphered. Hence, adequate care has to be taken while setting up a
genetic cross. The standard procedure that is followed in these cases is to use a
test cross. Figure 4.16 illustrates two possible test cross scenarios. The parental
genotypes are a critical feature of the test cross.

In scenario I depicted in Fig. 4.16, one of the parents is a double heterozygote

with a genotype AB/ab and consists of all four alleles. The genotypic notation AB/ab
indicates that alleles A and B are present in one of the homologs of a homologous
pair of chromosomes, and alleles a and b are located on the other homolog. Such
double heterozygotes are obtained by crossing pure-breeding strains, e.g., AB/AB 
ab/ab. A pure-breeding double homozygote is the second parent in this scenario
(Fig. 4.16), i.e., in this parent, both homologs of a chromosome pair consist of a and
b alleles, and the genotype is depicted as ab/ab.
The main objective of the analysis depicted in scenario I is to deduce genotypes of
gametes contributed by the double heterozygous parent and to determine the recom-
binant fraction among them. It can be envisioned that all gametes contributed by the
second double homozygous parent will consist of ab genotype, irrespective of
whether they are parental or recombinant gametes.
Since both alleles a and b are recessive, in effect, meiosis in this parent will
become invisible when genotypes of progeny are observed. This implies that
genotypes of the diploid progeny can be represented clearly as the genotypes of
gametes produced by the double heterozygous parent as shown in scenario I of
Fig. 4.16. Hence, a test cross enables direct examination of a single meiosis. Further,
this enables calculation of recombination frequency and map distance for the two
genes being analyzed. Therefore, it can be concluded that for analysis of heterozy-
gote genotypes (consisting of one dominant and one recessive alleles), the double
homozygous parent has to be recessive for alleles of both phenotypes.
In scenario II of Fig. 4.16, DNA markers have been used for the analysis. In case
of DNA markers, they can be codominant in nature, and therefore, the double
homozygous parent can have a genotype of homozygous alleles in any combination
182 R. Keshava

Fig. 4.16 Test cross for linkage analysis. (Left panel) Scenario I: genetic markers A and B, with
alleles A, a, B, and b, are being analyzed. The progeny of test cross are scored by examining their
phenotypes. Since the second parent is double homozygous recessive, no effective contribution is
made to the progeny phenotypes. Hence, phenotype of F1 individuals is same as the gametic
genotypes of the first parent. (Right panel) Scenario II: DNA markers A and B whose allelic
pairs are codominant are being analyzed. The figure shows the genotype of double homozygous
parent to be Ab/Ab. Direct detection of the alleles present in each of the F1 progeny is made possible
by PCR-based techniques, and hence genotypes of the gametes contributed by the first parent can be
deduced in each individual offspring

as follows: AB/AB, Ab/Ab, aB/aB, or ab/ab. In this case, the markers on each allele
are directly detected by PCR analysis and hence can be easily identified as depicted
in scenario II of Fig. 4.16.
4 Chromosome Mapping in Eukaryotes 183

• The second category involves organisms where it is not possible to conduct

planned breeding experiments, e.g., humans. In this case, family pedigrees will
have to be made use of for linkage analysis (see Sect. 4.3).

4.2 Gene Mapping with Recombination Frequencies,

Calculation of Map Distance, and Concept of Map Units

Morgan and Sturtevant’s work had established a correlation between physical

distances separating genes on chromosomes and their rates of recombination.
According to Sturtevant (see Sect. 4.1.4), the occurrence of crossover events was
random and was more likely to occur between two genes positioned far apart than
those located close to each other on a chromosome. It was projected that recombina-
tion frequencies between genes could provide a convenient means of estimating their
relative distances and positions (order of arrangement) on a chromosome. Hence, the
practice of constructing genetic maps using recombination frequencies was
introduced.
Distances on genetic maps are measured in map units, and are abbreviated as m.
u.; 1% recombination is equal to one map unit. Map units are represented in
centimorgans (cM), in honor of Thomas Hunt Morgan; one Morgan ¼ 100 m.u.
A simple illustration of a genetic map is given below: consider three hypothetical
genes A, B, and C, where distance between AB is 5 m.u., and BC is 10 m.u. Since
genetic distances measured using recombination frequencies are nearly additive,
distance between A and C is 15 m.u. According to this data, the location of B is in
between A and C. Hence, on the basis of given map distances, the genetic map for A,
B, and C can be constructed as follows:

The map can also be redrawn by interchanging the positions of the markers C
(left) and A (right):

Both maps are equivalent and correct because relative positions of only three
genes are known, and maximum information that could be deduced pertains to
predicting which one of the three markers occupies the central position with respect
184 R. Keshava

Table 4.4 Results of Gene pair Recombination frequency (%)

recombination between A,
A and D 8
B, and C markers with D
B and D 13
C and D 23

to their linear arrangement on the chromosome. If information about an additional

gene is obtained, then the relative positions of the two markers, in this case A and C,
can be deduced relative to the fourth marker. In this example, gene D is the fourth
marker. Through genetic crosses, the following recombination frequencies between
D and each of A, B, and C can be obtained:
From the data in Table 4.4, it is evident that C and D exhibit the highest
percentage of recombination; hence, C and D must be the farthest, with A and B
genes situated between them. With addition of data pertaining to D, the map can be
redrawn as follows:

By performing a series of genetic crosses between various gene pairs, genetic

maps can be constructed, and the linkage arrangements of several genes can be
shown. Two points of significance need to be considered while constructing genetic
maps based on recombination frequencies. Firstly, the recombination frequency
between two genes located on a chromosome cannot exceed 50% as the rate of
recombination for genes located on different chromosomes is also 50%. Therefore, it
is not possible to distinguish between genes situated on different chromosomes and
those located on the same chromosome but separated by larger distances. If 50%
recombination is exhibited by a pair of genes, then the utmost conclusion one can
derive is that they belong to different linkage groups, located either far apart on one
chromosome or on separate chromosomes.
Secondly, test cross results for two genes located on the same chromosome, but
relatively far apart, tend toward underestimation of the true recombination fre-
quency, because the double crossovers that might take place between the two
genes are not revealed (Fig. 4.17). Two separate crossover events occurring between
the same two loci on a chromosome result are termed as a double crossover. In a
single crossover, alleles on homologous chromosomes are swapped and
recombinants are produced. The occurrence of a second crossover between the
same two loci reverses effect of the first one and the original parental combination
is restored (Fig. 4.17).
Since double crossovers result in only nonrecombinant gametes, it is not possible
to distinguish between progeny produced without a crossing over and those that are
produced by double crossovers. Also, overall leads to underestimation of
4 Chromosome Mapping in Eukaryotes 185

Fig. 4.17 Double crossovers result in nonrecombinant gametes. A single crossover swaps alleles
on the homologous chromosomes and produces recombinants. But when a second crossover occurs
between same two loci, it reverses the effect of the first crossover, i.e., the original parental
combination is restored, leading to production of nonrecombinant gametes

recombination frequencies, because double crossovers go undetected. The frequency

of double crossovers is higher between far apart genes, and hence genetic maps
based on markers separated by short distances will always be more accurate than
those based on markers located at larger distances.

4.2.1 Gene Mapping Through Two-Point Test Cross

A series of test crosses can be performed selecting gene pairs, and the recombination
frequency obtained from them can be used for constructing genetic maps. When a
test cross is performed involving two genes, it is named as a two-point test cross or in
short called as two-point cross.
Consider the cross between wild-type females and males homozygous for vesti-
gial (vg) wing and black (b) body mutations of Drosophila (Fig. 4.18).
The vestigial wing and the black body mutations are autosomal mutations that
produce short wings and black body, instead of the wild-type long/normal wings
(vg+) and gray body (b+). All F1 offspring were gray bodied and long winged. This
indicated the dominant nature of the wild-type alleles (vg+ and b+). A two-point test
cross (Fig. 4.18) between the F1 females and vestigial, black males produced F2
progeny of four phenotypic classes, of which two were abundant and two were rare.
186 R. Keshava

Fig. 4.18 A Two-point

genetic cross involving two
linked genes. The two linked
genes analyzed in the cross are
the genes coding for two
mutant phenotypes vestigial
wings (vg) and black body (b)
in Drosophila

The phenotypes that were abundant were the same as parental phenotypes, and those
that were rare were recombinants. As the number of recombinants was much lesser
than 50% of the progeny, it clearly indicates the linkage relationship between
4 Chromosome Mapping in Eukaryotes 187

vestigial and black genes. The observed linkage also suggested that these two genes
are located on the same chromosome. The distance between these two genes can be
determined by approximating the average number of crossovers between these genes
in gametes of F1 double heterozygous females, by calculation of recombination
frequency in F2 flies. It is observed that each such recombinant fly has inherited a
chromosome that had crossed over once between vg and b. Therefore, the average
number of crossovers in the sample of progeny is as follows:

Nonrecombinants Recombinants
(0)  0.82 + (1)  0.18 ¼ 0.18

In the calculation shown above, the number within the brackets represents the
number of crossovers and the other number represents the frequency, for each class
(recombinants and nonrecombinants) of flies. The inclusion of the nonrecombinant
progeny in the calculation is for the purpose of calculating the average number of
crossovers. Hence, all data has to be considered and not just that of the recombinants.
The result of this analysis shows that, on an average, 18 chromosomes out of 100 had
a crossover between vg and b during meiosis. This can be further interpreted to
construct a genetic map wherein vg and b are 18 map units apart.

4.2.2 Gene Mapping Through Three-Point Test Cross

Data from test crosses including more than two genes can also be used for the
purpose of recombination mapping. C. B. Bridges and T. M. Olbrycht designed an
experiment in which they crossed wild-type male Drosophila with females homozy-
gous for three X-linked recessive mutations—scute (sc) bristles, echinus (ec) eyes,
and crossveinless (cv) wings. An intercross of the F1 progeny was performed to
produce F2 flies. The F2 flies obtained were further classified and counted. It is to be
noted that F1 females selected for the intercross were heterozygous for the three
recessive mutations, i.e., one of their X chromosomes carried the recessive
mutations, while the other X chromosome carried the respective wild-type alleles.
Additionally, F1 males possessed single X chromosome that carried the three
recessive mutations. Consequently, this intercross becomes equivalent to a test
cross where all the three genes in the F1 female are present in a coupling configura-
tion. F2 flies derived from the intercross consisted of eight distinct phenotypic
classes, two of which were parental and six were recombinant (Fig. 4.19). Among
these classes, the flies belonging to the parental types were the most numerous. The
recombinant classes were lesser in number and each represented a recombinant
chromosome of different crossover events (Fig. 4.19).
In order to find out which crossovers gave rise to each type of recombinant, it is
necessary firstly to determine the order of gene arrangement on the chromosome.
188 R. Keshava

Fig. 4.19 Bridges and Olbrycht’s three-point cross experiment. The three-point crosses were
performed with X-linked genes sc (scute bristles), ec (echinus eyes), and cv (crossveinless wings)
in Drosophila

4.2.2.1 Determining Gene Order

Considering the three X-linked genes, scute (sc) bristles, echinus (ec) eyes, and
crossveinless (cv) wings, these can be arranged in three possible ways.

1. sc—ec—cv
2. ec—sc—cv
3. ec—cv—sc

It is required to determine which one of the three orders is correct. In order to

determine this, the six recombinant classes have to be studied carefully. Four (classes
3, 4, 5, and 6) out of these six recombinants must have resulted from a single
crossover in either one of the two regions as shown in Fig. 4.20 top left and right
panel. The remaining two recombinants (class 7 and 8) must be products of a double
crossing over, i.e., one exchange has taken place in each of the two regions as shown
4 Chromosome Mapping in Eukaryotes 189

Fig. 4.20 Calculation of map distances between genes from three-point cross experiment of
Bridges and Olbrycht. By estimating the average number of crossovers between each pair of
genes, the distance between the genes is obtained

in Fig. 4.20 bottom left and right panel. As a double crossover swaps the position of
the gene located at the center position relative to genes located on either side, it can
be applied for determining the linear order of arrangement of these genes, i.e., their
positions relative to one another can be elucidated. Naturally, the frequency of
double crossovers is expected to be much lesser than the frequency of a single
crossover. As a result, it can be observed that out of the six recombinant types, two
are rare and hence must signify double crossover chromosomes. In the given
example, classes 7 and 8, i.e., sc ec+cv and sc+ec cv+, respectively, are the rare,
double crossover types, consisting of one fly each (Figs. 4.19 and 4.20). Comparison
with parental classes 1 and 2, i.e., sc ec cv and sc+ ec+ cv+, respectively, shows that
there has been a switch in the position of the echinus allele with respect to the scute
and crossveinless alleles. These observations indicate that the echinus gene is located
between the scute and crossveinless. Hence, it can be concluded that the correct gene
order is sc—ec—cv.

4.2.2.2 Determining Distance Between Genes

After determination of the gene order, the next step is to determine the distances
between these adjacent genes. The procedure involves computation of the average
number of crossovers in the chromosomal regions between each pair of adjacent
genes (Fig. 4.20).
Distances between pairs of genes can be derived by identifying recombinant
classes that are a result of crossover between the concerned pair of genes. Therefore,
the length of the region separating sc and ec can be calculated by considering the
following four classes: 3 (sc ec+cv+), 4 (sc+ ec cv), 7 (sc ec+ cv), and 8 (sc+ ec cv+).
Classes 3 and 4 involve single crossovers between sc and ec and classes 7 and
8 involve two crossovers. One of the crossovers occurs between sc and ec and the
other occurs between ec and cv alleles (Fig. 4.20). The average number of crossovers
between sc and ec can be estimated by using the frequencies of these four classes:
190 R. Keshava

Class 3 Class 4 Class 7 Class 8

163 + 130 + 1 + 1
Total ¼ 295/3248 ¼ 0.091

This result can be interpreted as follows: out of every 100 chromosomes going
through meiosis in the females of F1 generation, 9.1 have undergone a crossover
between sc and ec. Hence, distance between sc and ec genes is 9.1 map units or 9.1
centimorgans.
Similarly, the distance between ec and cv can be obtained. Recombinant classes
5 (sc ec cv+), 6 (sc+ ec+ cv), 7, and 8 involved a crossover between ec and cv. It is also
required to consider the double recombinants since one of their two crossovers has
occurred between ec and cv. Thus, by adding frequencies of all four classes, the
following is obtained:

Class 5 Class 6 Class 7 Class 8

192 + 148 + 1 + 1
Total ¼ 342/3248 ¼ 0.105

From the above calculations, the distance between ec and cv is 10.5 map units.
Thus, by combining data from both regions, a genetic map showing the relative
positions and distances between these genes can be constructed.
sc—9.1—ec—10.5—cv
The map distances calculated in this way are additive. Therefore, the distance
between sc and cv can be estimated by adding the map distance separating sc and ec
to the distance separating ec and cv. The calculation is as follows:

9:1 cM þ 10:5 cM ¼ 19:6 cM

The same can also be obtained by directly adding average number of crossovers
between these genes:

Non-crossover Single crossover Double crossover

classes classes classes
1 and 2 3, 4, 5, and 6 7 and 8
(0)  0.805 + (1)  0.195 + (2)  0.0006 ¼ 0.196

As observed in the above calculation, combined frequency of the different classes

has been multiplied with the total number of crossovers (written in parentheses) that
occurred within them. Hence, map distance is a product of the recombinant class
frequency and number of crossovers that occurred within it.
Bridges and Olbrycht, in their recombination experiment, studied seven X-linked
genes: sc, ec, cv, ct (cut wings), v (vermilion eyes), g (garnet eyes), and f ( forked
bristles). By calculating recombination frequencies between pairs of adjacent genes,
they constructed genetic map of an X chromosome segment (Fig. 4.21). In their map,
the extreme positions were occupied by sc and f. Each of the seven genes that
4 Chromosome Mapping in Eukaryotes 191

Fig. 4.21 Bridges and

s
ing
Olbrycht’s map of seven

sw

es
X-linked genes in Drosophila.

les
les

es

les

ey

ist
ey

ye
The map distances between

ist

ein

br
ing

te
br

us

ilio
these genes are given in

sv

d
e
e

hin

tw

rm

e
rn
os
ut

rk
centimorgans

ga
ec

cu

ve
sc

cr

fo
sc ec cv ct v g f

9.1 10.5 9.2 15.9 11.2 10.9

66.8
Drosophila X chromosome

Bridges and Olbrycht chose to study was markers representing a particular site on X
chromosome. The total length of the mapped X chromosome segment was obtained
by addition of map intervals between each pair of adjacent markers, and was
estimated to be 66.8 cM. Hence, the average number of crossovers in this X
chromosome segment was 0.668.

4.2.2.3 Interference and the Coefficient of Coincidence

Compared to two-point crosses, three-point crosses are more advantageous as they
make enable detection of double crossovers. This makes it possible to determine if
exchanges occurring in adjacent regions are mutually interdependent or indepen-
dent. For example, in the given three-point cross (Fig. 4.20), we will be able to
determine if occurrence of a crossover between sc and ec is independent of a
crossover between ec and cv or whether occurrence of one has an inhibitory effect
on the other. In general terms, it is possible to determine if crossover in one location
on a chromosome inhibits formation of another in a nearby site.
This can be analyzed by calculating the expected frequency of double crossovers,
by assuming that these events are independent and are done by multiplying the
crossover frequencies of two chromosomal regions that are adjacent to one another.
In the map constructed by Bridges and Olbrycht, the crossover frequency in the first
region (sc and ec) was (163 + 130 + 1 + 1)/3248 ¼ 0.091, and in the second region
(ec and cv), it was (192 + 148 + 1 + 1)/3248 ¼ 0.105. In case independence is
assumed then, 0.091  0.105 ¼ 0.0095 would be the expected double crossover
frequency between sc and cv. When this is compared with observed frequency
(2/3248 ¼ 0.0006), it can be seen that it is much lesser than the expected frequency.
Therefore, double crossovers between sc and cv are less frequent than would be
expected in case of independence. This indicated that occurrence of one crossover
inhibited occurrence of a second in the nearby vicinity, and this phenomenon is
called interference. The coefficient of coincidence (c) indicates the extent of inter-
ference and can be defined as a ratio of the observed frequency to the expected
frequency of double crossovers:

c ¼ Observed frequency of double crossovers

Expected frequency of double crossovers
¼ 0:0006
0:0095
¼ 0.063
192 R. Keshava

4.2.3 Mapping in Neurospora and Yeast: Haploid Mapping (Tetrad

Analysis)

The Ascomycetes fungi retain the four haploid (tetrad) meiotic products in a saclike
structure called an ascus and hence enable analysis of all products of meiosis,
thereby making it possible to determine certain fundamental facts such as occurrence
of DNA replication prior to crossing over and reciprocity of crossing over by
employing several techniques.
Primarily, two fungi have served as models for these analyses. One is the
Saccharomyces cerevisiae (common baker’s yeast) and the other is Neurospora
crassa (pink bread mold). Both these organisms retain the meiotic products as
ascospores (Fig. 4.22).

4.2.3.1 Phenotypes of Fungi

The phenotypes of microorganisms generally can be classified into three broad
types: colony morphology, nutrient requirements, and drug resistance. Several
microorganisms are culturable in supporting medium such as agar in test tubes and
petri plates (Fig. 4.22). Several substances can be added to the growth medium.
The wild-type Neurospora commonly grows in filamentous forms, while the
yeast cells form colonies. Several mutations that alter colony morphology have
been identified. One such alteration in the yeast (ade gene) results in red colored
colonies. Similarly, in Neurospora, colonial (col4), tuft (tu), fluffy ( fl), and dirty
(dir) are all basic growth form mutants. Additionally, wild-type Neurospora shows
sensitivity to sulfonamide, a sulfa drug, and also a mutant form of Neurospora (Sfo)
requiring sulfonamide for its survival and growth also exists. Similarly, yeast also
exhibits sensitivities to antifungal agents. The nutritional requirement phenotypes of
these microorganisms give insights into biochemical metabolic pathways and also
aid in genetic analysis. Growth of wild-type Neurospora can be observed in a
minimal medium consisting of sugar and a nitrogen source along with some salts,
organic acids, and biotin (a vitamin). Neurospora consists of several mutants, or
strains, that are unable to grow on minimal medium, and require addition of some
essential nutrients into the medium. For example, one such mutant strain of Neuros-
pora grows in minimal medium only when it is supplemented with the amino acid
arginine (Fig. 4.23).
It can be inferred that this mutant is deficient for one of the components
(an enzyme) involved in the biosynthesis pathway of arginine (Fig. 4.24), which is
functional in the wild-type Neurospora.
Several such nutritionally deficient strains with mutations in enzymes at various
steps, particularly in long biochemical pathways, can be found. For example, several
mutants at different loci for arginine biosynthesis are named as arg1, arg2, etc.
Several such biosynthetic pathways in Neurospora and yeast harbor such mutations,
and hence a variety of mutants exhibiting various nutritional requirements can be
4 Chromosome Mapping in Eukaryotes 193

Fig. 4.22 Spore isolation

technique in Neurospora

found. Mutations can also be induced by treatment with agents such as irradiation or
chemicals. Such mutants are applied as tools for the analysis and mapping of
chromosomes of microorganisms such as Neurospora and yeast.

4.2.3.2 Identification of Linkage and Mapping Genes in Yeast by

Analysis of Unordered Spores
The budding or baker’s yeast, Saccharomyces cerevisiae, occurs in both diploid and
haploid forms (Fig. 4.25).
Haploid form of yeast generally forms in conditions of nutritional stress or
starvation. With the return of optimal environmental conditions, haploid cells of a
194 R. Keshava

Fig. 4.23 Isolating nutritional requirement mutants in Neurospora

and α mating types undergo fusion resulting in the diploid yeast. Under conditions of
starvation, the haploid stage is again established by meiosis. All products of meiosis
in the yeast are contained in the ascus.
To understand mapping, the a and b loci can be taken as an example. When
spores or gametes consisting ab and a+b+ fuse, the diploid formed undergoes
meiosis. Such spores (products of meiosis) can be isolated and grown into haploid
colonies. These colonies can then be observed for phenotypes encoded by the two
loci. Only three patterns (Table 4.5) can occur in this case.
Therefore, there are three classes of spores. Class 1 spores are of two types, and
are identical to parental haploid spores, and such an ascus is called parental ditype
(PD). Class 2 consists of two types of recombinant spores and such an ascus is called
nonparental ditype (NPD). Class 3 called as a tetratype (TT) comprises all four
possible spore types.
Irrespective of the linkage status of the two loci, all three types of ascus can be
generated. As it can be seen in Fig. 4.26, parental ditypes arise due to the lack of
crossing over in case of linked loci, while nonparental ditypes arise due to double
crossovers involving all four chromatids, also termed as four-strand double
crossovers.
Therefore, it can be expected that the parental ditypes should be more in number
than nonparental ditypes with respect to linked loci. However, in case of unlinked
4 Chromosome Mapping in Eukaryotes 195

Fig. 4.24 Arginine

biosynthetic pathway in
Neurospora

loci, parental and nonparental ditypes can arise through independent assortment, and
in such a case, all types will be expected to arise in equal frequencies. Hence, by
analyzing number of parental and nonparental ditypes, linkage between the loci can
be determined. The parental ditypes will greatly exceed the number of nonparental
ditypes if the genes are linked. The next step would be to calculate map distance
between the given loci. From Fig. 4.26, it can be seen that all four chromatids in a
nonparental ditype are recombinant, whereas only half the chromatids in a tetratype
are recombinant. As mentioned earlier, 1% recombinant offspring is equal to 1 map
196 R. Keshava

Fig. 4.25 Yeast life cycle. Mating types are represented by a and α. The haploid stage is denoted
by n and the diploid stage is denoted by 2n

Table 4.5 Meiosis in a 1 2 3

dihybrid, aa+bb+, yeast and (PD) (NPD) (TT)
the resultant three ascus
ab ab+ ab
types
ab ab+ ab+
a+b+ a +b a +b
a+b+ a +b a+b+
75 5 20
4 Chromosome Mapping in Eukaryotes 197

Fig. 4.26 Types of asci resulting from meiosis in yeast. PD (parental ditype), NPD (nonparental
ditype), and TT (tetratype) asci formed in a dihybrid yeast by independent assortment or linkage at
meiosis. Centromeres are depicted as open circles

unit, and using the following formula, the map distance between the loci can be
calculated:
198 R. Keshava

Map ¼ ð1=2Þ the number of TT asciþthe number of NPD asci  100

Total number of asci
units
Thus, for the data of Table 4.5
Map ¼ ð1=2Þ20þ5  100 ¼ 10þ5
100
 100 ¼ 15
100
units

4.2.3.2.1 Mapping in Neurospora

Neurospora consists of ordered spores; life cycle of Neurospora is shown in
Fig. 4.27.
Mating types in Neurospora are referred to as A and a. Mating between these two
mating types results in fertilization and it takes place within an immature fruiting
body. The diploid nucleus of the zygote undergoes meiosis immediately. Due to the
narrow structure of the Neurospora ascus, the products of meiosis are arranged along

Fig. 4.27 Neurospora life cycle. The mating types are represented as A and a; n represents haploid
stage; 2n represents diploid stage
4 Chromosome Mapping in Eukaryotes 199

Fig. 4.28 Meiosis in Neurospora. The two mating types are denoted by A and a. Here A and
a represent centromeres of the chromosomes of the two mating types, and the spores are labelled
likewise. Note that, for convenience purpose only one pair of chromosomes is shown in the figure,
although Neurospora consists of seven pairs of chromosomes

the ascus’s longitudinal axis. Hence, the spores are said to be ordered (Fig. 4.28), i.e.,
if centromere of a chromosome of one mating type is labeled A and the other is
labeled a, then at meiosis I, one each of A and a will be present in a tetrad. At the
completion of meiosis, the four ascospores formed will be arranged in either one of
these two orders, a a A A or A A a a.
In Neurospora, a mitosis takes place in each nucleus prior to the maturation of
ascospores. This results in the formation of four pairs of spores instead of just four
spores. The pairs are always identical provided they do not show any genetic
phenomena such as a mutation or a gene conversion (Fig. 4.28). The ordered spores
in Neurospora make it possible to map genetic loci relative to their centromeres.

4.2.3.2.2 First- and Second-Division Segregation

The centromeres in an ascus of Neurospora undergo a 4:4 segregation. There are two
kinds of patterns that are observed among the loci on the chromosomes of spores and
are dependent on incidence of crossover between the given locus and its centromere
(Fig. 4.29). If a crossover did not take place between the given locus and its
centromere, then the allelic pattern is same as the centromeric pattern, and is called
first-division segregation (FDS), because the alleles separate from each other at
meiosis I. Alternatively, if a crossover occurred, then different types of segregation
patterns (2:4:2 or 2:2:2:2) called second-division segregation (SDS) can be
observed. However, centromeres will always follow a FDS pattern because of the
ordered nature of the spores in Neurospora, and hence will enable mapping distance
between the given locus and its centromere. Considering simplest circumstances
(Fig. 4.29), every SDS configuration will have four each of nonrecombinant and
recombinant chromatids (spores). Hence, in an SDS ascus, half of the chromatids
(spores) are recombinant, and the map distance can be calculated as follows,
considering 1% recombinant chromatid is equal to 1 map unit:
200 R. Keshava

Map distance ¼ ð1=2Þ the number of SDS asci  100

Total number of asci

Table 4.6 shows an example where this calculation is applied.

Three-point crosses can also be examined in Neurospora. Mapping of two loci
and their centromere can be taken as an example. The letters a and b are used to
denote two loci. Fusion of mycelia results in the formation of dihybrids (ab  a+b+),
which further undergo meiosis. Data is collected by analyzing 1000 asci, without

Fig. 4.29 Neurospora

ascospore patterns. Six
possible ascospore patterns
with respect to one locus are
depicted in the figure
4 Chromosome Mapping in Eukaryotes 201

Table 4.6 Meiosis in an a+a heterozygous Neurospora and the resulting genetic patterns (ten asci
are examined)
Ascus number
Spore number 1 2 3 4 5 6 7 8 9 10
1 a a a+ a a a+ a a+ a+ a+
2 a a a+ a a a+ a a+ a+ a+
3 a a a+ a+ a+ a+ a a a a+
4 a a a+ a+ a+ a+ a a a a+
5 a+ a+ a a+ a a a+ a a+ a
+
6 a a+ a a+ a a a+ a a+ a
+
7 a a+ a a a+ a a+ a+ a a
8 a+ a+ a a a+ a a+ a+ a a
FDS FDS FDS SDS SDS FDS FDS SDS SDS FDS
Map distance ðalocus to centromereÞ ¼ ð1=2Þ%SDS
Note: ¼ ð1=2Þ 40%
¼ 20 map units

changing the spore order. The data thus obtained are grouped as follows: since six
different patterns can be shown by each locus (Fig. 4.29), 36 possible spore
arrangements should be obtained when two loci are scored together (6  6).
Several of these 36 patterns are actually random variants of each other. This is so
because at the first meiotic division, either of the centromeres of a tetrad can separate
toward either of the poles (i.e., go left or right). Further, when splitting of
centromeres takes place at meiosis II, movement of the spores within the progeny
ascus is also random. Therefore, one single genetic event can yield up to eight
“different” patterns. One such possible arrangement of the spore chromatids post
crossing over is depicted in Fig. 4.30. Considering arrangements in which there is a
crossover between a and b loci, all eight arrangements that produce ascus patterns as
shown in Table 4.7 are equally possible. Then in that case, the 36 possible patterns
reduce to only seven unique patterns as shown in Table 4.8.

4.2.3.3 Gene Order Determination

After having obtained the data shown in Table 4.8, the distance between each locus
and the centromere and the linkage arrangement of two loci associated with the same
centromere can be determined. It can be established by inspection that two loci are
liked and also that they are linked to the same centromere. As shown in Table 4.8,
examination of classes 1 (PD—parental ditype) and 2 (NPD—nonparental ditype)
allows deduction of the linkage relationships among the loci and also that of the
centromere. If the two loci under consideration are unlinked, both categories 1 and
2 will represent two equally probable alternative events when crossing over does not
occur. Since in this case category 1 makes up almost 75% of all asci, it can be
202 R. Keshava

Fig. 4.30 Spore patterns in Neurospora. A single crossover between a and b loci can yield eight
possible random arrangements. Circular arrows indicate rotation of a centromere from its position in
the original configuration

deduced with certainty that the two loci are linked. In order to determine the distance
between each of the locus and the centromere, it is required that for each locus 50%
of SDS patterns are calculated. Classes 4, 5, 6, and 7 and classes 3, 5, 6, and 7 are the
SDS patterns for loci a and b, respectively. Therefore, for each locus, the distance to
the centromere, in map units, is calculated as follows:
4 Chromosome Mapping in Eukaryotes 203

Table 4.7 Eight out of 36 possible spore patterns in Neurospora, scored for loci, a and b (all are
random variants of the same genetic event)
Ascus number
Spore number 1 2 3 4 5 6 7 8
1 ab ab+ ab ab+ a +b + a+b+ a+b a+b
2 ab ab+ ab ab+ a +b + a+b+ a+b a+b
+
3 ab ab ab+ ab a +b a +b a+b+ a+b+
4 ab+ ab ab+ ab a +b a +b a+b+ a+b+
5 a +b a +b a+b+ a+b+ ab+ ab ab+ ab
6 a +b a +b a+b+ a+b+ ab+ ab ab+ ab
+ +
7 a b a +b + a +b a+b ab ab+ ab ab+
8 a +b + a +b + a +b a+b ab ab+ ab ab+

Table 4.8 Meiosis in a dihybrid Neurospora, ab/a+b+, giving rise to seven unique classes of asci
Ascus number
Spore number 1 2 3 4 5 6 7
1 ab ab+ ab ab ab ab+ ab
2 ab ab+ ab ab ab ab+ ab
3 ab ab+ ab+ a +b a+b+ a +b a+b+
4 ab ab+ ab+ a +b a+b+ a +b a+b+
+ +
5 a b a +b a+b+ a+b+ a+b+ a +b a+b
+ +
6 a b a +b a+b+ a+b+ a+b+ a +b a+b
+ +
7 a b a +b a+b ab+ ab ab+ ab+
+ +
8 a b a +b a+b ab+ ab ab+ ab+
729 2 101 9 150 1 8
SDS for a locus 9 150 1 8
SDS for b locus 101 150 1 8
Unordered PD NPD TT TT PD NPD TT

For locus a: (1/2) 9þ150þ1þ8

1000
 100
¼ 8.4 centimorgans
For locus b: (1/2) 101þ150þ1þ8
1000
 100
¼ 13.0 centimorgans

The two distances obtained in the above calculation do not allow determination of
gene order. As it can be seen in Fig. 4.31, there are two possibilities, i.e., the two loci
can either be 21.4 or 4.6 map units apart. However, it can be determined as to which
one of the two is correct. The solution for this is calculating distance between a and
204 R. Keshava

b using information on unordered spores. Therefore, the calculation for map distance
is as follows:

map units ¼ ð1=2Þ the number of TT asciþthe number of NPD asci  100
Total number of asci

¼ ð1=2Þ118þ3  100 ¼ 6.2

1000

Since the above calculated map distance (6.2 map units) is closer to the distance
expected if both loci were located on one side of the centromere, the second
alternative depiction in Fig. 4.31 is accepted.
Another way to select between the alternatives shown in Fig. 4.31 is by finding
what will be the status of b locus if a crossover occurs between a and its centromere.
If arrangement 1 is correct, then crossover between a and its centromere should not
alter b, and if arrangement 2 is correct, then most crossovers that move a relative to
its centromere will also move b.
The asci classes 4, 5, 6, and 7 (Table 4.8) comprise all SDS patterns pertaining to
a locus. In class 5 asci, 150 out of 168 have similar SDS patterns for b locus. Hence,
89% of the times, crossover between a and its centromere, also results in a crossover
between b and its centromere, which provides clear evidence supporting arrange-
ment 2 in Fig. 4.31.
The mapping procedure by tetrad analysis can be summarized as follows: for both
unordered and ordered spores, indication of linkage comes from the occurrence of
large numbers of parental ditypes compared with nonparental ditypes. With respect
to unordered spores, like those observed in yeast, the distance between any two loci
is equal to the sum of half the number of tetratypes and the number of nonparental
ditypes, divided by the total number of asci, expressed as percentage. In case of

Fig. 4.31 Possible arrangements of a and b loci with respect to centromere. The distances are
given in map units
4 Chromosome Mapping in Eukaryotes 205

ordered spores, like those in Neurospora, distance between a locus and its centro-
mere is half the percentage of SDS. The method of mapping distance between two
loci in ordered spores is similar to that applied for unordered spores.

4.2.3.4 Mapping of Centromere Using Linear Tetrads

In eukaryotic chromosomes, unlike other parts of the chromosome, mapping of
special DNA sequences that form the centromeres cannot be done by utilizing
recombination analysis as a tool. This is because they do not exhibit heterozygosity
which can be used as markers. Nevertheless, centromeres can be mapped particu-
larly in fungi such as Neurospora that produce tetrads that are linear. Neurospora is a
haploid fungus, in which the meiotic divisions occur along the ascus’s long axis,
such that each product of meiosis produces eight linearly arranged ascospores,
referred to as an octad. This octad comprises the tetrad (four products of meiosis)
and a mitosis following the meiosis. Centromere mapping takes into consideration a
gene locus and calculates the distance between this locus and the centromere. The
method depends on the fact that meiotic crossover between a locus and a centromere
within a tetrad results in different patterns of alleles, for example, an individual with
a allele when crossed with another individual with allele A at the same locus (a  A).
According to the Mendelian law of segregation, the octad should comprise eight
ascospores, including four each of genotype a and A. Hence, if a crossover did not
occur between centromere and locus a/A, then the linear octad will consist of four
ascospores of each allele placed adjacently in sets (Fig. 4.32). In case a crossover
occurred between centromere and locus a/A, then the octad will consist one of four
patterns, in which each of the alleles is arranged in sets of twos. Data from a
particular cross of A  a have been given in Table 4.9.
In the given data in Table 4.9, the first two columns are products of meiosis
without crossover between locus A and the centromere and are the FDS patterns
(MI). The remaining four patterns are the SDS patterns (MII), which are all products
of meiotic crossover between A and centromere (Fig. 4.33).
Hence, in this case, there has been no FDS, but the A and a alleles are segregated
into separate nuclei during meiosis II. The formation of one of the MII patterns is
depicted in Fig. 4.33. The other patterns observed are also produced in a similar
fashion but vary in the chromatids that move to the different poles in the second
division of meiosis (Fig. 4.34).
The frequency of MII pattern octads will be proportional to the distance between
a/A region and centromere and hence can be used as a measure of distance between
these points on the chromosome. In the given example, MII frequency is equal to
14% (42/300). Using this frequency, the number of map units between the locus and
the centromere can be calculated. The SDS frequency of 14% denotes the percentage
of meiosis, whereas a map unit is the recombinant chromatids percentage produced
during meiosis. Since meiotic crossover yields only 50% recombinants (4 out of 8;
see Fig. 4.33), the MII frequency of 14% must be divided by 2 to convert to map units
(a frequency of recombinant chromatids). Therefore, in the given example, the
distance between locus A and its centromere is 7 map units. These measurements
can be further used to construct a chromosomal map.
206 R. Keshava

Fig. 4.32 Allelic segregation at meiosis in Neurospora. (a) Mitosis of the tetrad derived from
meiosis produces octads which are present within the ascus. (b) A meiocyte with genotype A/a
undergoes meiosis followed by mitosis, giving rise to equal number of A and a products, indicating
law of segregation
4 Chromosome Mapping in Eukaryotes 207

Table 4.9 Data from a Octads

particular cross of A  a
A a A a A a
A a A a A a
A a a A a A
A a a A a A
a A A a a A
a A A a a A
a A a A A a
a A a A A a
126 132 9 11 10 12
Total ¼ 300

4.3 Construction of Genetic Maps of Human Genome

As explained in Sect. 4.1.5.1, linkage analysis in humans cannot rely on planned

breeding experiments. In this case, family pedigrees will have to be made use of for
linkage analysis. Primarily, disease alleles have been analyzed with the help of
pedigrees.

4.3.1 Mapping by Pedigree Analysis: X-Linkage

The traits encoded by the X chromosome have unique inheritance patterns, and the
gene loci on the X chromosome can be easily identified. Over 400 loci have been
identified on the X chromosome. By using several different methods, it has been
estimated that human chromosomes consist of between 50 and 100,000 loci.
Initially, the X chromosome has been mapped using the pedigree analysis.
Once a gene is determined to be X-linked, it is necessary to determine the position
of that gene on the X chromosome. It is also required to determine the distance (map
units) of the gene and other loci on the X chromosome. Proper pedigrees can be used
to determine position and map distances, if occurrence of crossing over can be
ascertained. One such example is depicted in Fig. 4.35 and is called the “grandfather
method.” In the given example, the grandfather expresses one of the traits in
consideration, which is color-blindness in this case. It can be observed that his
grandson is glucose-6-phosphate dehydrogenase (G-6-PD) deficient, which
indicates that the grandson’s mother is a dihybrid for these two alleles. The alleles
in the grandson’s mother are in trans configuration, i.e., she has received the color-
blindness allele from her father on one of her X chromosomes and an allele for G-6-
PD-deficiency on her other X chromosome from her mother. Therefore, among the
grandsons (Fig. 4.35), two are nonrecombinant (left), and two are recombinant
(right). In theory, the map distance can be calculated by dividing the sum of the
recombinant grandsons by the total number of grandsons. The same method could be
208 R. Keshava

Fig. 4.33 Second-division segregation pattern in Neurospora. Segregation of A and a into separate
nuclei takes place during the second meiotic division as a result of a crossover between the A locus
and the centromere

applied in case the grandfather was both G-6-PD deficient and color-blind. In this
case, the mother would be a dihybrid with cis configuration, and the sons would have
a reverse arrangement. Here it is important to note that the grandfather’s phenotype
leads to the inference that the mother was a dihybrid, and reveals information on the
cis-trans configuration of the alleles. Further, this allows the scoring of her sons as
either nonrecombinant or recombinant.
4 Chromosome Mapping in Eukaryotes 209

Fig. 4.34 Four SDS patterns in linear asci. Centromeres randomly attach to the spindle during the
second meiotic division, resulting in the four arrangement patterns shown in the figure. The four
arrangements occur with equal frequency

Fig. 4.35 Pedigree analysis for X-linkage. “Grandfather method” of crossover determination
between genetic loci on human X chromosome. Color-blindness and G-6-PD alleles are considered
for analysis

4.3.2 Mapping by Pedigree Analysis: Autosomal Linkage

Mapping of sex chromosomes through pedigree analysis is a relatively easier task.

But when it comes to the autosomes, it is a much more complex process, because
there are 22 autosomal linkage groups and it is practically impossible to use simple
pedigrees to determine on which chromosome any two given loci are located
on. With the help of pedigrees, it is possible to deduce if there is linkage between
two gene loci, but the chromosomal location of the same cannot be deduced.
For example, nail-patella syndrome is a dominant trait whose symptoms include
abnormal nail growth along with underdevelopment or absence of kneecaps, among
other effects. In the given pedigree (Fig. 4.36) of this disorder, the male in generation
II is a dihybrid, with the nail-patella allele (NPS1) associated with allele A of ABO
blood group system and the normal nail-patella allele (nps1) associated with B allele
of the ABO blood group system. Consequently, only one out of eight children (III-5)
is a recombinant and the actual map distance is about 10%. Generally, because a
greater number of crossovers occur in females, the map distances appear to be
greater than in males.
210 R. Keshava

Fig. 4.36 Pedigree for autosomal linkage analysis. Analysis of the linkage between ABO blood
group loci and the nail-patella syndrome

4.3.3 Using Lod Score Method to Assess Linkage in Human

Pedigrees

In humans thousands of phenotypes are transmitted via the autosomes. Due to

various reasons, the progress in mapping of the gene loci on human chromosomes
was slow initially. The most important reason is that unlike in animals, performing
controlled crosses in humans is impossible. Therefore, calculation of recombinant
frequencies (RFs) had to be restricted to the occasional dihybrids that were produced
by chance mating. Test cross equivalent mating is extremely rare. Another reason is
that generally human matings produce smaller number of progeny. The smaller
number of progeny limits availability data and hence making it difficult to derive
statistically significant map distances. The third limitation is the vastness of the
human genome and hence the distances between known genes, on an average, are
very large.
Large sample sizes are essential in order to obtain reliable RF values. Neverthe-
less, even in cases of individual mating where the number of progeny is small,
combining results of several such identical matings are essential to obtain a more
reliable estimate. Calculation of Lod scores is a standard way of doing this. LOD is
the acronym for “log of odds.” To obtain a set of results in a given family, the LOD
method employs calculation of two different probabilities. The first probability is
calculated by assuming independent assortment, while the second is calculated by
assuming a specific degree of linkage. This is followed by the calculation of the ratio
(odds) between the two probabilities, and a logarithm of this ratio is called as the Lod
value. Lod scores derived from different mating, but involving the same genetic
markers, can be added as the logarithms are exponents. Therefore, a cumulative data
set indicating presence or absence of a particular linkage value is obtained.
4 Chromosome Mapping in Eukaryotes 211

Fig. 4.37 Pedigree equivalent to a dihybrid test cross. D/d are alleles of a disease gene; M1 and M2
are alleles of a molecular marker. P indicates parental (nonrecombinant); R indicates recombinant

An example of how this calculation works is as follows: assume a hypothetical

situation where a family is the product of a dihybrid test cross. Also, it needs to be
assumed that for a dihybrid individual, the input gametes can be deduced, and hence
the individual’s gametes can be assessed for recombination events. The dihybrid in
consideration is heterozygous for a dominant disease (Dd) allele and a molecular
marker (M1M2). It is assumed that the dihybrid individual is a male, and is formed
by union of gametes with genotypes (parental genotypes) D•M1 and d•M2. Consider
this individual is married to a woman with genotype d/d•M2/M2. The six children of
this marriage are shown in a pedigree depicted in Fig. 4.37. The pedigree also shows
the parental and recombinant categories with respect to gamete contributed by the
father.
Out of the six offspring, two are recombinants that would give a 33% RF. It can
be considered that the genes are undergoing independent assortment and the off-
spring form a nonrandom sample. The probability of this outcome can be calculated
under several hypotheses. The expected proportions of parental (P) and recombinant
(R) genotypes are shown in Table 4.10 under independent assortment and under
three RF values:
When assorted independently, in which case RF is equal to 50%, the probability
of obtaining results is computed as follows:

Table 4.10 Expected RF

proportions of parental
0.5 0.4 0.3 0.2
(P) and recombinant
(R) genotypes under three P 0.25 0.3 0.35 0.4
RF values and under inde- P 0.25 0.3 0.35 0.4
pendent assortment R 0.25 0.2 0.15 0.1
R 0.25 0.2 0.15 0.1
212 R. Keshava

0.25  0.25  0.25  0.25  0.25  0.25  B

¼ 0.00024  B

where B is the number of possible birth orders for the two recombinant and four
parental individuals.
When the RF is 0.2, the probability is as follows:

0.4  0.1  0.4  0.4  0.1  0.4  B

¼ 0.00026  B

The ratio of both the values is 0.00026/0.00024 ¼ 1.08 (it can be seen that B gets
cancelled out). On the basis of these data, it can be inferred that the hypothesis of an
RF equal to 0.2 is 1.08 times as possible as the hypothesis of independent assort-
ment. Lastly, in order to obtain the Lod value, the logarithm of the ratio needs to be
taken. Table 4.11 lists some ratios and their corresponding Lod scores:
These data indicate 30–40% RF because the largest Lod scores are generated by
these hypotheses. However, these data alone are insufficient to credibly support any
linkage model. In practice, for a specific RF value, a Lod score of at least 3 (obtained
by adding scores from several matings) is considered as a convincing support. It can
be noted that Lod score 3 represents RF that is 103 (1000) times as likely as the no
linkage (independent assortment) hypothesis.

4.3.4 Assigning Genetic Loci to Chromosomes

The gene locus for the Duffy blood group, located on chromosome 1, was the first
gene locus to be definitely assigned to a specific chromosome. Banding of
chromosomes and somatic cell hybridization are the two techniques that have been
very important for mapping of autosomes.

4.3.4.1 Chromosomal Banding

Around the 1970s, certain techniques to stain chromosomes were evolved. These
techniques used certain histochemical stains that could stain the chromosomes,
producing reproducible banding patterns on them. One such example was the
Giemsa staining technique and the bands that formed were called G-bands. Before
the advent of these staining techniques, mammalian chromosomes, including that of
humans, were grouped into categories based on their general size, because

Table 4.11 Ratios and RF

their corresponding Lod
0.5 0.4 0.3 0.2
scores
Probability 0.00024 0.00032 0.00034 0.00026
Ratio 1.0 1.33 1.41 1.08
Lod 0 0.12 0.15 0.03
4 Chromosome Mapping in Eukaryotes 213

differentiating them was a difficult task. The banding techniques enabled identifica-
tion of each human chromosome arranged in a karyotype.

4.3.4.1.1 ISCN Mapping System

A consistent numbering system is required for mapping genes on chromosomes and
constructing relevant idiograms. The International System for Cytogenetic Nomen-
clature (ISCN) is a mapping that is used for this purpose. The numbering for a
chromosome starts at the centromere according to the ISCN scheme. Based on
centromere position, chromosomal short (p) and long (q) arms are identified; like-
wise, regions on the short arm will be designated p, and those on long arm with q. By
convention, the p arm is always depicted on top in a karyotype.
Chromosomal arms are divided into regions, and starting from the centromere,
these regions are assigned numbers in an increasing order according to the distance
from the centromere. Specific morphological features such as a Giemsa band
characterize these regions, and these are consistent with the chromosomes. On the
short and long arm, the regions are labeled p1, p2, etc. and q1, q2, etc., respectively.
Depending on resolution of staining procedures, within each region, additional
bands may be detected, and these are further designated by adding another digit to
the existing number of that region (Fig. 4.38), again in the increasing order of the
distance from the centromere.

4.3.4.2 Somatic Cell Hybridization

Ability to distinguish the chromosomes allows application of somatic cell
hybridization. It is a technique in which hybrid cells are formed by in vitro fusion
of mouse/hamster cells with human cells.
Fusion of two cells initially results in a heterokaryon (a cell comprising nuclei
from different origins). The different nuclei eventually fuse and a hybrid cell which
tends to preferentially lose human chromosomes in the following generations is
formed. After stabilization, it results in a cell consisting of the hamster or mouse
chromosomes along with one or more human chromosomes. These chromosomes
can be identified by applying banding techniques. The hybrid cells can be screened
for human specific phenotypes, e.g., enzyme products, and these phenotypes can be
assigned to particular human chromosomes present in those cells.
To map human genes, two particular tests are employed. One of the tests is called
as the synteny test. Synteny means the same linkage group. The synteny test verifies
the belongingness of two gene loci to the same linkage group. Two genes are
considered syntenic if phenotypes encoded by them are always present or absent
together in several hybrid cell lines. The other test, called the assignment test,
determines chromosomal location of a particular gene locus. This is done by
observing concordance between presence of a phenotype and a particular chromo-
some, or absence of a phenotype and a particular chromosome is in a hybrid cell line.
In 1970, the first synteny test for autosomes was performed, and it was
demonstrated that the B locus of peptidase (PEPB) was linked to the B locus of
lactate dehydrogenase (LDHB). Both enzymes required subunits for their formation,
214 R. Keshava

Fig. 4.38 Idiogram for chromosome 12. The chromosome is mapped as per the ISCN mapping
system. (Left) A low-resolution map, obtained by staining of a metaphase chromosome with a stain
like Giemsa. (Right) A high-resolution map, obtained by staining of a prometaphase chromosome.
The number depicted on top of the ideogram represents the number of bands visualized

and the subunits were regulated by two loci, A and B, which were shown to be
located on chromosome 12.
Another example of assignment test includes localization of tissue factor III, a
blood-coagulating glycoprotein to chromosome 1. Results of the assignment test
performed for the localization of the coagulating factor are depicted in Table 4.12.
Twenty-nine human-mouse hybrid cell lines, or clones containing human
chromosomes, and their tissue factor score, have been shown. Using the table
location of the gene coding for tissue factor III can be clearly determined to be
human chromosome 1. The concordance between the presence of human
4 Chromosome Mapping in Eukaryotes 215

Table 4.12 Use of human-mouse hybrid cell lines for assigning the gene for blood-coagulating
factor III to chromosome 1 of humans

Source: Reprinted with permission from S.D. Carson, et al., “Tissue Factor Gene Localized to
Human Chromosome 1 (after 1p21),” Science, 229:229–291.
Copyright # 1985 American Association for the Advancement of Science
a
A translocation in which only part of the chromosome is present
b
Discord refers to cases in which the tissue factor score is plus, and the human chromosome is
absent, or in which chromosome is present but the score is minus

chromosome 1 and the tissue factor III in the cell line is clearly established.
Likewise, the absence of human chromosome 1 also corresponds to absence of
tissue factor. Therefore, it can be said that that there is 100% concordance or zero
discordance. None of the other chromosomes showed similar pattern with respect to
the tissue factor III. Hence, it was established that the tissue factor III is located on
chromosome I.
The task of determining the exact position of a particular locus on a chromosome
is facilitated by particular cell lines which can be developed to incorporate broken
chromosomes, which may lack certain parts or those parts may become incorporated
216 R. Keshava

in other chromosomes. These rearrangements reveal new linkage relationships that

allow determination of the chromosomal region in which a particular locus is
situated.

4.3.5 Chi-Square Test in Linkage Analysis

An objective statistical test called the chi-square (χ 2) test can be used to determine
the presence or absence of linkage between two genes. If the recombination fre-
quency (RF) is less than 50%, it can be inferred that the two genes are linked and are
positioned on the same chromosome. It cannot be ascertained how much less than
50% RF signifies linkage; hence, it is not possible to directly test for linkage, as there
is no precise linkage distance. Presence or absence of independent assortment is the
only genetic criterion that can be precisely used to predict the presence or absence of
linkage. Therefore, it becomes necessary to test the hypothesis for lack of linkage. If
the observed results reject no linkage hypothesis, then it can be inferred as presence
of linkage. Such a hypothesis is called null hypothesis. Since it allows precise
experimental prediction that can be verified, it is generally of use in χ 2 analysis.
For example, the following specific data set can be tested for linkage using
χ 2 analysis. Assume that a cross has been made between pure-breeding parents of
genotypes A/A ∙ B/B and a/a ∙ b/b. A dihybrid A/a ∙ B/b has been obtained, which is
test crossed to a/a ∙ b/b. A total of 500 progeny are obtained and classified as follows
(depicted as gametes obtained from the dihybrid):

142 A∙B Parental

133 a∙b Parental
113 A∙b Recombinant
112 a∙B Recombinant
500 Total

The recombinant frequency calculated from these data is 225/500 ¼ 45%. The RF
value 45% is less than 50% as expected from independent assortment, and hence
appears to be a case of linkage. However, the lower percentage of recombinant
classes could also be a chance occurrence. Therefore, it is required that a χ 2 test be
performed in order to calculate the likelihood that this result is based on chance.
Calculation of the expectations (E) for each class is the first step in a χ 2 test. The
hypothesis that needs to be tested here is that the two loci assort independently or in
other words there is no linkage. Gametic E values are calculated by making simple
predictions based on the first and second laws of Mendel as follows:
4 Chromosome Mapping in Eukaryotes 217

Hence, it might be asserted that if the dihybrid allele pairs are independently
assorting, the gametic types should be in the ratio 1:1:1:1. Therefore, considering 1/4
of 500, i.e., 125, as the expected proportion of each gametic class seems rational.
Nevertheless, it is important to note that 1:1:1:1 ratio can be expected only if all
genotypes are equally viable. Often it is observed that genotypes are not equally
viable due to the presence of certain alleles that affect the survival of individuals.
Hence, allele ratios such as 0.6 A:0.4 a or 0.45 B:0.55 b may be observed rather than
the 0.5:0.5 depicted above. These ratios can be used to predict independence. The
observed genotypic classes from which the allelic proportions can be clearly
observed are shown below:

Observed values Segregation of A and a

Segregation of B and b A a Total
B 142 112 254
b 113 133 246
Total 255 245 500

It can be seen that the allele proportions are 255/500 for A, 245/500 for a, 254/500
for B, and 246/500 for b. By multiplying allelic proportions, expected values under
independent assortment can be calculated. For example, expected number of A B
genotypes is obtained as follows:

Expected (E) value for A B

¼ (255/500)  (254/500)  500 ¼ 129.54

The entire grid of E values can be calculated using this approach, as follows:

Expected values Segregation of A and a

Segregation of B and b A a Total
B 129.54 124.46 254
b 125.46 120.56 246
Total 255 245 500
218 R. Keshava

The value of χ 2 is calculated as follows:

Genotype O E (O  E)2/E
AB 142 129.54 1.19
ab 133 120.56 1.29
Ab 113 125.46 1.24
aB 112 124.46 1.25
Total (equals the χ 2 value) ¼ 4.97

The χ 2 table is used further to find the corresponding probability ( p) for

the obtained χ 2 value (4.97). This requires calculation of the degrees of freedom.
The number of nondependent values, in a statistical test, is generally referred to as
the degrees of freedom. The product of number of classes denoted in rows minus one
and number of classes denoted in columns minus one is equal to the number of
degrees of freedom. In the given example, the degrees of freedom df ¼ (2–1)  (2–1)-
¼ 1. Hence, χ 2 value is located along the row corresponding to one degree of
freedom in a χ 2 table. The value 5.021 is the closest to the χ 2 value of 4.97 in the
χ 2 table and this corresponds to the probability 0.025, or 2.5%. Probability value
p indicates the deviation from expectations. In the given example, probability is less
than 5%, and hence the independent assortment, i.e., no linkage hypothesis, is
rejected. Since the no linkage hypothesis is rejected, a probable linkage between
the given loci is inferred.

4.4 Comparison of Genetic Map and Physical Map

Conventionally, genome mapping methods are grouped into two categories.

• Mapping that is based on genetic techniques is called genetic mapping. Maps are
constructed using genetic techniques such as cross-breeding experiments or
analysis of family histories, i.e., pedigrees in the case of humans. Position of
genes and other genome sequence features are shown on these maps.
• Mapping wherein molecular biology techniques are used is called physical
mapping. Using these techniques, DNA molecules are directly examined and
maps are constructed. These maps show positions of genome sequence features,
including genes.

4.4.1 Genetic Mapping

Genes were the first markers to be used in mapping of chromosomes. The first
genetic maps were constructed for organisms such as the fruit fly, during the early
twentieth century, and genes were used as the markers for this purpose (refer to Sect.
4.1.4). It is required that a heritable characteristic should exist in at least two
alternative phenotypes, such that it can be used in genetic analysis, for example,
4 Chromosome Mapping in Eukaryotes 219

Table 4.13 Biochemical Marker Phenotype

phenotypes used in
ADE2 Requirement of adenine
mapping yeast
chromosomes CAN1 Canavanine resistance
CUP1 Resistant to copper
CYH1 Cycloheximide resistance
LEU2 Requirement of leucine
SUC2 Ability for sucrose fermentation
URA3 Requirement of uracil

the tall and dwarf pea plant phenotypes. Each variant phenotype is encoded by
different alleles of a gene.
At the beginning, only genes that specified visually distinguishable phenotypes
could be studied. For example, fruit fly chromosome maps that were initially
constructed showed positions of genes that coded for phenotypes such as the body
color, eye color, wing shape, etc. All of these phenotypes could be easily observed
by visual examination of the flies either by the naked eye or by using a low-power
microscope. Although this approach was useful in the early days, the number of
visual phenotypes was the limitation, and also the analysis was complex in several
cases as more than one gene can affect a single phenotype. In order to make gene
maps more comprehensive, the biochemical characteristics of the phenotypes were
used to distinguish the phenotypes. Biochemical phenotypes have been particularly
significant in gene mapping of microbes and humans. The biochemical phenotypes
used in mapping yeast chromosomes have been listed in Table 4.13.
Although visual characteristics have been used in humans, genetic variation
studies have largely relied on biochemical phenotypes, identifiable via blood typing.
In addition to the standard blood groups (such as ABO), serum and immunological
protein variants, for example, the HLA (human leukocyte antigen) system, were also
used for this purpose. These markers have an added advantage, because several of
them have multiple alleles. Since gene mapping in humans is based on pedigree
analysis, this becomes very relevant. Presence of multiple alleles increases the
chance of marriage between individuals possessing allelic variants, and their inheri-
tance patterns with the family lineages can be studied to derive useful information
for the mapping purpose.

4.4.2 DNA Markers for Genetic Mapping

Even though the genes are very useful as markers, they are certainly not adequate.
Particularly, in organisms such as the vertebrates and flowering plants which possess
larger genomes, the maps cannot be based entirely on genes. This is because genes
are widely spaced with large intergenic regions in most eukaryotic genomes. There-
fore, using only genes for mapping will result in a map that is not very detailed. In
addition, the fact that only a fraction of the total genes possess easily distinguishable
220 R. Keshava

allelic forms limits its use as a sole marker for mapping. Hence, very comprehensive
gene maps cannot be constructed.
This clearly suggests that other types of markers are required. Markers other than
genes that are utilized for mapping purposes are called DNA markers. Similar to
gene markers, DNA markers should also comprise a minimum of two alleles to
qualify for mapping purposes.
Three types of DNA sequence features, such as RFLPs (restriction fragment
length polymorphisms), SSLPs (simple sequence length polymorphisms), and
SNPs (single nucleotide polymorphisms), satisfy these requirements and hence are
suitable for mapping purposes.

4.4.2.1 RFLPs (Restriction Fragment Length Polymorphisms)

The first DNA markers to be studied were the RFLPs. The digestion of a specific
DNA molecule with a particular restriction enzyme will result in cleavage at specific
sequence sites and produce same pattern of fragments reproducibly. However, this
may not be the case with respect to genomic DNA, due to the polymorphic nature of
the restriction sites. Some restriction sites will exist in two allelic forms, one
consisting of the correct recognition sequence, whereas the other consisting of an
altered restriction sequence, such that the enzyme can no longer recognize the
restriction site. As a result, on digestion with that particular restriction enzyme,
variable lengths of the restriction fragments will be generated, thus seen as a length
polymorphism (Fig. 4.39).
It is estimated that the human genome consists of about 105 RFLPs. Each RFLP
consists of only two alleles, i.e., either with presence or absence of the restriction
site. Similar to mapping gene markers, inheritance pattern of RFLP alleles is
analyzed and their position on the genome map is determined. Despite the number
of RFLPs that can be available, their utility for gene mapping is limited due to

Fig. 4.39 RFLP marker. Allele 1 (left) consists of a polymorphic restriction site (indicated with an
asterisk), while it is absent in Allele 2 (right). Restriction digestion with a specific restriction
enzyme reveals this restriction fragment length polymorphism (RFLP) and is observed as variation
in the pattern of fragments produced. In this example, Allele 1 (left) generates four fragments,
whereas Allele 2 (right) produces three fragments
4 Chromosome Mapping in Eukaryotes 221

Fig. 4.40 RFLP scoring methods. (a) Scoring of RFLP by Southern hybridization. Involves
restriction digestion of DNA with an appropriate restriction enzyme, separation of fragments on
an agarose gel, transfer onto a nylon membrane, and analysis using a probe spanning the polymor-
phic restriction site. If the restriction site is absent, a single restriction fragment is detected (lane 2);
presence of the restriction site yields two restriction fragments (lane 3). (b) Alternatively, PCR can
also be used for RFLP analysis. Amplification is carried out with primers annealing on either side of
the polymorphic restriction site, followed by restriction digestion of the PCR products, and agarose
gel electrophoresis. Presence of the restriction site yields two bands, while its absence yields a
single band

frequent absence of variation in a RFLP targeted for analysis among members of a

family. Also, adding to the limitation is the tedious task of identifying one or two
individual restriction fragments among several irrelevant fragments for a given
restriction enzyme. For example, EcoRI, which recognizes 6-bp recognition
sequence, is estimated to cut at about once every 46 ¼ 4096 bp and hence should
produce nearly 800,000 fragments when human DNA is digested with it. Use of a
probe that spans the polymorphic restriction site and analysis using Southern
hybridization is one of the methods for scoring an RFLP (Fig. 4.40a). However,
more recently, PCR-based methods are being used for scoring RFLPs. For this
purpose, primers complementary to sequences on both ends of the polymorphic
site are designed. RFLP analysis involves restriction digestion of amplified
fragments followed by agarose gel electrophoresis (Fig. 4.40b).

4.4.2.2 SSLPs (Simple Sequence Length Polymorphisms)

SSLPs consist of an array of repeat sequences, with variable length. Alleles vary
with respect to the number of repeat units (Fig. 4.41). In contrast to the RFLPs which
consist of only two alleles, SSLPs can consist of multiple alleles wherein each allele
222 R. Keshava

Fig. 4.41 Typing of SSLPs. (a) The figure represents two alleles of a SSLP. The motif “GA” is
repeated three times in allele 1, whereas it is repeated five times in allele 2. (b) SSLP typing by PCR.
Lane A consists of amplified products of the region surrounding SSLP. Lane B consists of DNA
markers, representing size of the PCR amplified bands of the two alleles. It can be observed that the
band in lane A is the same size as that of the larger bands in the marker lane, thus indicating that the
tested DNA contained allele 2

can possess a different number of repeat units, thus can be of varying lengths. These
kinds of repeat sequences are called microsatellites. It is estimated that the human
genome consists of about 6.5  105 microsatellites.

4.4.2.3 SNP (Single Nucleotide Polymorphism)

SNPs are genomic positions which vary in a single nucleotide, for example, a G may
be present in some individuals, while in others a C may be present (Fig. 4.42). Every
genome consists of vast numbers of SNPs. Some of these SNPs also give rise to the
RFLPs. About 1.42 million SNPs are estimated to be present in the human genome,
and of these only 100,000 results in an RFLP.
Potentially, each SNP can exist as four alleles, due to the four possible
nucleotides. But most of them exist in two forms, and hence pose the same limitation
as the RFLPs with regard to their utility in human genetic mapping. Further, it is also
4 Chromosome Mapping in Eukaryotes 223

Fig. 4.42 SNP (single

nucleotide polymorphism).
The figure depicts a SNP,
where a position in the
sequence of Allele 1 is
occupied by nucleotide G and
Allele 2 consists of nucleotide
C in the corresponding
position

Fig. 4.43 Oligonucleotide hybridization for SNP typing. Highly stringent conditions are
maintained during the assay. Due to the stringent condition, stable hybrids are formed only if the
oligonucleotide completely base-pairs with the target site. Hybrids do not form even if there is a
single mismatch. Stringent hybridization conditions are maintained by regulating incubation tem-
perature and maintaining it a little below Tm (melting temperature) of the oligonucleotide;
temperatures above Tm make even completely base-paired hybrids unstable, whereas temperatures
more than 5  C below the Tm may make even mismatched hybrids stable

possible that a particular SNP does not show any variability in a particular family
chosen for the study. Due to their abundant numbers and availability of typing
methods not requiring slow and labor-intensive gel electrophoresis make SNPs
advantageous. These are rapidly detected by oligonucleotide hybridization analysis
(Fig. 4.43), which is a very specific method enabling discrimination between two
SNP alleles. DNA chip technology and solution hybridization (Fig. 4.44) are some
of the screening strategies developed.
224 R. Keshava

Fig. 4.44 Solution

hybridization method of SNP
detection. The oligonucleotide
probe consists of two
end-labels, one is a fluorescent
dye, and the other is a
quenching compound. Base
pairing of the two ends of the
oligonucleotide result in
quenching of the fluorescent
signal by the quencher. But
when the oligonucleotide
hybridizes with the target
DNA, the fluorescence is
emitted since the quencher is
separated from the
fluorescent dye

4.4.3 Physical Mapping

Genetic maps were insufficient for directing the genome sequencing projects, due to
two reasons. The number of crossovers scored determines the resolution of the
genetic map. With respect to microorganisms, this does not pose a problem because
microorganisms can be cultured to obtain large numbers and thus several crossovers
can be studied. This enables construction of a highly detailed genetic map, in which
markers are just a few kb apart. For example, 1400 markers comprise the
Escherichia coli genetic map at the rate of one per 3.3 kb on an average, and were
adequate for directing the sequencing program, and an extensive physical mapping
was not required. Similarly, for the Saccharomyces cerevisiae genome sequencing
project, a fine-scale genetic map became available. It approximately comprised 1150
genetic markers, i.e., one per 10 kb on an average.
But with most other eukaryotes and humans, only few meioses can be studied
because it is not possible to obtain large numbers of progeny. Hence, in these cases,
linkage analysis will have limited resolving power. This implied that for such cases
alternative mapping procedures will be required to supplement the genetic maps
before large-scale DNA sequencing. To address this problem, several physical
mapping techniques have been developed. The most important ones are as follows:

• Restriction mapping, which allows location of relative positions of restriction

sites on a DNA molecule
• Fluorescence in situ hybridization (FISH), which allows marker localization to
specific chromosomes using probes that hybridize to intact chromosomes
• Sequence-tagged site (STS) mapping, which involves mapping positions of short
sequences with the help of PCR and/or hybridization analysis
4 Chromosome Mapping in Eukaryotes 225

4.4.3.1 Restriction Mapping

Using RFLPs as DNA markers for genetic mapping, the polymorphic restriction site
positions can be located within a genome. Since very few restriction sites are
polymorphic, several of them cannot be mapped by this technique (Fig. 4.45).
Restriction mapping aims to increase the marker density on a genome map by
mapping even non-polymorphic restriction sites using alternative methods.

4.4.3.1.1 Basic Methodology Involved in Restriction Mapping

In its simplest way, restriction maps are constructed by digesting DNA with two
different restriction enzymes (each recognizes a different target sequence), and then
comparing the sizes of the fragments obtained after digestion. For example, using
restriction enzymes EcoRI and BamHI (Fig. 4.46), DNA is first digested with each of
the enzymes individually, and the size of the fragments obtained is characterized by
agarose gel electrophoresis. Hence, the number of restriction sites pertaining to each
of these enzymes present in the given DNA molecule can be worked out by these
single digests. Further, the DNA can be digested by both these restriction enzymes
together. This double restriction enzyme digestion enables determination of their
relative positions in the given DNA molecule (Fig. 4.46).

4.4.3.1.2 Examining DNA Molecules Directly for Restriction Sites

Methods other than electrophoresis, such as optical mapping, can also be used for
mapping restriction sites in DNA. In this the location of the restriction site on the
given DNA, fragment is directly visualized with the help of a microscope. For this
purpose, by using certain methods, such as gel stretching (Fig. 4.47) and molecular
combing (Fig. 4.49), DNA is first attached to a glass slide in a stretched manner.

Fig. 4.45 Polymorphic

restriction sites are important
DNA markers used for
mapping of genomes. All
restriction sites are not
polymorphic and techniques
for mapping non-polymorphic
sites have been developed.
The above figure depicts the
distribution of polymorphic
and non-polymorphic
restriction sites in a molecule
of DNA
226 R. Keshava

Fig. 4.46 Restriction mapping. The figure represents the basic restriction mapping procedure. In
this example, the DNA molecule of size 4.9 has been digested using EcoRI and BamHI represented
as E and B, respectively. The fragments obtained by single and double restriction digestion are
shown at the top. By comparing with single digests, the results of double digests can be interpreted
to develop two alternative maps as described in the middle panel. Three restriction sites can be
mapped using double restriction data. There are two alternative possibilities for the larger EcoRI
fragment, as it consists of two BamHI sites. This can be solved by a partial restriction of the original
DNA molecule with only BamHI by either using a suboptimal incubation temperature or incubating
the reaction for a short duration of time. As shown in the bottom panel, the inclusion of 2.7 kb
fragment in the products of the partial restriction shows MapII to be the correct one
4 Chromosome Mapping in Eukaryotes 227

Fig. 4.47 Gel stretching and

direct visualization of a
restriction site. DNA
molecules targeted for
analysis are suspended in
molten agarose and pipetted
onto a slide coated with
restriction enzyme.
Solidification of the gel results
in stretching of DNA
molecules. At this stage, the
enzyme is inactive due to the
absence of magnesium ions,
which is required for enzyme
activity. The enzyme is
activated by washing with a
solution of magnesium
chloride. The DNA molecules
are cut by the enzyme, and the
cut locations become visible
as gaps as the DNA molecules
gradually coil up. The
visualization of DNA and
hence the gaps is made
possible by the addition of
fluorescent dyes such as
4,6-diamino-2-phenylindole
dihydrochloride (DAPI). The
restriction site locations can
be examined with a high-
power fluorescence
microscope

4.4.3.2 FISH (Fluorescence In Situ Hybridization)

FISH is the second type of physical mapping procedure. Similar to the optical
mapping technique, FISH allows direct visualization of the position of a marker
on an extended DNA molecule or a chromosome. In FISH, a DNA sequence marker
is visualized after hybridization with a fluorescent probe (Fig. 4.48).
A fluorescent signal within a metaphase chromosome obtained by FISH is
mapped by measuring its position relative to the end of chromosomal short arm
(the FLpter value). The highly condensed nature of the metaphasic chromosome is a
disadvantage and allows only a low-resolution (markers have to be at least 1 Mb
apart) mapping. However, determination of the chromosome on which a new marker
228 R. Keshava

Fig. 4.48 FISH

(fluorescence in situ
hybridization). A sample of
dividing cells is dried onto a
slide, and treated with
formamide to denature the
chromosomes. The
characteristic morphologies of
the metaphase chromosomes
are retained. The required
fluorescently labeled probe is
added to the chromosomes on
the slide. The position on the
chromosomal DNA to which
the probe hybridizes is
visualized by detection of the
fluorescent signal that is
emitted

is present has been one of the main applications of metaphase FISH. This provides a
preliminary indication of its map position and can precede a fine scale mapping
using various high-resolution FISH techniques. These techniques involve
improvements in the chromosome preparation methods, leading to use of more
extended chromosomes. There are two such methods:

• Mechanically stretched chromosomes—this involves inclusion of a centrifuga-

tion step in isolation of chromosomes, thus generating a shear force which causes
chromosomes to stretch up to 20 times their normal length. The resolution is
significantly improved (markers that are 200–300 kb apart can be distinguished).
• Non-metaphase chromosomes—chromosomes are highly condensed only dur-
ing metaphase. Chromosomes are in an extended form at other stages of the cell
cycle. Nuclei at prophase stage, consisting of adequately condensed and distin-
guishable chromosomes, have been used and have been found to be equivalent to
mechanically stretched chromosomes without further advantage. Chromosomes
at the interphase have been found to be more useful because chromosomes are
most unpacked at this stage and a resolution down to 25 kb is possible. However,
loss of chromosome morphology leads to lack of external reference points for
mapping probe positions. Therefore, preliminary map information is obtained by
4 Chromosome Mapping in Eukaryotes 229

Fig. 4.49 Molecular combing. A solution containing the DNA molecules of interest is taken and a
cover slip is dipped into it. The DNA molecules become attached by their ends to the cover slip. The
cover slip is removed from the solution at a rate of 0.3 mm s1 resulting in a “comb” consisting of
parallel DNA molecules

using this technique, which is usually used to determine order of a series of

markers in small chromosomal regions.

To increase FISH resolution to more than 25 kb, an approach called fiber-FISH,

which utilizes purified DNA, is employed. For this purpose, DNA is prepared by gel
stretching (Fig. 4.47) or molecular combing (Fig. 4.49) and enables a higher resolu-
tion, such that markers less than 10 kb apart can be distinguished.

4.4.3.3 STS (Sequence-Tagged Site) Mapping

Ideally, a rapid and technically undemanding high-resolution mapping procedure is
required to generate a detailed physical map of a large genome. Neither restriction
mapping nor FISH meet these requirements. In spite of the rapidness, ease, and
230 R. Keshava

Fig. 4.50 Collection of fragments for STS mapping. The figure shows a set of DNA fragments that
are appropriate for STS mapping. These are fragments spanning an entire chromosome, wherein
each point on the chromosome is represented in about five fragments on an average. Markers shown
in blue are closely located, and hence there is a high probability of finding them together on the
same fragment. The two markers shown in green are further apart and hence there is less probability
of finding them on the same fragment

usefulness of restriction mapping in obtaining detailed information, it is not suitable

for large genomes. Although large genomes can be mapped using FISH and fiber-
FISH, these are procedurally complex with slow rate of data accumulation because
per experiment only three or four markers can be analyzed. Therefore, for
constructing detailed physical maps, more powerful techniques are required. In
this regard, the most powerful physical mapping technique is STS mapping. This
has been used to generate high-resolution maps of large genomes.
An easily recognizable short (100–500 bp) sequence of DNA, occurring only once
in a chromosome or genome, is called as a sequence-tagged site (STS). A set of STSs
are mapped using a collection of overlapping DNA fragments (Fig. 4.50) derived from
a single chromosome or whole genome. Data for construction of the map is obtained
by determining fragments containing the STS, by hybridization analysis or PCR. PCR,
however, is the preferred method as it is faster and amenable to automation.
How closely located two STSs are in a given genome determines the probability
of these two STSs being located on the same fragment. If they are located close to
one another, there is a higher probability that they will always be present on the same
fragment. On the contrary, if they are located further apart, they may or may not be
present on the same fragment with varying frequencies (Fig. 4.50). This data can be
applied further for calculation of distance between markers, similar to mapping via
linkage analysis (Sect. 4.1.5.1). As map distance is calculated using crossover
frequency between two markers in linkage analysis, in STS mapping, map distance
relies on break frequency between markers.
An STS is any unique DNA sequence. Two criteria must be satisfied by a DNA
sequence to qualify as an STS. Firstly, it must be a known sequence, such that its
4 Chromosome Mapping in Eukaryotes 231

Fig. 4.51 Map of the polytene X chromosome. The figure represents the map of the left end of the
Drosophila polytene X chromosome. It shows a comparison between the genetic map (above) and a
physical map (below). The genes represented are as follows: yellow body ( y), white eyes (w),
echinus eyes (ec), cut wings (ct), and singed bristles (sn). It can be seen that w and ec are closer on
the physical map than on the genetic map of the chromosome, whereas y and w are far apart on the
physical map, but closer on the genetic map

presence or absence on various DNA fragments can be assayed by PCR. Secondly,

an STS is required to occupy a unique chromosomal or genome location. If an STS
sequence is present at more than one position, an ambiguity will be created in the
mapping data. It has to be ascertained that the STSs should not be located within the
repetitive DNA of a chromosome/genome.
The most common sources of STSs are the ESTs (expressed sequence tags),
SSLPs, and random genomic sequences.

• ESTs (expressed sequence tags): ESTs are short sequences representing genes.
cDNA clones of protein-coding gene mRNAs are analyzed to obtain ESTs.
• SSLPs: In physical mapping, SSLPs can also be used as STSs. Polymorphic
SSLPs, previously mapped using linkage analysis, are particularly valuable, as
these establish a direct correlation between genetic and physical maps.
• Random genomic sequences: Sequences derived by sequencing random cloned
genomic DNA fragments are called random genome sequences. Alternatively,
sequences already deposited in the databases can be downloaded.

4.4.3.4 Genetic Distance vs Physical Distance

The occurrence of crossing over between homologous pairs of chromosomes forms
the basis of all procedures involved in genetic distance measurement and construc-
tion of recombination maps. It can be expected that more frequent crossovers occur
in long chromosomes than short ones. Hence, it can also be expected that this
relationship will be shown in their genetic map lengths. Although this assumption
is true for a large part, it has been observed that relatively more crossing over can
occur in some regions of the chromosome than others. Therefore, distances
232 R. Keshava

represented on the genetic map do not show exact correspondence to the physical
distance along the physical map of a chromosome (Fig. 4.51). Moreover, regions
around the centromere and the ends of a chromosome are less likely to undergo
crossing over; as a result, these regions appear condensed on a genetic map.
Likewise, regions which undergo frequent crossovers are expanded in the genetic
map. In spite of the lack of a uniform relationship between genetic and physical
distances, there is a collinearity between genetic and physical maps of a chromo-
some, i.e., the loci on the chromosomes are present in the same order. Hence,
mapping using the recombination frequency shows the exact order of the genes on
a given chromosome. However, by using such a map, the actual physical distances
between genes cannot be estimated.

Box 4.1 Scientific Concept: Estimation of Distances and Map

Construction Using Radiation Hybrids: William Newell
Radiation hybrid (RH) mapping is a powerful technique applied for the
purpose of mapping unique DNA sequences in the genomes. It is one of the
preferred techniques for localization of new markers to specific regions of
genomes. Markers such as the ESTs and STSs are used for analysis. The
technique uses sampling of irradiation-induced chromosome fragments. This
technique is originally developed by Goss and Harris (1975), and was further
improvised for practical large-scale application by Cox et al. (1990) and
Walter et al. (1994).
Goss and Harris (1975) irradiated human fibroblasts and the fragments were
fused with recipient rodent cells. Scoring for the coretention of markers in a set
of hybrid cells allowed the ordering of the markers on chromosomes 1 and
X. This was based on the assumption that markers located far apart are more
probable to become separated by irradiation than markers that are close, and
hence are independently segregated in the component hybrid cells of the panel.
Figure 4.52 depicts the marker distances for a given chromosome obtained by
analysis of a panel of 100 hybrids.
Cox et al. (1990) extended this technique further by irradiating donor
somatic cell hybrids, which consisted of a single copy of one human chromo-
some. The fragments obtained were fused with rodent cells. This particular
technique was applied to map markers located on proximal and distal regions
of chromosome 21. Subsequently, markers around disease loci, for example,
BRCA1, neurofibromatosis type 2, Huntington’s disease, and incontinentia
pigmenti 1, in localized areas of the genome have been mapped using this
technique.
A summary of chromosome-specific RH panels has been compiled.
Recently, whole genome RH panels have been constructed and genome
maps consisting of 100 s or 1000 s of markers have been developed. RH
panels represent a sample of the donor genome. It specifically signifies a

(continued)
4 Chromosome Mapping in Eukaryotes 233

Box 4.1 (continued)

sample of radiation events that occurred in several donor chromosomes to give
rise to the observed marker retention patterns in the RH panel.
Several statistical methods have been utilized for RH data analysis. Fig-
ure 4.53 shows the raw data showing the retention and breakage frequencies of
the markers analyzed in a panel of hybrids. Presently, four main software
packages, RHMAP, RHMAPPER, SAMAPPER, and MultiMap, have been
used for the construction of complete maps from RH data. Retention of
different chromosomal fragments in the different clones comprising an RH
panel is modeled by each of these methods. The parameters in these models
include breakage frequencies between all marker pairs, and retention
frequencies of different fragments, which may depend on chromosomal loca-
tion (Fig. 4.53). A chromosome map built on the distance data depicted in
Fig. 4.52 is shown in Fig. 4.54. The raw data which forms the basis of the map
is shown in Fig. 4.53.
This technique has proved to be powerful and has led to the construction of
several good maps. However, the methods of analysis are being improved
consistently. Particularly, none of the methods of analysis considered the
probable irradiation induced physical rearrangements of chromosomes. It is
suggested that several of the errors that have been encountered could be a
result of such events. Studies in Tradescantia by Catcheside et al. (1946) have
reported several different rearrangements in the chromosomes in addition to
simple breaks. Similar studies involving human cells might improvise the
models used for estimation of relative marker locations. For example, several
conditions such as the temperature, stage in cell cycle, etc. are shown to
influence the chromosome breakage efficiency and the types of alterations
that occur (such as inversions, chromatid exchanges, isochromatid exchanges,
etc.). It is also possible that some changes induced by the X-irradiation may
prevent incorporation of human DNA into the rodent genome. It is required
that these changes are quantified in various conditions and then incorporated
into more accurate stochastic models.
234 R. Keshava

Fig. 4.52 Estimated vs. known distances for a panel of 100 hybrids, 100 markers, chromosome
length 3 R. The distance between markers is measured in centirays (cR), where for each unit, there is
a 1% probability of X-ray-induced breakage for a specific dosage in rads

4.5 Summary

• Construction of a series of descriptions depicting position and spacing of charac-

teristic, identifiable biochemical markers, such as genes and other DNA sequence
features comprising a chromosome, is called as chromosome mapping.
• Linkage or genetic linkage can be defined as the tendency of genes closely located
on a chromosome to be frequently inherited.
• Test cross is an extremely important tool used to reveal the gametic composition
of a heterozygous offspring of the F1 generation.
• The crossing over among homologous chromosomes results in production of
recombinant gametes.
• It is during the late prophase of meiosis I, chiasmata, the cytological evidence for
crossing over can be clearly visualized.
• Distances on genetic maps are measured in map units, and are abbreviated as m.
u.; one map unit ¼ 1% recombination.
• Map units are denoted by centimorgans (cM); one Morgan ¼ 100 m.u.
• When a test cross is performed involving two genes, it is named as a two-point
test cross or in short called as two-point cross.
• Distance between gene pairs can be obtained by identifying recombinant classes
resulting from crossover between concerned pair of genes.
4 Chromosome Mapping in Eukaryotes 235

Fig. 4.53 Raw data used for the map (black) present and (white) absent. The retention frequencies
are plotted to the left. (Bottom) Relative numbers of markers and breaks observed in each hybrid
cell of the panel
236 R. Keshava

Fig. 4.54 Map constructed from the distances in Fig. 4.52. The optimal order is to the left of the
names, the two-dimensional configuration at the extreme left, and the original (known) positions at
the right
4 Chromosome Mapping in Eukaryotes 237

• Linkage analysis in humans cannot rely on planned breeding experiments. In this

case, family pedigrees will have to be made use of for linkage analysis. Primarily,
disease alleles have been analyzed with the help of pedigrees.
• Mapping that is based on genetic techniques is called genetic mapping. Maps are
constructed using genetic techniques such as cross-breeding experiments or
analysis of family histories, i.e., pedigrees in the case of humans. Position of
genes and other genome sequence features are shown on these maps
• Mapping wherein molecular biology techniques are used is called physical
mapping. Using these techniques, DNA molecules are directly examined and
maps are constructed. These maps show positions of genome sequence features,
including genes.
• Genes were the first markers to be used in mapping of chromosomes. The first
genetic maps were constructed for organisms such as the fruit fly, during the early
twentieth century, and genes were used as the markers for this purpose.
• Even though the genes are very useful as markers, they are certainly not adequate.
Particularly, in organisms such as the vertebrates and flowering plants which
possess larger genomes, the maps cannot be based entirely on genes. This is
because genes are widely spaced with large intergenic regions in most eukaryotic
genomes. Therefore, using only genes for mapping will result in a map that is not
very detailed. In addition, the fact that only a fraction of the total genes possess
easily distinguishable allelic forms makes it even more difficult to be used as a
sole marker for mapping. Hence, gene maps cannot be made very comprehensive.
• RFLPs (restriction fragment length polymorphisms), SSLPs (simple sequence
length polymorphisms), and SNPs (single nucleotide polymorphisms) are the
DNA sequence features used for mapping.
• Genetic maps were insufficient for directing the genome sequencing projects as
they have a low resolution. Several physical mapping techniques such as restric-
tion mapping, fluorescence in situ hybridization (FISH), and sequence-tagged site
(STS) mapping have been developed to obtain maps of higher resolution.

References
Catcheside DG, Lea DE, Thoday JM (1946) Types of chromosome structural change induced by the
irradiation of Tradescantia microspores. J Genet 47:113–136
Cox DR, Burmeister M, Price ER, Kim S, Myers RM (1990) Radiation hybrid mapping: a somatic
cell genetic method for constructing high-resolution maps of mammalian chromosomes. Sci-
ence 250:245–250
Goss SJ, Harris H (1975) New method for mapping genes in human chromosomes. Nature 255:
680–684
Walter M, Spillett D, Thomas P, Weissenbach J, Goodfellow P (1994) A method for constructing
radiation hybrid maps of whole genomes. Nat Genet 7:22–28
Study of Chromosome
5
Dhruti Patwardhan, S. A. Varshini, and Latha Galoth

5.1 Overview of Chromosome

All living things on earth have evolved from a single primordial ancestor which
arose about 3.5–4 billion years ago. A consequence of this is that all organisms share
a common system for storing and retrieving biological information. A genetic code
consisting of deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) is utilised.
The same four nucleotides of either RNA or DNA are seen and the genetic code also
remains constant in almost all organisms. Chromosomes are the way in which DNA
is packaged and stored inside the cell. The method of packaging is different in
prokaryotes and eukaryotes.
In eukaryotes, DNA is coiled around a set of proteins known as histones. This
complex of DNA and histones is referred to as chromatin. The chromatin also coils
around itself and creates a compact mass of DNA that can fit into the nucleus of a
cell. This packaging does limit the accessibility of genes for expression and genes
that need to be expressed are often unwound from this organisation. DNA in archaea
is also organised around histones but they are different from the histones found in
eukaryotes. Bacteria lack histones and their DNA is therefore not as compactly
organised as those of eukaryotes. Gene expression, therefore, also becomes simpler
in bacteria.
Chromosomes are very complex structures and have multiple levels
of organisation. As stated above, the first level of organisation is the winding of
DNA double helix around an octamer of histone proteins. This octamer consists of
two copies each of H2A, H2B, H3, and H4 histones, also called the core protein. The

D. Patwardhan
Indian Institute of Science, Bangalore, India
S. A. Varshini (*) · L. Galoth
Ramaiah University of Applied Sciences, Bangalore, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 239
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_5
240 D. Patwardhan et al.

DNA wraps twice around this core protein which consists of 145–147 bp of DNA.
This arrangement of DNA coiled around an octamer of histone proteins is called a
nucleosome. This is the fundamental unit of the chromosome and occurs repeatedly.
Another histone known as the H1, present outside the nucleosome, clamps around
the 20–22 bp of entry and exit points of the DNA and stabilises this structure. The
nucleosome together with its associated H1 protein is called a chromatosome. Two
chromatosomes are separated by 30–40 bp of linker DNA. The length of linker DNA
may vary between different cell types. Together the nucleosomes and linker DNA
appear as beads on string. The chromatosomes are then packed tightly together to
form a fibre of 30 nm diameter. This fibre then forms loops of varying lengths
averaging about 300 nm in length and anchored at its base to nuclear scaffold
proteins. This 300 nm fibre is further compressed and folded to form 250 nm wide
fibre. This is then supercoiled to form the chromatid which is 700 nm in width.
During DNA replication, another copy of the chromatid is produced which is
anchored together at the centromere. This structure is called the chromosome
(Fig. 5.1).
In essence, each chromosome consists of a single strand of DNA which is tightly
coiled and compactly packed in the cell with the help of histone proteins. Under
normal circumstances, chromosomes are difficult to observe. However, during cell
divisions, chromosomes condense further and become thick structures which can be
observed. Chromosomes consist of three elements: centromere, telomeres and
origins of replication.
Telomeres are the tips of the chromosomes. The ends of the chromosomes are
vulnerable and may get degraded over time. Telomeres provide stability to the
chromosomes and prevent useful information situated at its ends from getting
degraded. Telomeres shorten in length with each cell division. They therefore play
important roles in determining cell senescence, cancer and aging. Origins of replica-
tion are the points at which DNA replication is initiated. These cannot be easily seen
under the microscope. Before mitosis can begin, each chromosome replicates itself
to create an identical copy. This copy is called the sister chromatid and they are held
together at the centromere (Fig. 5.2).
Centromere appears as a constricted structure which does not stain very well. This
is the point at which protein assemblies called kinetochore form during cell division.
Spindle microtubules attach to the kinetochore during cell division. These spindles
pull the sister chromatids towards opposite ends of the cell. A single cell separates
into two daughter cells, each of which inherits one of the sister chromatids. Thus,
centromeres are very important to ensure correct separation of chromosomes into
each cell during cell division. Chromosomes lacking centromeres are not attached by
spindle fibres and are lost during cell division. Based on the position of centromere,
chromosomes are divided into four types: metacentric, submetacentric, acrocentric
and telocentric. In metacentric chromosomes, centromere is present at its centre. The
chromosome arms on either side of a metacentric chromosome are roughly equal in
length. In a submetacentric chromosome, the centromere is present slightly off
centre, due to which one arm of the chromosome is slightly short ( p) and one arm
of the chromosome is slightly longer (q). When the centromere is present closer to its
5 Study of Chromosome 241

1
At the simplest level,chromatin 2 nm
is a double-stranded helical DNA double helix
structure of DNA.

3
Each nucleosome consists of
eight histone proteins around
which the DNA wraps 1.65 times.
2
DNA is complexed with histones
to form nucleosomes. Nucleosome core of
4
eight histone molecules A chromatosome consists
H1 histone of a nucleosome plus the
6 H1 histone.
...that forms loops averaging
300 nm in length.

300 nm 11 nm Chromatosome
5
The chromatosomes
fold up to produce
a 30–nm fiber....

30 nm
250–nm–wide fiber
700 nm

7 8 1400 nm
The 300–nm fibers are Tight coiling of the 250–nm
compressed and folded to fiber produces the chromatid
produce a 250–nm–wide fiber. of a chromosome.

Fig. 5.1 Complex organisation of chromatin: The double helix of DNA is wound around an
octamer of histone proteins (nucleosome) which, along with the H1 protein, forms a chromatosome.
The chromatosome coils up to form a 30 nm fibre which loops up to produce a 300 nm fibre. This
fibre is further folded up to form a 250 nm wide fibre which ultimately supercoils to form the
chromosome (Annunziato 2008)

end than to its centre, the chromosomes are called acrocentric. The p arm of such
chromosomes is very short and q is long. In a telocentric chromosome, the centro-
mere is present on one end of the chromosome. This chromosome therefore has only
one arm (Fig. 5.3).
Acrocentric human chromosomes like chromosomes 13, 14, 15, 21 and 22 have a
secondary constriction other than the centromere in its short arm. These are called
chromosomal satellites and are composed of repetitive DNA and tandem copies of
ribosomal RNA genes. The position of these secondary constrictions remains con-
stant, and they serve as markers for identification of these chromosomes. Due to the
presence of large ribosomal RNA genes in this area, they form the nucleolar
organising regions (NORs). Nucleoli are the site of ribosome biosynthesis. It has
been seen that NORs are major hotspots for mutations in cancer which means that
242 D. Patwardhan et al.

Fig. 5.2 Structure of an eukaryotic chromosome: Chromosomes replicate before cell division and
the two sister chromatids are held together at its centromere. Kinetochore assembly takes place at
the centromere and spindle microtubules attach to the kinetochore. Telomeres are present at
chromosome terminals and help stabilise them (Pierce 2010)

the NOR region is prone to recombination in cancer. NORs have also been
implicated in ageing and senescence.

5.1.1 Chromosome Number

Organisms vary in the number of chromosomes they possess. Bacteria typically

possess a single chromosome, potatoes have 48 chromosomes and humans have
46 chromosomes. The complexity of organism has no relation with the number of
chromosomes it possesses per cell. Eukaryotes possess chromosomes in pairs. This
is because during sexual reproduction, organisms inherit one set of chromosomes
from the male parent and another from the female. Thus, out of the 46 chromosomes
5 Study of Chromosome 243

Fig. 5.3 Types of

chromosomes: Chromosomes
can be classified into four
types based on the position of
their centromeres. Telocentric
chromosomes have their
centromeres at the edges.
Acrocentric chromosomes
have centromere closer to one
end of the chromosome.
Metacentric chromosomes
have centromere at its centre
and submetacentric
chromosomes have
centromere closer to the centre
than to its edges. Some
acrocentric chromosomes
have secondary constrictions
besides the centromeres which
are known as satellite
chromosomes (Pierce 2010)

present in humans, 23 are inherited from the mother and 23 from the father. Each
chromosome therefore has a pair which is referred to as the homologous chromo-
some. The pair of chromosomes are generally similar in shape, size and genes that
they possess. If gene A is present on chromosome 1 at 1600 bp which codes for
protein A, another gene also coding for protein A will be present on the same
location in a homologous pair. These genes, however, may or may not be identical.
The gene on homologous pair may contain some variation. These different forms of
the same gene are called alleles. Genes may have multiple alleles. Such cells,
possessing pairs of chromosomes, are called diploid. Most cells are diploid except
cells which are involved in forming gametes, i.e. sperm or egg. The sperm and egg
possess only one set of chromosomes and are called haploid. The fusion of single set
of chromosome each from sperm and egg leads to the formation of a diploid
organism. Chromosomes can be stained and visualised under a microscope. This is
called a karyotype and it is useful in detecting the number and shape of
chromosomes and chromosomal defects if there are any. Figure 5.4 shows a normal
human karyotype and abnormal karyotype of a person having Down syndrome.

5.1.2 Autosome

Of the 23 pairs of chromosomes in humans, 22 pairs follow Mendelian pattern of

inheritance and segregate equally between all offspring. These chromosomes are
called autosomes. The remaining pair is called the sex chromosome because com-
position of this pair determines the sex of an individual. In human, females have two
244 D. Patwardhan et al.

Fig. 5.4 Karyotype can give indication about chromosomal abnormalities: Karyotype is the
complete set of chromosomes present in a eukaryotic cell. A. Humans normally have 23 pairs of
chromosome—22 autosomes and 1 pair of sex chromosomes which can be either XX for females or
XY for males. B. Karyotype for person having Down syndrome. The individual possesses an extra
chromosome 21 shown by the red arrow. The total number of chromosomes in this individual is
therefore 47 instead of the normal 46. Courtesy: National Human Genome Research Institute

X chromosomes and males have one X and one Y chromosome. Due to this, the
inheritance of genes on these chromosomes may not follow Mendelian inheritance
and may preferentially be inherited by daughters or sons.

5.1.3 Sex Chromosome

The term X chromosome comes from the term X body used by Hermann Henking
while describing a structure that he observed in the nuclei of male insects. McClung
recognised that this structure was a chromosome while studying grasshoppers and
termed it as accessory chromosome, but it became generally known as the X
chromosome. He stated that accessory chromosome determined sex of the individual
based on the observation that female cells had one extra chromosome compared to
males. In 1905, Nettie Stevens and Edmund Wilson, based on their observation in
grasshoppers and other insects, showed that female cells have two X chromosomes,
while male cells have only one X chromosome. In some insects, they also observed
that both males and females had the same number of chromosomes, and males had
another shorter chromosome instead of the second X chromosome. This was termed
as the Y chromosome.
They also showed that females formed gametes containing only the X chromo-
some, while males formed gametes half of which had the X chromosome and the
other half had Y chromosome. Thus, when a male sperm/gamete containing the X
chromosome fused with the egg, it formed a female individual with XX chromo-
some. When the sperm containing Y chromosome fused with the egg, it led to the
formation of a male individual with XY chromosome. This showed that sex of an
organism is determined by the composition of its chromosomes. The X and Y
chromosome are therefore called sex chromosomes and the nonsex chromosomes
are called autosomes. Although X and Y chromosomes differ in their size and genes
that they carry, they can pair during meiosis and get segregated. Pairing happens at
5 Study of Chromosome 245

the tips of X and Y chromosomes which are similar and carry the same genes. These
regions are called pseudoautosomal regions.
Some organisms, like grasshoppers, lack the Y chromosome. In this case, sex
determination occurs through the XX-XO system. That is, females have two X
chromosomes and males have a single X chromosome. During gametogenesis,
females produce gametes, all of which have the X chromosome. In males, half the
gametes receive X chromosome, while the other half contains no sex chromosome.
Since males in both XX-XO and XX-XY systems produce two types of gametes,
they are referred to as the heterogametic sex. Females in both cases produce a single
type of gamete and are referred to as the homogametic sex.
In some birds, moths and amphibians, the female is heterogametic and the male is
homogametic. To prevent confusion with the XX-XY system, the chromosomes in
these organisms are referred to as the Z and W chromosomes. Thus, the females have
a ZW composition and on gametogenesis produce half of the gametes having Z
chromosome and half of them having W chromosome. The males have a genetic
composition of ZZ and produce sperms having the chromosome Z.
Some insects like bees, wasp and ants have no sex chromosome. Sex is deter-
mined from the number of sets of chromosomes it possesses. If the organism has a
single set of chromosome (haploid), it is a male. If the organism has two sets of
chromosome (diploid), it is female. Males can produce gametes through mitosis as
they are already haploid. Females undergo meiosis to form gametes. A fusion of
male and female gametes leads to a female offspring. An unfertilised female gamete
can develop into a male offspring. Thus, genetically, males have a 50% chance of
similarity since two types of gametes can be developed from a diploid organism. On
the other hand, all females inherit the same male gametes and have a 50% chance of
inheriting the same female gamete. In Drosophila, or fruit fly, the sex of the
organism is determined by the ratio of sex chromosomes to autosomes. This is
because the X chromosome contains female determinants and the autosomes contain
the male determinants. Thus, if a fly has two sets of autosomes and one X chromo-
some (1X, 2A), the fly is male. In contrast, if the fly has two sets of autosomes and
2 X chromosomes (2X, 2A), it is female. In Drosophila, the Y chromosome is not
involved in determination of sex. It contains genes essential for forming sperm in
adults.

5.2 Chromosomal Basis of Heredity

Walther Flemming, while studying cell division, was able to stain the chromatin and
published his results in 1878. Later, Heinrich Wilhelm Gottfried von Waldeyer-
Hartz, in 1888, coined the term chromosome. However, the link between
chromosomes or genes and heredity was not known. Independent observations by
Walter Sutton and Theodor Boveri in 1902–1903, as well as rediscovery of Mendel’s
theories, led to the proposition of chromosomal theory of inheritance. Boveri while
studying the development of sea urchins discovered that embryonic development
was hampered if all chromosomes were not present. Sutton discovered that
246 D. Patwardhan et al.

chromosomes are present in pairs in grasshopper and they separate during meiosis
and stated that this may constitute the physical basis of Mendel’s principles of
heredity. The chromosomal theory of inheritance states that genes are present on
chromosomes and they constitute the genetic material responsible for Mendelian
inheritance.

5.2.1 Proof That Inheritance Linked to Chromosome

Thomas Hunt Morgan provided proof for the chromosomal basis of heredity through
his experiments on fruit flies. Inspired by the rediscovery of Mendelian principles,
Morgan started working on conducting genetic experiments initially on mice. He
later moved on to work with Drosophila melanogaster or the fruit fly. His lab did not
have very sophisticated instruments. The room was very small and untidy and
contained just some bottles for rearing the flies and lens or microscopes to observe
them. A lot of extremely important experiments in the field of genetics occurred in
this primitive fly lab.
While conducting his experiments, Morgan came across a male Drosophila
having white eye which stood out from the other flies which had red eyes. He
conducted a series of genetic crosses to study the inheritance of this trait. Morgan
started by crossing this white eyed male with a red eyed female. As expected, almost
all the progeny had red eyes. Morgan actually found 3 white eyed males in a progeny
of 1237 flies. He assumed that the white eyes had arisen due to spontaneous
mutation. Thus, it seemed to be following Mendel’s principles where red eye was
the dominant character and white eye was the recessive character. He then crossed
the F1 progeny with each other. Morgan expected to obtain a ratio of 3 red eyed
progeny:1 white eyed progeny. Instead, he observed that all females had red eyes
while half the males had red eyes and half had white eyes. Since the inheritance of
the trait differed between males and females, Morgan hypothesised that the trait was
linked to the X chromosome. Since females had two X chromosomes, the allele for
white eyes would get masked and all female would have red eyes. In males, since
only one X chromosome was present, they would automatically express the charac-
ter dictated by the inherited allele on X chromosome.
If eye colour was indeed an X-linked trait, Morgan predicted that a reciprocal
cross would yield different result. If a white eyed female was crossed with a red eyed
male, all the F1 females would be red eyed and all the F1 males would be white eyed.
The males would inherit X chromosome from the mother and Y chromosome from
the father. Since the female is homozygous recessive, all males would inherit allele
for white eyes from the mother. When the F1 generation is crossed with each other,
half the F2 generation in both males and females would be red eyed and the other
half would be white eyed. This becomes clearer in Fig. 5.5. Morgan obtained results
that were in accordance with his expectations, and he therefore concluded that eye
colour in Drosophila was an X-linked character.
5 Study of Chromosome 247

Fig. 5.5 Morgan’s

experiments on fruit flies
showed that eye colour was an
X-linked trait. When red eyed
females were crossed with
white eyed females, all F1
progeny had red eyes. On
crossing them with each other,
it was observed that all
females in F2 had red eyes.
Males in F2 had a ratio of
1 red eye:1 white eye.
Reciprocal cross yielded
different results. When white
eyed females were crossed
with red eyed males, all F1
females had red eyes and all
F1 males had white eyes. The
F2 progeny in both males and
females in this cross had a
ratio of 1 red eyes:1 white eye.
w, allele for white eye; w+,
wild type allele for red
eye (Pierce 2010)
248 D. Patwardhan et al.

5.2.2 Nondisjunction: Proof of Chromosome Theory

As mentioned previously, Morgan encountered some white eyed flies during his first
cross between red eyed females and white eyed males. Since the number of these
flies was very small, he attributed their presence to development of new mutations.
However, such flies with unexpected phenotype continued to appear even in the
succeeding crosses. One of Morgan’s students, Calvin Bridges, worked on under-
standing the genetic basis of these exceptions. We can denote the allele for red eyes
as Xw+ and that for white eyes as Xw. When white eyed females (XwXw) were
crossed with red eyed male (Xw+Y), we would expect that all males (XwY) would be
white eyed and all females (XwXw+) would be red eyed. It was, however, seen that
2.5% of the males were red eyed and 2.5% of the females were white eyed.
Bridges came up with a hypothesis to explain this phenomenon. He assumed that
this might be due to an error in the segregation of chromosomes during meiosis.
Instead of two X chromosomes getting separated and forming two gametes with one
X chromosome each, they failed to separate, forming gametes with one having two
X chromosomes and another devoid of any X chromosome. The failure of
chromosomes to separate was called nondisjunction. Due to nondisjunction, two
types of eggs will be produced in the given example: one with XwXw and one with
no X chromosome. Both of these can combine with a sperm bearing either Xw+
chromosome or Y chromosome. Thus, four different combinations are possible—
XwXw Xw+, XwXwY, Xw+O, or YO. O denotes absence of sex chromosome. XwXwY
will develop into a white eyed female because sex in Drosophila is determined by
the ratio of X chromosome to number of sets of autosomes. Since the ratio here is
1, embryo bearing this genotype will develop into a female. Xw+O will develop into
a red eyed male. Both XwXw Xw+ and YO genotypes are lethal and these embryos
will die. Nondisjunction can therefore explain the unexpected appearance of a small
number of white eyed females and red eyed males in a cross between white eyed
females and red eyed male flies. Bridges also examined the chromosomes of the flies
in question and found them to be the same as he had predicted (Fig. 5.6).

5.2.3 Chromosomal Basis of Mendelian Theory

The success of Bridge’s experiment is not limited to the fact that he was able to
predict the genotype of a fly showing unexpected phenotype in his culture. This
actually meant that the phenotype expressed by an organism is dependent on the
genotype. It established that genes are carried on chromosomes and these will dictate
the phenotype of an organism. Thus, the ‘units’ defined by Mendel in his hypothesis
which were later defined as genes were carried by chromosomes in the cell. The
chromosomes contain hereditary information, and the experiments by Morgan and
Bridges provided proof for chromosomal basis of heredity. They showed that the
gene for eye colour was a sex linked because it was present on the X chromosome.
5 Study of Chromosome 249

Fig. 5.6 Nondisjunction led to the appearance of unexpected phenotypes. (a) A cross between
white eyed female and red eyed male yields red eyed females and white eyed males. (b) If there is
nondisjunction during meiosis and the chromosomes fail to separate, one gamete has both X
chromosomes and the other lacks a sex chromosome. In this case, the F1 generation will consist
of white eyed females and red eyed males (Pierce 2010)

5.3 Sex-Linked Genes: Human

Sex-linked genes are also present in humans. Essentially, these are the genes which
are either present on the X or Y chromosome or both. Their inheritance will vary
among males and females. Disorders caused due to mutations of genes on X
chromosomes are called X-linked disorders. Many more males than females are
generally affected in an X-linked recessive disorder. Another feature of these
disorders is that the affected father will never transmit the disorder to his son.
X-linked recessive disorders are caused due to a mutation in a gene present on the
X chromosome, and they are inherited in a recessive manner. That means, that one
defective copy is usually not enough to cause the disease as a copy on the other pair
of the chromosome can make up for its loss. However, males possess a single copy
of the X chromosome, and most of the genes present on this chromosome are absent
on the Y chromosome. The defective copy of a gene on X chromosome remains
uncompensated for in males, and they express the disorder in spite of carrying only
one defective copy. This makes them more vulnerable to X-linked recessive disorder
(Fig. 5.7). In the example below, if the father carries a mutation in the X chromo-
some, the gene is passed on to the daughters. The daughters do not express the
250 D. Patwardhan et al.

X-Linked Recessive
Parents

Y Y
mutation
X XX X XX

Father Affected Mother Unaffected Father Unaffected Mother Carrier

Children

Y Y Y Y

X XX X XX X XX X XX
Son Daughter Son Daughter Son Daughter Son Daughter
Unaffected Carrier Unaffected Carrier Affected Carrier Unaffected Unaffected
NIH U.S. National Library of Medicine

Fig. 5.7 Probability and pattern of inheritance of X-linked recessive disorders. In case of X-linked
recessive disorders, if the father carries a mutation in the X chromosome, he will pass the
chromosome to his daughters. Sons will not be affected since they will only inherit Y chromosome
from their father. Since the daughters carry only one X chromosome with the affected gene, they
will only be carriers and will not express the disorder. In case the mother carries a mutant gene on
the X chromosome, she may pass it to both her sons and daughters. However, males being
hemizygous will express the disorder if they inherit the mutant gene. Daughters will remain
carriers (Courtesy of MedlinePlus from the National Library of Medicine)

disorder and are instead just carriers since they carry a single defective copy. The
sons will remain unaffected as they will never inherit the X chromosome from their
father. If the mother carries a mutation in the X chromosome, the gene may be passed
equally between the sons and daughters. The daughter will be a carrier but the son
will express the disorder. A female will only express an X-linked recessive disorder
if she inherits a defective X chromosome from both the parents.

5.3.1 Haemophilia: X-Linked

Haemophilia is a disorder where clotting of blood is affected. Several proteins act in

a cascade leading to clotting of blood. Mutations in any of these may cause
haemophilia. One of the most common causes is mutation in a protein called factor
VIII. Although there are medications available now, this disorder was thought to be
potentially fatal earlier as the affected person would bleed profusely causing his or
her death. Haemophilia is inherited in a recessive manner. Only one defective copy is
not sufficient to cause the disease but both copies need to be mutated for the disease
5 Study of Chromosome 251

to manifest. Haemophilia is sometimes referred to as the royal disease because many

members of European royalty suffered from this disease in the nineteenth and
twentieth century. The mutation for haemophilia seems to have originated from
Queen Victoria who passed on the defective gene to two of her daughters who were
carriers. That is, they were not affected by the disorder because they had a normal
copy of the gene on the other X chromosome. However, they did carry the genetic
defect and passed it to their offspring. Queen Victoria’s son also suffered from the
disease. Since the male progeny inherits the X chromosome from the affected mother
and Y chromosome from the father, he may inherit the allele with the genetic
mutation. Since he also lacks another X chromosome to compensate for the mutation
in the loci, he will suffer from the disorder.

5.3.2 Colour Blindness: X-Linked

Another example of X-linked recessive disorder is colour blindness. Colour blind-

ness is a disorder in which individuals are unable to distinguish between some
colours like blue and yellow or red and green. In humans, the retina in the eye
consists of cone cells which aid in the perception of light. The cone cells consist of
one of three different pigments which absorb light at different wavelengths. One type
of pigment absorbs blue light, another absorbs red light, and a third absorbs green
light. We are able to detect only three colours—red, blue and green, but processing
of these signals by the brain allows us to perceive a host of various colours. The three
pigments are coded by different genes. Gene for blue pigment is present on chromo-
some 7, while the gene for red and green pigment is present on the X chromosome.
Colour blindness can occur if there are mutations in the loci coding for the red and
green pigments. These mutations are generally recessive and inherited as an
X-linked characteristic.
We can denote the normal allele as X+ and the defective allele as X
. If an
affected male (X
Y) were to mate with an unaffected female (X+ X+), their
daughters would be carriers (X+ X
) but remain unaffected, while their sons
would be normal (X+Y). On the other hand, if an affected female (X
X
) mates
with an unaffected male (X+Y), their daughters would be carriers but remain
unaffected (X+ X
), while all the males will be affected (X
Y). Sons inherit the
condition from their mothers due to the fact that they are hemizygous. Males are
hemizygous for X-linked genes because they have a single X chromosome and
therefore a single allele. Carrier daughters may pass on the condition to their sons. It
may be noticed that the condition can pass from mothers to sons or it can be passed
from fathers to grandsons through their daughters. X-linked recessive inheritance
therefore shifts between sexes from one generation to another. For this reason, it also
sometimes referred to as a criss-cross inheritance (Fig. 5.8).

5.3.2.1 X-Linked Dominant Disorders

Just as there are autosomal dominant disorders, there are also examples of X-linked
dominant disorders. This means that the gene responsible for causing the disease is
252 D. Patwardhan et al.

II

III

IV

Fig. 5.8 Pedigree showing inheritance of an X-linked recessive disorder. Shaded boxes show
affected individuals. More males exhibit this disorder than females. Females having a shaded centre
are carriers and carry the mutated gene on one of the X chromosomes. This pedigree clearly exhibits
criss-cross inheritance of disorder from carrier mother to affected son and from affected father to
carrier daughter (Griffiths et al. 2002)

present on the X chromosome and is inherited in a dominant manner which means

one copy of the mutant gene is sufficient to express the disorder. In these disorders,
inheritance is not necessarily more common in males than in females unlike the
X-linked recessive disorders. The pattern of inheritance depends on whether the
father or mother carries the mutated gene. If the father carries the mutant gene, the
trait will be exclusively passed on to his daughters. This is because sons will only
inherit Y chromosome from the father and so the X chromosome is inherited by the
daughters. In addition, being a dominant disorder, presence of one mutated copy is
sufficient to cause a phenotype. In contrast, if the mother carries the defective X
chromosome, the trait can be passed to both sons and daughters. It should be noted
from this that a son having an X-linked dominant disorder will have necessarily
inherited it from his mother (Fig. 5.9).

5.3.2.2 Rett Syndrome

Rett syndrome is an example of X-linked dominant disorder. It is caused due to the
mutation in MeCP2 gene. It is a developmental disorder and symptoms may start
appearing after 6–18 months of age. They develop microcephaly (brain size is small)
and breathing abnormalities. They may also have abnormal eye movements, irrita-
bility, seizures and scoliosis. They have language and communication problems and
tend to make repeated movements. Rett syndrome is often seen in girls. This is
because males having a mutation in MeCP2 generally die in infancy. Rett syndrome
belongs to a spectrum of disorders and it can range from mild to severe and atypical
form of the disorder. The mutations in MeCP2 may affect synapses and communi-
cation between neurons. MeCP2 protein is believed to regulate activity of genes
involved in brain function. It may lead to the disruption in neuronal functioning. It is
however unclear how the MeCP2 mutations lead to symptoms seen in Rett
syndrome.
5 Study of Chromosome 253

X-linked dominant, affected mother X-linked dominant, affected father

Unaffected Affected Affected Unaffected

father mother father mother

XY XX XY XX
Unaffected Unaffected
Affected Affected

XY XX XY XX XY XX XY XX

Unaffected Affected Affected Unaffected Unaffected Affected Unaffected Affected

son daughter son daughter son daughter son daughter
U.S National Library of Medicine U.S National Library of Medicine

Fig. 5.9 Probability and pattern of inheritance of X-linked dominant disorder. If the mother carries
an X chromosome with a mutated gene, both sons and daughters have an equal probability of
inheriting the mutated gene and expressing the disorder. If the father carries an X chromosome with
a mutated gene, his sons will remain unaffected. The daughters will inherit and express the
disorder (Courtesy of MedlinePlus from the National Library of Medicine)

5.3.2.3 Fragile X Syndrome

Fragile X syndrome is another example of an X-linked dominant disorder. It causes
severe developmental abnormalities including learning disabilities and cognitive
impairment. Affected children may have delayed development and difficulty
maintaining attention and focusing on a particular task. They may have difficulty
in social interactions and communication. Typical features associated with fragile X
syndrome is a long, narrow face and prominent jaw and forehead, large ears, flexible
fingers and flat feet. It is more common in males than in females. Fragile X syndrome
is caused by a mutation in the FMR1 gene which codes for the FMRP protein. The
FMR1 gene has trinucleotide CGG repeats which can range from 5 to 40 in normal
individuals. In affected individuals, there may be more than 200 repeats. This leads
to a silencing of the gene, reducing the levels of FMRP protein. FMRP protein is
thought to be involved in formation of synapses which helps neurons communicate
(Fig. 5.10).

5.3.3 Genes on Y Chromosome

Genes on Y chromosome are passed exclusively from male to male. Especially genes
that are present on the differential region of the Y chromosome, that is, the region on
the Y chromosome which is not similar to the X chromosome, are present only in
254 D. Patwardhan et al.

Fig. 5.10 Pedigree showing inheritance of X-linked dominant disorder. An X-linked dominant
disorder can be inherited without any bias between males and females within a family. We can see
that a father will pass the mutated gene on X chromosome to all his daughters. Sons may inherit the
disorder from their mother carrying the mutated gene on the X chromosome

males. One of the primary genes that influence maleness is the sex-determining
region Y (SRY) gene which is sometimes also referred to as the testis-determining
factor. This gene is located on the differential region of the Y chromosome. The SRY
protein is a transcription factor which regulates the expression of a number of
different genes. It signals the foetus to develop male reproductive organs like testis
and prevents the formation of female reproductive structures like uterus and
fallopian tubes. Mutations on Y chromosome are associated with sterility. But this
condition is not heritable and is caused by spontaneous mutations in the Y
chromosome.

5.3.4 Genes Both on X and Y Chromosomes

Certain regions present on the ends of the X and Y chromosomes share common
genes and can therefore pair up and split during meiosis. Since these genes are
present on both chromosomes, they follow an autosomal form of inheritance rather
than a sex-linked pattern of inheritance. For this reason, they are called
pseudoautosomal regions or PAR. The PAR present on the short arms of X and Y
region is called PAR1 and consists of about 2.6 Mb of DNA. PAR 2 present on the
long arm of the chromosomes is shorter and spans about 320 kb. Pairing of PAR1
region is important for spermatogenesis. About 24 genes have been identified in the
PAR1 region and they play a variety of roles. For example, CD99 is involved in T
cell adhesion process. ASMT is involved in the synthesis of melatonin. CXYorf3 is a
regulator of alternate splicing. There are a few genes for which function is not yet
known.
5 Study of Chromosome 255

Mutations in SYBL1 located in PAR2 region are found to be associated with

bipolar affective disorder. It codes for a member of synaptobrevins which play a role
in cellular exocytosis. IL9R also located in PAR2 region has been linked to devel-
opment of asthma. It belongs to the hematopoietin receptor subfamily. The SHOX
(short stature homeobox-containing gene) is implicated in short stature of Turner
syndrome. This gene is located in the PAR1 region.

5.4 Concept of Sex Determination

The sex of human embryos is not developed until the seventh week of gestation.
Genes present on the Y chromosome signal for development of the testis at this
point. In the absence of Y chromosome, the embryo develops into a female. The Y
chromosome is therefore important for development of male characteristics.

5.4.1 Y Chromosome: Maleness

As stated before, regions on the tips of Y chromosomes called pseudoautosomal

regions pair with the X chromosomes and segregate during gamete formation. The
remaining region on the chromosome does not pair with the X chromosome and is
referred to as the male-specific region of the Y (MSY). The MSY consists of both
euchromatin and heterochromatin regions. Euchromatin near the PAR at the short
end of the chromosome contains a gene called SRY. As stated in Sect. 5.3.3, this gene
plays an important role in stimulating male gonad development in the embryo. It has
been observed that there are males with two X chromosomes and no Y
chromosomes. However, they do have the region of the Y chromosome containing
the SRY gene attached to one of their X chromosomes. There are also cases where a
female develops even when she has a genotype of XY. In such cases, the Y
chromosome usually lacks the SRY region. These examples show that SRY gene is
essentially responsible for male determination. It is not very clear how SRY triggers
male development. It acts as a transcription factor which activates expression of a
cascade of genes, some of which are present on the autosomes eg Sox9 on chromo-
some 17 and WT1 on chromosome 11. The action of these genes and their down-
stream partners promotes male gonad development and aids in blocking of female
gonad development.
The Human Genome Project helped in getting a better picture of the genes present
on Y chromosome. This information changed the perception of Y chromosome as
the carrier of only a few genes. The MSY region is about 23 Mb long and consists of
three regions—X transposed region, X degenerative region and ampliconic region.
The X transposed region is derived from the X chromosome during the course of
human evolution. It is similar to the Xq21 region of the modern X chromosome and
has two genes. This region consists of 15% of the MSY region. The X degenerative
region is more distantly related to the X chromosome. Many of the genes in this
region are pseudogenes and are non-functional. There are 14 genes in this region that
256 D. Patwardhan et al.

Fig. 5.11 The human Y

chromosome. PAR is the
pseudoautosomal region
present on both the tips of the
chromosome. MSY is called
the male-specific region of the
Y chromosome which does
not pair with the X
chromosome during meiosis.
The SRY gene is located in the
MSY near the PAR on the
short arm of the chromosome.
The MSY consists of both
euchromatin and
heterochromatin (Klug et al.
2007)

are capable of getting transcribed and SRY is one of them. This region forms 20% of
the MSY. The ampliconic region has about 60 transcription units which are divided
into nine gene families. Most of them have multiple copies. The members of each
family are nearly identical to each other. Each repeat unit, called an amplicon, is
present on the euchromatin region of the Y chromosome. Most of these genes are
related to the development of the testis and its function. Mutations in these regions
may lead to sterility in males. The ampliconic region forms about 30% of the MSY
region (Fig. 5.11).

5.4.1.1 Klinefelter Syndrome

Individuals with Klinefelter syndrome have an underdeveloped testis which is
unable to produce sperm. They have male genitalia and internal ducts. They are
tall and have long limbs with larger hands and feet. There might be slight breast
enlargement and presence of rounded hips. It was discovered later that these
individuals had an abnormal karyotype. They had one Y chromosome and multiple
X chromosomes ranging from 47, XXY to 49, XXXXY. The number here denotes
the total number of chromosomes followed by the composition of the abnormal
chromosomes. Here, 47, XXY means that the individual has the normal
44 autosomes and then has two X chromosomes and a Y chromosome instead of
one X and one Y chromosome. More X chromosomes led to more severe symptoms.
5 Study of Chromosome 257

This condition may arise due to nondisjunction of chromosomes during meiosis. The
presence of Y chromosome allows the formation of male genitalia but it is abnormal
and incomplete. This syndrome is present in 2 out of every 1000 male births.

5.4.1.2 Turner Syndrome

The affected female in Turner syndrome has underdeveloped ovaries but develops
external female genitalia. These individuals are short, have underdeveloped breasts
and have skin flaps on the back of the neck. They may also have a broad chest. They
generally lack one X chromosome and are 45, X. Since these individuals lack a Y
chromosome, they develop as females though this development is abnormal due to
the lack of another X chromosome. Turner syndrome may also be a result of
mosaics. In this case, the embryo is normal in the beginning. Mutations may occur
in some cell during early development leading to a loss of one of the chromosomes.
This mutated cell can divide and give rise to more abnormal cells. The individual
thus has both the normal and abnormal cells and is a mosaic of these two cell types.
The most common chromosome combinations are 45, X/46, XX and 45, X/46,
XY. Turner syndrome affects 1 in every 2000 female births. This frequency is
much lesser than the Klinefelter syndrome. One reason for his may be that many
more of the 45, X foetuses get spontaneously aborted and die in utero.

5.5 Analysis of Sex-Linked Traits

5.5.1 X-Linked Recessive Inheritance

Expression of X-linked recessive inheritance is mostly seen in males as they have

only one X chromosome and most of the alleles are hemizygous. Presence of
defective allele on X chromosome is expressed in males, but it’s not expressed in
females as they have corresponding normal allele to mask the effect of defective
allele.
The following are some criteria for understanding X-linked recessive inheritance
(Adkison 2012):

1. Males who are not affected do not pass the disease on to their offspring.
2. Affected males’ daughters are all heterozygous carriers.
3. Heterozygous women pass the mutant allele on to 50% of their sons (who are
affected) and 50% of their daughters (who are heterozygous carriers).
4. Half of an affected male’s sons would be affected if he marries a heterozygous
woman, giving the false impression of male-to-male transmission (Fig. 5.12).

X-linked recessive inheritance is seen in case of transmission of haemophilia A. It

is a blood disorder in which gene for clotting factor VIII is defective causing
abnormally delayed clotting. It is mostly seen in males who receive defective
genes from their unaffected mothers.
258 D. Patwardhan et al.

Fig. 5.12 X-linked inheritance of haemophilia A among descendants of Queen Victoria (1–2) of
England

Fig. 5.13 Structure of the FVlll molecule linear structure

FVlll is a 300 kDa glycoprotein which is produced by the liver. FVlll gene is a
dimeric protein containing a light chain of 80 kDa and heavy chain of 90–250 kDa.
FVIII is an essential cofactor for the conversion by activated FIX (FIXa) of the
zymogen factor X (FX) into active FXa (Fig. 5.13).
Mutations responsible for mild/moderate haemophilia A and resulting in reduced
stability of the A2 domain are localised at or near the interface between the A
domains.
A series of mutations in residues either only partially surface-exposed or located
in the core of the FVlll molecule were predicted to result in impaired stability or
folding of the FVlll molecule. Such alterations were predicted to result in accelerated
intracellular degradation and poor secretion of a large number of FVlll variants.
5 Study of Chromosome 259

5.5.2 X-Linked Dominant Inheritance

X-linked dominant inheritance occurs in both males and females, and only one
mutant allele is involved. This type of mutation can be transmitted by a female to
both male and female offspring; but males can transmit it only to females. Affected
females transmit the affected allele to 50% of offspring, whereas males transmit
mutant allele to 100% of females (Fig. 5.14).
In this case, heterozygous females are less affected than males as they have a
normal, unmutated allele.

5.5.3 Y-Linked Inheritance

Y chromosome is smaller and carries lesser genes unlike X chromosome. It is

transmitted from a father to a son. Y chromosome carries genes for:

• ASMTY (acetylserotonin methyltransferase)

• TSPY (testis-specific protein)
• IL3RAY (interleukin-3 receptor)
• SRY (sex-determining region)
• TDF (testis-determining factor)
• ZFY (zinc finger protein), PRKY (protein kinase, Y-linked)
• AMGL (amelogenin)
• CSF2RY (granulocyte-macrophage colony-stimulating factor receptor, alpha
subunit on the Y chromosome)

It also inherits the genes for hypertrichosis of ears, webbed toes and
porcupine man.
In hypertrichosis, a prominent amount of hairs of atypical length grow from
the ears.

Fig. 5.14 Inheritance of X-linked dominant trait

260 D. Patwardhan et al.

5.6 Techniques for Studying Chromosome

Through the study of chromosomes, one can infer the number and morphology of
chromosomes for the construction of karyotype and recombination studies through
meiosis. To start with, the tissue sample is to be collected which can be anthers from
bigger flowers or whole inflorescence from smaller flowers, and in the case of study
of somatic chromosomes, root or shoot tips can be collected and fixed. To increase
the number of metaphase cells, the somatic tissues may be pretreated before fixation.
Furthermore, pretreatment increases chromosome condensation and makes morpho-
logical detection easier (Singh 2003). Followed by pretreatment and fixation, the
cells are subjected to staining. For preparation of smear, the sample is placed over a
slide and crushed in a drop of stain. Crushing of samples ensures spreading of cells
and easy observation.

5.6.1 Cytological Fixatives

Cytological fixatives are chemical substances used to preserve or stabilise biological

samples. A good fixative should have all of the following characteristics: rapid
penetration strength, freezing the cell state without dissolving or distorting it,
shrinking or swelling the cell, pathogen killing capacity and autolytic enzyme
inactivation. One of the most important characteristics of a successful fixative is
that it sensitises various cell constituents to particular stains so that they can be
detected. Fixation helps to preserve cellular architecture and composition of cells in
the tissue to allow them to withstand subsequent processing (Thavarajah et al. 2012).
The process of fixation involves halting all the activities in a cell or a tissue in order
to study cell activities across a time range. Data from different stages are united in
order to study a particular process of cell. The fixative used is determined by the
mechanism being studied, with the main focus being on the structure of the chromo-
some and its activity without interference from other cellular components.
On the basis of their oxidation potential, fixatives are classified into metallic and
non-metallic compounds (Table 5.1). One of the most important characteristics of a
good fixative is that it sensitises various cell constituents to particular stains,
allowing them to be examined more easily. Since both of these properties cannot
be present in a single fixative, a combination of one or more compatible chemicals
must be combined to achieve successful results. Baker (1944) proposed that a

Table 5.1 Some common fixatives grouped on the basis of their oxidation potential
Reductants Oxidants
Ethanol (C2H5OH) ‘Osmium tetroxide (OsO4)’
‘Chromium trioxide (CrO3)’
‘Potassium dichromate (K2Cr2O7)’
‘Formaldehyde (HCOH)’
Mercuric chloride (HgCl2) ‘Acetic acid (CH3COOH)’
5 Study of Chromosome 261

Table 5.2 Some widely used fixatives are classified based on their reaction with albumin, with
subgrouping based on their metallic appearance
Coagulants Non-coagulants
Metallic fixatives Non-metallic Metallic fixatives Non-metallic
fixatives fixatives
Mercuric chloride Ethanol Osmium tetroxide (OsO4) Formaldehyde
(HgCl2) (HCOH)
Chromium trioxide Potassium dichromate Acetic acid
(CrO3) (K2Cr2O7) (CH3COOH)

fixation mixture consisting of basic non-precipitant chemicals combined with a

non-fixative salt would be the safest.
Coagulants and non-coagulants are the two types of fixatives depending on their
reaction with albumin (Table 5.2). Non-coagulants form a meshwork, while
coagulants react with albumin to form a coagulum. Non-additive fixatives add
atoms to the protein backbone and interlink chains (Wolman 1955), while additive
fixatives add atoms to the protein backbone and interlink chains (Baker 1966); some
precipitate, while others do not.

5.6.1.1 Mercuric Chloride (HgCl2)

Mercuric chloride is a compound that has been used for tissue fixation for a long
time. It is a good protein precipitant since it reacts with amines, amides, amino acids
and sulphydryl groups. It repairs nucleoproteins by reacting with the phosphate
groups of nucleic acids. Mercuric chloride as a fixative has drawbacks in that it is
corrosive in nature, and mercury, which is extremely poisonous, can be ingested
through the skin and is a cumulative poison. Chromosomes pick up dyes well after
fixation with mercuric chloride which is compatible with many fixatives, but one of
the serious drawbacks is that it leaves behind metallic needle-like precipitates
(Mayer 1918).

5.6.1.2 Potassium Dichromate (k2Cr2O7)

At pH greater than 4.6, potassium dichromate acts as a coagulant, interacting with
chromic acid to preserve chromosome structure at lower pH. It has a non-coagulant
effect. It interacts with unsaturated lipids to render them insoluble, which is why it’s
used in mitochondrial research. The oxidation of proteins is combined with the
interaction of reduced chromate ions to form cross links in the fixation reactions.
Chromium ions react with the carboxyl and hydroxyl side chains of proteins, leaving
the amino group free, allowing acidic dyes to stain the protein.

5.6.1.3 Chromic Acid (H2CrO4) or Chromium Trioxide (CrO3)

Chromic acid is a strong oxidising agent and highly corrosive in nature. Reaction of
chromic acid with proteins occurs in two steps. In the first step, coagulation and
precipitation of protein occurs and in the second step, hardening due to HCrO4—
occurs. The nucleus and the chromosomes are well fixed, while the cytoplasm is
262 D. Patwardhan et al.

coarsely coagulated. Since it reacts violently with organic reducers like ethanol and
formalin, chromic acid should not be combined with them.

5.6.1.4 Osmium Tetroxide (OsO4)

Osmium tetroxide is a highly radioactive and volatile crystalline solid found in
nature. It must be treated with caution due to its high volatile property, as it can
repair the conjunctiva of the eye and nasal mucosa. Intercalating between unsatu-
rated bonds of lipids and phospholipids is part of the osmium tetroxide fixation
reaction. Most proteins lack several double bonds, but certain amino acids, such as
tryptophan and histidine, have double bonds in their rings, making them reactive
with osmium tetroxide and forming a dark precipitate. Proteins that have been
treated with osmium tetroxide are no longer reactive to acidic dyes. Osmium
tetroxide is soluble in lipids and becomes black when exposed to ethanol. Due to
the reaction with double bonds, it also blackens unsaturated lipids (Altmann 1894).
Since it does not precipitate DNA, it cannot be used to analyse interface
chromosomes. The disadvantages of using osmium tetroxide as a fixative include
its high cost and the fact that it blackens tissues as it reacts with formaldehyde and
ethanol to form reduced osmium dioxide, which is dark in colour.

5.6.1.5 Ethanol (C2H5OH)

70–100% ethanol is most commonly used as a chemical fixative. It acts as a
dehydrating agent but does not react with macromolecules. It precipitates proteins
and DNA by removing water molecules from them and making them insoluble in
water. Its staining ability is unaffected because it does not react with macromolecules
like DNA. Since it is a reductant that is oxidised to acetaldehyde and then to acetic
acid, it is incompatible with oxidising fixatives including metallic oxides. The
disadvantages of using ethanol as a fixative are that it shrinks and hardens the cells
excessively. As a result, ethanol must be combined with other fixatives that reverse
this effect.

5.6.1.6 Acetic Acid (CH3COOH)

Acetic acid is a weak acid that works well as a solvent. It dissolves both polar and
nonpolar compounds, making it ideal for dye dissolution. It’s known as glacial acetic
acid in its anhydrous form, and vinegar in its 4–8% form. It precipitates nucleic acid
and dissolves histones, but it does not fix proteins, according to Pischinger (1937). It
is understood that it causes excessive swelling of cellular elements, including
chromosomes, but not nuclear protein objects. It prefers to fix pachytene
chromosomes, but it can also fix metaphase and anaphase chromosomes. The
stainability of cells is affected by acetic acid fixation. The cytoplasm is more
sensitive to acidic dyes, while the metaphase and anaphase chromosomes are more
sensitive to simple dyes. Since acetic acid has the ability to swell cells, it must be
used in conjunction with a fixative that has the ability to shrink the cytoplasm.
5 Study of Chromosome 263

5.6.1.7 Formaldehyde (HCHO)

Formaldehyde is often used to preserve specimens. It hydrates to form methylene
glycol, a glycol (hydrated formaldehyde) in aqueous solution. Because of its ability
to react with proteins, it is used as a fixative. It cross-links proteins by reacting with
terminal and secondary amino groups, as well as immino groups. It forms bridges
between the –CO and –NH groups at pH 4, but at pH 8, it reduces the sulphur bridges
(-S-S-) to the –SH groups and then reacts to form the methylene bridge (-S-CH2-S-)
(Middlebrook and Phillips 1942) The property of bridge formation hardens the
tissue, making tissue analysis easier. It isn’t recommended for chromosome studies
because it doesn’t fix or precipitate DNA.

5.6.1.8 Propionic Acid (C2H5COOH)

It’s for species with a limited chromosome count. It has a structure and solvent
properties identical to acetic acid, but unlike acetic acid, it does not swell the
chromosomes, so it is widely used as a fixative in chromosome research.

5.6.1.9 Chloroform (CHCl3)

It is highly soluble in alcohol, ether or acetone and moderately soluble in water.
Since it dissolves fat, it’s mainly used on tissues with an oily or waxy coating. Since
it dissolves the upper layers, it aids fixative penetration. It converts to carbonyl
chloride in the presence of air and light.

5.6.1.10 Fixing Mixtures

None of the above-mentioned fixatives can be used alone as they have drawbacks.
Hence, it is recommended to mix two or more fixatives together to overcome the
drawbacks of each other and enhance the process of fixation.

5.6.1.11 Flemming’s Weak Fluid (1882) and Flemming’s Strong Fluid

(1884) (Table 5.3)

Flemming’s solution contains both coagulating agent chromium and additive

fixative osmium tetroxide, hence considered a good fixative. The drawback of
shrinking of cells due to osmic acid is overcome by the swelling effect of acetic
acid. Osmium tetroxide aids in fixing the outer layer of cells due to its property of
slow tissue penetration, whereas acetic acid penetrates the tissue well. Osmic acid
causes blackening of tissues; hence, care should be taken not to keep the tissue in the
fixative for a longer time.

Table 5.3 Composition of Flemming’s weak fluid and Flemming’s strong fluid
Flemming’s weak fluid (1882) Flemming’s strong fluid (1884)
Osmic acid 0.1% Chromium trioxide, 5% aq 0–3 mL
Chromic acid aqueous 0.25% Osmium tetroxide, 2% aq 0–4 mL
Glacial acetic acid 0.1% Acetic acid, 20% aq 0–5 mL
Distilled water 0–8 mL
264 D. Patwardhan et al.

5.6.1.12 Carnoy’s Fluid (1886) or Carnoy’s Fixative 1 (Table 5.4)

It is the most commonly used fixative and used for almost all plant samples. It is
also mostly recommended for nuclear and mitochondrial organelles. The drawbacks
of using ethanol alone as a fixative is that it fixes the cytoplasm alone and shrinks the
tissue but does not fix the nucleus. This drawback is overcome by acetic acid which
fixes nucleus and does not fix cytoplasm. This fixative aids in studying both squash
and smear preparations. It is effective for both meiotic and mitotic studies of
chromosomes.

5.6.1.13 Carnoy’s Fluid II (1886) (Table 5.5)

It’s mostly used to fix flower buds. Proportions of 1:3:4 and 1:1:3 are used in the
Asteraceae and other families (Turner 1956). Since chloroform is present, the
fixative is able to penetrate the waxy coating on the flower buds.

5.6.1.14 Navaschin’s Fluid (Navaschin 1925) (Table 5.6)

Solutions A and B are prepared and stored separately as chromic anhydride

present in solution A is an oxidiser and formaldehyde present in solution B is a
reducer, hence, if mixed together, can cause oxidation reaction that can modify the
fixation properties. It has a high penetration property. Hence, it requires lesser time
for fixation, i.e. 3–4-h treatment is enough to act on a tissue. Since acetic acid can
cause swelling up of cells, it must be used in lesser proportion for study of details of
the chromosomes.

Table 5.4 Composition of Glacial acetic acid 1 part

Carnoy’s fixative
Ethanol 3 parts

Table 5.5 Composition of Glacial acetic acid 1 part

Carnoy’s fixative 2
Chloroform 3 parts
Absolute ethanol 6 parts

Table 5.6 Composition of Solution A

Navaschin’s fluid
Chromic anhydride 1.5 g
Glacial acetic acid 10 mL
Distilled water 90 mL
Solution B
Formaldehyde aq. sol. 40 mL
Distilled water 60 mL
5 Study of Chromosome 265

5.6.2 Cytological Stains

Live cells are transparent in nature; hence, it is difficult to observe them under the
microscope. Stains and dyes are usually used to overcome this problem. According
to Baker (1960), three factors influence the staining capacity of cellular components:
(1) dye-component affinity, (2) component density, and (3) dye permeability within
the same component. A chromophore is a group of atoms or electrons that causes an
organic molecule to be coloured, and auxochromes are additional groups attached to
chromophores that are known to increase the colour strength. Depending on the
charge of the chromophore (basic or acidic), the dye is classified as basic or acidic. If
the chromosome is positively charged, the chromophore is negative and vice versa.
These can be cationic, with a positive charge that attracts the cell’s basic
components, or anionic, with a negative charge that attracts the acidic components.
The ability of stain to stain is due to interatomic interactions such as double bond
resonance. The majority of dyes are based on the delocalised electron structure of an
aryl ring. Counterstaining is when one or more stains are used and each stain
interacts differently with various cellular components.

5.6.2.1 Carmine
Carmine is a basic stain used to stain plant chromosomes. It is crimson red in colour
having anthraquinone linked to glucose unit as colouring matter. It is obtained from
an insect Coccus cacti. Carminic acid is the dye’s main ingredient (C22 H20O13). It
stains chromatin as a basic dye at acidic pH, while it stains chromatin as an acidic dye
at basic pH. As a consequence, staining plant cells with carmine dissolved in acetic
acid is favoured. Adding a few drops of ferric hydroxide as a mordant improves the
stainability. Certain hard tissues should be stained with warm acetocarmine in HCl
because it softens the tissue (Figs. 5.15 and 5.16).

5.6.2.2 Orcein
Orcein is derived from a variety of lichen types. The crude part, orcinol, is extracted
and converted to orcein in the presence of air by ammonia. Orcein’s chemical
components were only discovered in the 1950s (Musso 1961 in Henwood 2003).
It’s made up more of alpha-amino orcein phenoxazone derivatives (Fig. 5.17). It is
reddish brown in colour and readily soluble in ethanol, but less soluble in water.
Dyer (1963) used a solution of 2 g natural orcein dissolved in 100 mL of a 1:1
mixture of lactic and propionic acids and diluted to 45% with water to analyse fresh
pollen mother cells. It was also found to be ideal for preparing root tip chromosomes

Fig. 5.15 Structure of

anthraquinone
266 D. Patwardhan et al.

Fig. 5.16 Structure of carminic acid

Fig. 5.17 Structure of alpha-

amino orcein

quickly for studying detailed morphology (Dyer 1963). Until mounting in acetic-
orcein dye, acetic-orcein is combined with HCl for hydrolysis in root tip and shoot
tip tests (Tijo and Levan 1950; Sharma and Sharma 1957).

5.6.2.3 Crystal Violet

It’s a triarylmethane dye that’s used for Gram’s staining and histological staining.
It’s mostly used to describe bacteria. Alfred Kern was the first to synthesise it in
1883. The three triarylmethane groups attached to nitrogen (Fig. 5.18) serve as
auxochromes, while the aryl rings function as chromophores. At neutral pH, it is
bluish purple in hue, colourless at alkaline pH and yellow at acidic pH. It can’t be
used to research chromosomes because of its poor penetration.

5.6.2.4 Acetocarmine, Aceto-orcein (Table 5.7)

Procedure
• Dissolve 1 g carmine/orcein in 100 mL of 45% glacial acetic acid.
• Add aluminium granules and reflux for 24 h.
• Filter and store in dark bottle.
• Staining can be improved by adding 5 mL of 10% ferric chloride to 100 mL of
solution.
5 Study of Chromosome 267

Fig. 5.18 Structure of crystal

violet

Table 5.7 Composition of Sl. no. Constituents

acetocarmine/aceto-orcein
1. Orcein, carmine 1g
2. Glacial acetic acid 45 mL
3. Distilled water 55 mL

Table 5.8 Composition of Constituents

Schiff’s reagent
Basic fuchsine 0.5 g
Distilled water 100 mL
1 N HCl 10 mL
Potassium metabisulfite 0.5 g
Activated charcoal 0.5 g

5.6.2.5 Crystal Violet

With continuous stirring, 1 g of dye is added to 100 mL of boiling distilled water.
Filter through Whatman filter paper and set aside for a week to mature.

5.6.2.6 Schiff’s Reagent/Fuchsine Reagent (Table 5.8)

Procedure
• In 100 mL of boiled distilled water, 0.5 g of dye is added.
• Cool to 58  C and strain into an amber bottle using Whatman filter paper.
• At 26  C, add 10 mL of 1 N HCl and 0.5 g of potassium metabisulfite.
• Close the cover and set aside for 24 h; then wait until the solution becomes straw
coloured or bleach it with a small amount of charcoal (0.25–0.5 g).
• Filter after a comprehensive shake.
268 D. Patwardhan et al.

5.6.3 Cytological Pretreating Agents

Prior to fixation, tissues are pretreated with agents to achieve well-spread, transpar-
ent and condensed chromosomes for studying their structure and behaviour.
Pretreatment, according to La Cour (1935), is important for studying the spiral
structure of chromosomes. The following modifications arise in mitotic cells as a
result of tissue pretreatment:

• Increase the number of cells in metaphase.

• The chromosomes are constricted longitudinally.
• It elucidates the restraints.
• Increase the cytoplasm’s viscosity.
• Allows for quick fixative penetration.

5.6.3.1 Colchicine
It is a water-soluble alkaloid having formula C22 H25 NO6 (Fig. 5.19) and molecular
weight 399.43 g/mol. Pelletier and Caventou were the first to isolate it in 1820.
Geiger purified and named it in 1833, but O’Mara was the first to use it in 1939.
Colchicine binds to microtubules and prevents tubulin polymerisation by combining
with it (Molad 2002). The mitotic apparatus is made up of microtubules and is
responsible for separating chromatids during division (Palevitz 1993). Colchicine
pretreatment increases the number of metaphase cells, which increases the chances
of seeing chromosomes in extremely condensed form.

Fig. 5.19 Structure of

colchicine
5 Study of Chromosome 269

5.6.3.2 a-Bromonaphthalene
With a molecular weight of 207.07 g, it is a bromine derivative of naphthalene with
the molecular formula C10 H7Br (Fig. 5.20). Similar to colchicine, it prevents the
development of spindle fibres and stops dividing cells at metaphase.

5.6.3.3 8-Hydroxyquinoline
It has the molecular mass of 145.16 g/mol and the formula C9H7NO (Fig. 5.21).
Owing to an increase in cytoplasm viscosity, it affects spindle formation and causes
metaphase arrest, resulting in chromosome immobilisation, similar to other
pretreating agents. It’s best for plants with a limited number of chromosomes. For
different plant species, different amounts of this chemical and different periods of
time are suggested. For example, aloe vera (Vig 1968) received a 0.2% solution for
30 min, and orchid species received a 0.002 molar solution for 4–8 h (Pridgeon et al.
1999).

5.6.3.4 p-Dichlorobenzene (p-DCB)

It is a colourless solid with the molecular weight of 147 g/mol and the formula C6H4
Cl2 (Fig. 5.22). It induces chromatid separation, chromosome fragmentation and
chromosome bridges at an early stage (1943, Carey and McDonough).

Fig. 5.20 Structure of

α-bromonaphthalene

Fig. 5.21 Structure of

8-hydroxyquinoline
270 D. Patwardhan et al.

Fig. 5.22 Structure of

p-dichlorobenzene

5.6.4 Analysis of Mitotic Chromosome

• The nucleosome fibre is the basic unit of chromatin organisation. It entails

packing DNA into the nucleosome as a negative supercoil, resulting in a 10 nm
diameter interface chromatin fibre. Histone octamer is wrapped twice with
double-stranded DNA (H2a, H2b, H3, and H4).
• The nucleosome fibre folds into a solenoid (zigzag or crossed) to form the 30 nm
chromatin fibre. The supercoiling is aided by histone H1.
• Based on protein-protein interaction or protein-DNA interaction between adja-
cent chromatin fibres, further folding or coiling of chromatin fibres to create
higher-order domains that could fold into increasingly larger structures
(Fig. 5.23).
• Non-histone chromosomal proteins form a scaffold that tightly packs the chro-
matin fibre into metaphase chromosomes.
• Scaffolds are non-histone proteins that vary depending on the organism’s species
and developmental stage. It creates a mechanism that keeps chromosome struc-
ture intact. Scl and Scll have been identified as two main scaffold proteins.
• Scl is related to topoisomerase II, which is present in the axial area of extended
mitotic chromosomes but not in the loop domains.
• Scll is a protein that helps preserve the structural integrity of chromosomes. It is
found in the middle of the long axis of mitotic metaphase chromosomes’
chromatid arms.
• LFM-1 (Licensing Factor Model 1) is involved in cell cycle activities.
• The scaffolding network includes the kinetochore, actin-binding protein and
tropomyosin.
• Condensin and cohesin are members of the SMC family and are required for sister
chromatid pairing, DNA repair and replication and gene expression regulation.
• Condensin is found along the chromosome arms’ central axis (Fig. 5.24).
• Chromosomes are most often studied during mitotic metaphase as they are
shortest, thickest and most condensed. Mitosis is the phase of the cell cycle
where sister chromatids are separated by spindle fibres.
5 Study of Chromosome 271

Fig. 5.23 Mitotic chromosome organisation. Diagram showing different levels of chromatin
condensation, the 11 nm nucleosome is condensed into a 30 nm chromatin fibre which is then
segregated into supercoiled domains or loops by attachment to chromosome scaffolds composed of
non-histone proteins

• The cell copies its DNA during interphase, and chromatin is at its least
condensed, that is, hundreds of thousands of times less condensed than during
mitosis. This is why the enzyme complexes that copy DNA have the most access
to chromosomal DNA, which is where the bulk of gene transcription takes place.

5.6.4.1 Prophase
During prophase, chromosomes begin to condense by enlisting the aid of condensin
(reorganises chromosomes into their highly compact mitotic structure). Cohesin, a
protein complex that binds replicated sister chromatids together, is largely removed
from the arms of the sister chromatids, allowing them to separate. At the centromere,
it is held. During prophase, the spindle starts to develop, and the two pairs of
centrioles travel to opposite poles, while microtubules polymerise from the
duplicated centrosomes.

5.6.4.2 Prometaphase
The nuclear envelope is broken up into tiny vesicles that are shared by future
daughter cells. Since centrosomes are located outside the nucleus in animal cells,
expanding cells’ microtubules do not have access to them until the nuclear
272 D. Patwardhan et al.

Fig. 5.24 Molecular architecture and actions of condensin. (a) Composition of condensin com-
plex, (b) supercoiling assay, (c) knotting assay, (d) interaction of condensin with DNA
5 Study of Chromosome 273

membrane tears apart. The chromosomes are bound to the mitotic spindle during
mitosis. The kinetochore is a specialised chromosome area where both chromatids of
each chromosome bind to the spindle.

5.6.4.3 Metaphase
During metaphase, chromosomes are in the most compacted state. The
chromosomes align themselves across the equator of the mitotic spindle in the centre
of the cell. The kinetochore microtubules pull the sister chromatids back and forth
until they align along the cell’s equator, known as the equatorial plane. The cell
ensures that it is able to divide during mitosis by passing through the metaphase
checkpoint. Anaphase can only be reached by cells with properly constructed
spindles.

5.6.4.4 Anaphase
Owing to the degradation of cohesin molecules that were joining the sister
chromatids by protease separase, the duplicated genetic material in the nucleus of
the parent cell is divided into two identical daughter cells during anaphase. The
mitotic spindle separates the chromosomes. During anaphase, there are two types of
movements. The chromosomes travel towards the spindle poles as the kinetochore
microtubules shorten, and the spindle poles separate as the non-kinetochore
microtubules move past each other in the first section, and the spindle poles separate
as the non-kinetochore microtubules move past each other in the second part.

5.6.4.5 Telophase and Cytokinesis

The chromosomes reach the poles during telophase. In their interface confirmations,
the nuclear membrane reforms and chromosomes begin to decondense, which is
accompanied by the cytoplasm being split into two daughter cells of the same
genetic make-up.

5.6.5 Analysis of Human Karyotype

Karyotyping is the method of pairing and ordering all of an organism’s

chromosomes, resulting in a genome-wide snapshot of the chromosomes of an
individual. They are used to detect genetic modifications such as chromosome
number, deletion, replication, translocation and inversion using normal staining
techniques.
Mitotic cells that have been arrested in metaphase or prometaphase are used to
make karyotypes. Colchicine, which poisons the spindle formation, prevents cells
from entering metaphase. The cells are then exposed to a hypertonic solution, which
causes the cells’ nuclei to swell and burst. After that, the nucleus is fixed, dropped on
glass and stained with various stains to help in the analysis of the chromosome.
The majority of the karyotypes are stained with Giemsa staining, which provides
a more consistent preparation and better individual band resolution and can be
examined with a standard bright-field microscope. In G banding, AT-rich or
274 D. Patwardhan et al.

Fig. 5.25 Chromosome banding pattern by different staining techniques. (a) Giemsa banding, (b)
Q-banding (c) R-banding and (d) C-banding are shown

Table 5.9 Classification of chromosomes based on size

Group. Chromosomes Description
A 1–3 Largest; 1 and 3 are metacentric. But 2 is submetacentric
B 4,5 Large; submetacentric with two arms very different in size
C 6–12, X Medium size; submetacentric
D 13–15 Medium size; acrocentric with satellites
E 16–18 Small; 16 is metacentric, but 17 and 18 are submetacentric
F 19, 20 Small; metacentric
G 21, 22, Y00 Small; acrocentric with satellites on 21 and 22 but not on Y

heterochromatic regions appear to stain darker. GC-rich areas, on the other hand,
tend to stain lightly (Fig. 5.25).
Human autosomes are numbered 1 to 22 and arranged in descending order by size
in a karyogram. With the exception of chromosomes 21 and 22, all other
chromosomes are present. Chromosome number 21 is the smallest. Sex
chromosomes are usually found at the end of the chromosomes (Table 5.9).
Karyogram can be used to detect chromosomal abnormalities. Aneuploidy is
caused by the absence or addition of a chromosome, e.g. trisomy 21 (Down
syndrome).
5 Study of Chromosome 275

5.6.6 Cytological Variation

Differences between members of the same species or between species are referred to
as genetic variation. Allelic differences are caused by mutations in specific genes.
Significant variations in chromosome structure are referred to as chromosomal
aberrations. Usually, these have an effect on several genes. Typically, these have a
multigene effect. They’re also known as chromosome mutations.
Deletions, duplications, inversions and translocations are four types of chromo-
somal aberrations that can be observed under a microscope cytologically. However,
certain cytological shifts are too subtle to identify. Missing chromosome fragments
are referred to as deletions. Homozygous deletions may be lethal, while heterozy-
gous deletions may be nonlethal or lethal, and can express recessive genes that were
previously unknown. Duplications can trigger a genetic imbalance, resulting in
phenotypic effects in the organism and a greater range of gene functions.
Inversions are caused by a 180-degree turn of a chromosome fragment. Inversion
heterozygotes also have pairing problems during meiosis, resulting in the creation of
inversion loops. When loops are crossed, the outcome is normally unviable. For
pericentric and paracentric inversions, the crossover products would be distinct. A
chromosomal segment is relocated to a different location in the genome during a
translocation. Translocations generate duplication-deletion meiotic products in the
heterozygous state, which may result in unbalanced zygotes and new gene linkages.

5.7 Monosomy: Cri-du-Chat Syndrome

Cri-du-chat syndrome, also known as 5p-syndrome or cat cry syndrome, is a rare

genetic disorder caused by a deletion (missing piece) of genetic material on chromo-
some 5’s small arm (the p arm).
A high-pitched catlike scream, mental retardation, slow growth, distinct facial
features, small head size (microcephaly), widely spaced eyes (hypertelorism), low
birth weight and poor muscle tone (hypotonia) in infancy are some of the signs. Over
time, the catlike cry becomes less noticeable.

5.8 Trisomy

Trisomies are aneuploids; the addition of an extra chromosome (2n + 1) belongs to

any one of the different chromosomes of a haploid complement. The addition of a
chromosome defines that there are three homologous copies, so the total number of
attainable trisomics is equal to the haploid set of chromosome number. This extra
chromosome directs its type: normal (primary), isochromosome (secondary) and
translocate (tertiary) or interchange forms. In the nomenclature system, a human
chromosome complement (normal) is 46-XX (female) and 46-XY (male). Where a
male with an extra X chromosome will be 47, XXY, and a female with trisomy
21 will be 47; XX, +21, such triplications are trisomies.
276 D. Patwardhan et al.

The presence of one extra chromosome results due to meiotic irregularities like an
abnormal association during metaphase I and unequal segregation of chromosomes,
bridge/laggard formation during anaphase I. This results in the occurrence of
unbalanced gametes (i.e. n + 1) in trisomic individuals. Usually, autosomal
aneuploids are miscarriage, except for aneuploids of some autosomes like chromo-
some 21. These chromosomes carry lesser genes and have a small size where the
occurrence of extra copies is less liable than for bigger chromosomes. Apart from
trisomy 21 results in human live births, Edwards syndrome (trisomy 18) and Patau
syndrome (trisomy 13) are only human trisomies. The following are the description
of viable human aneuploids.

5.8.1 Down Syndrome

Down syndrome or trisomy 21 is the most habitual genetic disorder that impacts
foetal development, affecting 1 in 800 to 1 in 1000 every live-born infant. It was
discovered by John Langdon Down in 1866. It is normally associated with trisomy in
chromosome 21 in the G group of the acrocentric region of the smallest human
autosome. The extra chromosome 21, i.e. there are 47 chromosomes, including two
X chromosomes as well as the extra chromosome 21. The karyotype of the individ-
ual (Fig. 5.26a) is 47, XX, +21. Individuals with Down syndrome typically share
similar features, and they show an obvious resemblance to one another (Fig. 5.26b).
Due to an epicanthic fold (each eye); a flat/flush face with a round head; large
tongue-swollen or protruding; underdeveloped and small ears; characteristically
short; stubby fingers; Physical, psychomotor; while life expectancy is reduced to
an average of about 50 years.

Fig. 5.26 (a) The karyotype; three representatives of the G group chromosome 21 are present. (b)
A child with Down syndrome
5 Study of Chromosome 277

Fig. 5.27 HAS-21 regions linked to a specific Down syndrome phenotype. HSA21 short and long
arm (yellow and blue), G-banding (light and dark region). The centromere is shown in red; numbers
mean distance in Mb (megabases) from the HSA21 (distal end) short arm. Alzheimer disease (AD);
acute megakaryoblastic leukaemia (AMKL)/transient myeloproliferative disorder (TMD); atrioven-
tricular septal defect (AVSD); Down syndrome critical region; Hirschsprung disease (HD); imper-
forate anus (IA)/duodenal stenosis (DST); mental retardation (MR); recombinant DNA

Approximately 95% of cases of Down syndrome are because of the existence of

one extra (third) copy of HAS-21 (human chromosome 21), while in some cases,
trisomy of a part of HAS-21 directs to Down syndrome. In some smaller cases,
mosaicism (state of being composed of cells of two genetically different types) of
HAS-21 can lead to Down syndrome. Most often, the defect of paired homologues to
disjoin in anaphase I or II, due to this chromosome composition, will be gametes
with n + 1. Seventy-five per cent of these errors attributes to nondisjunction during
maternal meiosis I leading to Down syndrome, and there is an evident maternal age
effect for the birth of an individual with Down syndrome. There are high chances
that an older mother will give birth to a baby with Down syndrome. Thus, the huge
majority of Down syndrome individuals are conceived from older mothers.
Human segmental trisomies were analysed and have shown several dispersed
HAS-21 sites linked to a certain Down syndrome phenotype (Fig. 5.27). A
278 D. Patwardhan et al.

composite interaction of these causative genes (AVSD, AD, IA/DST, MR, HD) may
exert a pathological effect at various stages of maturity or development. On the
contrary, genes in charge for Down syndrome perhaps clustered into one chromo-
some region indicated as DSCR (Down syndrome critical region).
Genetic counselling early in certain pregnancies (certain women who get preg-
nant late during their reproductive years) is highly recommended. Diagnosis during
pregnancy provides positive test outcome, or at high risk of having a newborn with
Down syndrome. CVS (chorionic villus sampling) is a prenatal test where chronic
villi (cervix; transcervical and abdominal wall; transabdominal) are taken from the
placenta and used to analyse the foetal chromosome. The test is usually performed
during the first trimester between 11 and 14 weeks of pregnancy. Another approach
is amniocentesis (insertion of a needle into the mother’s uterus to sample the fluid
surrounding the foetus). Early detection of infants and children with Down syn-
drome makes a major difference in improving their quality of life. Because each
newborn with Down syndrome is unique, treatment mainly depends on the need of
the individual. Also, various stages of life may require numerous services.

5.8.2 Patau Syndrome

Patau syndrome is an infrequent genetic disorder for having an additional/extra copy

of chromosome 13 (trisomy 13) in a few or entire body cells. Each cell usually
involves 23 pairs of chromosomes, but an individual with Patau syndrome carries
three copies of chromosome 13 (Fig. 5.28a, b), instead of two. Patau syndrome was
first described by Klaus Patau in the year 1960. This affects 1 in every 5000 births
and the risk of having a newborn with Patau syndrome is directly linked with the
mother’s age. More than nine out of ten children born with Patau syndrome die
during the first year, whereas children with partial or mosaic trisomy 13 live for more

Fig. 5.28 (a) Karyotypic and phenotypic illustration of an individual with Patau syndrome. Three
sets of chromosome 13 (D group) are present (43, 13+). (b) Infant with Patau syndrome
5 Study of Chromosome 279

than a year. Individuals with Patau syndrome possess a wide range of health
problems: holoprosencephaly (brain often divided into two half), cleft lip and cleft
palate, microphthalmia (small eyes), anophthalmia (absence of one or both eyes),
microcephaly (smaller in size than the normal head size); cutis aplasia (absence of
skin from the scalp), ear malformation, deafness, capillary haemangiomas (red
birthmarks), the presence of abnormal cysts in the kidney, reduced penis, enlarged
clitoris in girls and polydactyly.

5.8.3 Edwards Syndrome

Edwards syndrome, called trisomy 18, is a rare but serious condition. The syndrome
affects the survivability of an individual, and most often individuals will die before
or shortly after being born. About 3 in 100 individuals born alive with Edwards
syndrome will survive past their first birthday. It was first described by John
H. Edwards and his colleagues. Every cell in our body carries 23 pairs of
chromosomes, but an individual having Edwards syndrome carries three pairs of
chromosome number 18 (instead of 2) (Fig. 5.29). Mosaic Edwards syndrome
consists of extra chromosome 18 in some cells, whereas in some only a part of the

Fig. 5.29 Karyotype of infants having Edwards syndrome. Three members of chromosome
18 (trisomy 18), creating the 47, 18+ condition
280 D. Patwardhan et al.

extra chromosome 18 in the cells is present, called partial Edwards syndrome. It is

characterised by multiple impairments, low sets of ears, small subsiding lower jaw,
clamped fingers, cardiac malformations and deformation of the face, skull and feet.

5.9 Polyploidy

We are very familiar with haploids and diploids (n and 2n). Often, the whole set of
chromosomes fail to differentiate during meiosis or mitosis. Tetraploid offspring is
occasionally produced by diploid parents. The chromosome doubling in the zygote
results in the polyploid formation, or from cytologically non-reduced female and
male gametes that will result in the development of functional tetraploid (3n)
zygotes. Polyploidy involves triploids (3n), tetraploids (4n) and pentaploids (5n),
including a higher number of chromosome sets. Polyploidy is most commonly
present in plants like ferns and flowering plants, for example, Hibiscus rosa-sinensis,
wheat and many tetraploids which are agriculturally most important plants (genus
Brassica), and may occur during the division of cells, either during metaphase I in
meiosis or mitosis. Also, polyploidy formation in human tissues is highly
differentiated (heart muscle, liver, placenta and bone marrow) and occurs in a
somatic cell of few animals: salmon, goldfish and salamander. Here we consider
two major types of polyploids: auto-polyploidy, the multiple set of chromosomes,
similar to the normal n complement of the related species; allopolyploidy, in which
chromosome complements are from multiple species; and endoploidy, which is a
repeated division of the chromosome in the absence of subsequent division of cell/
nucleus.

5.9.1 Autopolyploidy

A type of polyploidy wherein the extra chromosome set is derived from an identical
parental species or a parent. The cell or organism in autopolyploidy condition is
called an autopolyploid. Common examples of autopolyploids in plants are Tolmiea
menziesii (piggyback plant) and Acipenser transmontanum (white sturgeon). The
nondisjunction of chromosomes during mitosis (which produces double (4n) the
number of the chromosome) and meiosis (fusion of diploid gamete with normal
haploid gamete to form triploid (3n) zygote) produces autopolyploid and is depicted
in Fig. 5.30. In other words, the binding/fusion of diploid gametes (2n) results in
offspring either showing triploid (3n ¼ n + 2n) or tetraploid (4n ¼ 2n + 2n) number.
General results of autopolyploidy are seedlessness as in watermelon and bananas and
inducing sterility in salmon and trout farming.
How polyploidy appears naturally is of considerable interest to the geneticist.
Research on A. thaliana (Arabidopsis thaliana) explained that during the process,
deviation from doubling of mRNA/set of RNA transcripts (transcriptome) in
A. thaliana autotetraploid resulted due to increased size of the cell, wherein the
size of the cell and associated phenotype effectively influenced the content of DNA
5 Study of Chromosome 281

Fig. 5.30 Autotetraploid

containing a double number of
chromosome (2C), with the
doubling content of DNA
(4C). Diploid progenitors
(2C) and autotetraploid
(4C) resulting in larger cells
and nuclei (shaded circles and
nested circles with dotted
lines)

rather than being completely determined by genotype. A separate chromosome state

is maintained by the individual duplicated chromosome in the doubled nucleus. An
increase in nuclear volume is directly related to the genome size, but doubling
volume produced less genome doubling, which affects chromatin condensation
and by altering the contacts between chromosomes (Fig. 5.31). As a result, autotet-
raploid showed increased inter-chromosomal interactions and reduced intra-
chromosomal interaction. Overall, around 12% of the genome displayed position
difference between structural domains such as loose vs condensed (LSD-CSD) that
is between largely euchromatic and heterochromatic (A and B) regions commonly
found in animals and plants.

5.9.2 Allopolyploidy

A type of polyploid strain or individual consisting of a chromosome set composed of

more than two chromosome sets extracted more or less complete from variant
species. Allopolyploid species that originated from a seed-setting shoot of a sterile
F1 hybrid between P. floribunda Wall. and P. verticillata Forssk. represents a well-
known case of new allopolyploid formation through somatic chromosome doubling.
The production of unreduced gametes provides three possible ways: first, F1 hybrid
individuals are formed through interspecies crossing; second, genome doubling
takes place in a one-step process, that is, the union of unreduced gametes that are
produced by the F1 hybrid; and this allopolyploid speciation undergoes the third
pathway, the ‘hybrid genome doubling’ pathway (Fig. 5.32).
The new allopolyploids have a complete set of disomic chromosomes derived
from the parental species. Karyotypic changes through recombination between
homologous chromosomes may occur and lead to chromosomal diploidation.
Triticum and Aegilops are wheat species which are the best systems for studying
allopolyploid speciation. These species have two distinctive features: the first one is
282 D. Patwardhan et al.

a b c d
2C

CT
Inter-chromosomal Intra-chromosomal
interaction interaction
4C

Fig. 5.31 Autopolyploid showing genetic and epigenetic effects in A. thaliana with its diploid
progenitor (Col-0). (a–c) A. thaliana showing epigenetic response to autopolyploid. Within the
diploid (2n) nucleus during interphase, every chromosome specifically occupies a space or a region
inside the nucleus which is referred to as chromosome state or territory (CTs); in the autotetraploid,
the nucleus of interphase wherein the chromosomes are of the doubled complement and the whole
set appear to occupy the separate chromosomal state. The nucleus of Arabidopsis is also divided
into transcriptionally oppressed and transcriptionally active structural domains, i.e. compact struc-
tural domain (repressed CSD-dark shades at the bottom of a nucleus) and active loose structural
domain (LSD-unshaded interior part). Around 12% of autotetraploid shows chromatin switch states
between these structural domains. Panels a and b shows the gametic number, 5-diploid and
10-tetraploid chromosomes. White circles represent the folding of chromatin structure producing
intramolecular interactions. And blue circles represent intermolecular interactions. (c) Gene activity
alters the chromatin reconstructions. FLC gene overexpression is correlated with the autotetraploid
with increased accumulation of local chromatin loop along with decreased methylation of
H3K-27me-3 represented in grey circles across the entire length of the gene. (f) FLC gene
overexpression is related to late flowering, suggesting the possible link between the reconstruction
of chromatin and its evolutionary aspects

Stage I
Stage II
Parental
species 1 Stage III
(AA)
F1 hybrid Allotetraploid Established
X (AABB)
(AB) species
Parental (AABB)
species 2
(BB)

Fig. 5.32 Three major steps of allopolyploid speciation through the hybrid genome doubling
pathway. Stage I, where F1 hybrid individuals are formed through interspecies crossing, forms the
allopolyploid speciation. Stage II—actual doubling of genome; allopolyploidy individuals are
formed through the union of unreduced gametes from F1 hybrids. Stage III—the new allopolyploid
individuals formed via hybrid genome doubling start to propagate as reproductively isolated entities
and finally become established as species
5 Study of Chromosome 283

paternal and maternal lineages (known for Triticum and Aegilops polyploid species)
and the second one is artificial interspecific crosses are easily made in those genera.

5.9.3 Endopolyploidy

Endopolyploidy refers to the augmentation of nuclear DNA within the cell.

Endoreduplication is the most common process in plants. This involves the chromo-
somal DNA replication without the involvement of mitosis and no condensation/
decondensation of chromatin structures which stays united either at the entire length
(rarely) or at the centromeres. This kind of occurrence is most uneven in plants as
shown in Fig. 5.33. Polyploidy may also occur in few tissues of diploid animals, like
muscle tissues in humans. Prokaryotes lack nuclei as seen in Epulopiscium fishelsoni
bacterium. And it is also most common in angiosperms such as flowering plants,
where endopolyploidy is detected in few tissues at a higher level. Endopolyploidy
mechanism involves internal and external factors, which can be observed as the key
role of the developmental flexibility of plants.

5.10 Chromosome Shuffling

Change in the chromosome morphology refers to chromosome shuffling which

results in structural change and the difference in gene sequence and number with
or without altering gene structure or ploidy. Breaking of the chromosomal segment is
known as chromosomal aberrations, that is, the chromosomal loss or gain or union

Fig. 5.33 During cell division, DNA replication at the S phase and chromosome segregation and
cells divide in M phase producing two daughter cells. Endomitosis; partial M phase where cells
enter the mitosis stage and perform segregation but exit before the division of the cell takes place.
As a result, there will be the formation of 4C and 4n (tetraploid). Also, during the S phase,
replication is present at a specific part of chromosomal DNA, which ends up showing the increase
in DNA content (partially) 2C
2n + P
284 D. Patwardhan et al.

Fig. 5.34 Overview on the five different types of chromosomal aberration: gain, loss or rearrange-
ment of chromosome segments

with different or same chromosomes. This aberration usually occurs spontaneously

but physical and chemical factors are mainly influencing these structural changes.
Structural changes can exist within single or non-homologous or homologous
chromosomes. This includes deletion (loss) or duplication (gain) of a chromosomal
segment, or the translocation (exchange) of the chromosomal segment with different
segment. In an inversion, segment of a chromosome gets deleted, turns 180  C and is
repositioned at the exact same position on the chromosome. Gene balance is
disrupted by deletion and duplication, thus changing or altering the characters of
an organism. In translocations, shifted segment involving genes gets movement and
inserted into different linkage groups, whereas in inversion gene sequence is
changed in the chromosome. Gene balance or altering the phenotypic character of
organisms is neither by translocation nor inversion (Fig. 5.34).
In heterozygotes, meiosis occurs containing one normal chromosome and one
chromosome with inversions. The chances of these chromosomes coming together
are difficult, and crossing over between them will not take place or is uncommon.
Aneuploidy formation is often occurring, and consequently, heterozygotes are
comparatively less fertile than homozygotes due to which both pairs of
chromosomes are either inverted or normal. Moreover, heterozygotic chromosomes
also undergo translocation by producing the number of aneuploid gametes.
5 Study of Chromosome 285

Chromosomal aberrations show direct link in evolution of organisms: increase in

number of gene is mainly due to duplication. Inversions as well as translocations
may result in formation of individuals (genetically isolated homozygous condition)
that are more fertile than heterozygotes. In all chromosomal aberrations, the gene
position effect is sometimes observed, in which a gene gets shifted to a new site on a
chromosome exerting a different effect on the organism’s phenotype.

5.10.1 Deletions

Deficiency occurs when deletion of part of a chromosome results in loss of one or

more chromosomal genes. It was first observed by Bridges in 1917 in Drosophila.
Deficiency arises from chromosomal breakage occurring at random position in either
both or only one chromatid of a chromosome. The deletion caused by two breaks in
the chromosome will result in an interstitial deletion (Fig. 5.35a). If there is only one
break that occurs at the end of the chromosome, it is terminal deletion (Fig. 5.35b).
Sometimes break occurs at both ends of a chromosome. Detection is based on the
unpaired region of the normal chromosome that produces a loop during the pachy-
tene stage (Fig. 5.35c). The deletion in wild-type allele of heterozygous organism
may produce a mutant phenotype, while the same deletion in one allele of a
homozygous wild-type organism would produce a normal phenotype. And also,
deletion of the centromere results in an acentric chromosome that is lost, usually with
serious or lethal consequences.
Deletion can be caused by the loss of bases during translocation, during unequal
crossing over or during chromosomal crossovers within a chromosomal inversion.
Deletions are responsible for an array of genetic disorders, and small deletions are
less likely to be fatal; large deletions are often fatal depending on which gene is lost,
whereas medium-sized deletion led to recognisable human disorders, e.g. Williams
syndrome. Deletion of part of the short arm of chromosome 5 results in cri-du-chat
syndrome. Deletion in the SMA-encoding gene causes spinal muscular atrophy. And
some deletions are highly conserved (hCONDELs) which are responsible for evolu-
tionary differences present among closely related species.

5.10.2 Duplications

Duplication is attachment of a chromosomal fragment resulting in the addition of

one or more genes to a chromosome. This was first observed by Bridges in 1919.
Whenever there is a duplication in a chromosome, there is a corresponding deletion
in another chromosome. The origin of duplication is due to unequal crossing over
during meiosis. This type of aberration also belongs to the category of unbalanced
rearrangements as a segment of a chromosome is repeated. The segment can be
repeated once or several times; the orientation of the repeat may be the same as the
original segment (tandem repeat) or it may be in the opposite orientation (inverted
duplication). The known human disorder is Charcot-Marie-Tooth disease type 1A
286 D. Patwardhan et al.

Fig. 5.35 Origins of (a) terminal deletion and (b) intercalary deletion. (c) For the formation of
synapsis which occurs between a chromosome with a normal homologue and a large intercalary
deficiency. The normal homologue having unpaired region must loop out of the linear structure into
a compensation loop or deletion

which is caused by duplication of the gene encoding peripheral myelin protein

22 (PMP22) on chromosome 17.
The duplicated segment of a chromosome is present in another chromosome of
the genome. Duplication (inter-chromosomal duplication) occurs in two forms: one
5 Study of Chromosome 287

a b c d e f g e f g h i

Normal chromosome

l m n o p q e f g
A

a b c d e f g h i
B

e f g

Interchromosomal Duplication

a b e f g c d e f g h i
C

a b c d e f g h e f g i
D

a b c d e f g e f g h i
E

a b c d e f g g f e h i
F
Intrachromosomal Duplication

Fig. 5.36 Diagrammatic representation of some of the possible duplication types. (a) Duplicated
segment is present in a non-homologous chromosome. (b) Duplicated segment is present separately
as an acentric region. (c) Duplicated segment is present in the other arm of the same chromosome.
(d) Duplicated segment is present in the same arm but is removed from the original segment. (e)
Direct tandem duplication. (f) Reverse tandem duplication

is the segment of a chromosome which is duplicated is incorporated into a

non-homologous chromosome, and other is the duplicated segment of a chromo-
some is present as a separate chromosome and the duplicated chromosome segment
remains in the same chromosome (intra-chromosomal duplication) present either in
the different arm or in the same arm but removed from the original segment
(Fig. 5.36). If the segment remains in the same arm and next to the original segment,
it is known as tandem duplication which results in direct tandem (gene order of the
duplicated segments the same as that of the original segment) and reverse tandem
(gene order of the duplicated segment is inverted).
The duplication was extensively studied in Drosophila, B locus (barred eye) of
the X chromosome. This is the condition where the eye is narrower as compared to
the normal eye shape. This is due to a part of the chromosome that is duplicated. The
region of duplication is 16A (X chromosome).
288 D. Patwardhan et al.

a D

F
15 16A
16 16A
A 16A
16A
16A
B
Bar Bar - double
C
Normal
(wild type)

Wild type Homozygous Homozygous

(779 facets) Bar (68 facts) double Bar
(25 facets)

Fig. 5.37 (a) Ultra-bar formation from bar due to unequal crossover. (b) Position effect. (c)
Duplication in the normal eye. Bar eye and a double bar eye (corresponding with chromosomal
segment)

Individuals with barred eye develop ultra-bar (UB) (16A,16A (B)-16A,16A,16A)

and normal wild form due to unequal crossing over (Fig. 5.37a, b). The different
phenotypes in the homozygous bar and heterozygous UB individuals and each
casing segment of 16A remain the same. And this is known as the position effect
(Fig. 5.37c).

5.10.3 Inversion

Inversion shows the reverse gene order of a chromosome. Inversion is created when
part of the chromosome turns 180 , detached or reinserted in such a manner that the
genes are reversed in order. During meiotic prophase and chromosome break,
entanglements of threads are formed which are presumable inversions. For instance,
the segment is broken at two random places, and the two breaks can be nearby due to
the formation of a loop in the chromosome. When they rejoin, the wrong ends get
5 Study of Chromosome 289

Fig. 5.38 Inversion

connected. One side of the loop connects with a different broken end from the one
which is originally connected by joining the remaining two broken ends resulting in
inverted or turned around loops.
During meiosis pairing between inverted chromosome and non-inverted (known
as inversion heterozygous) chromosome results in the formation of inversion loop, if
crossing over occurs within inversion loop, producing abnormal chromatids. If
crossing over does not occur, homologues will segregate and form normal and two
inverted chromatids. The mechanism is depicted in Fig. 5.38. Inversions are usually
of two types: paracentric inversion (where inverted segments do not include centro-
mere) and pericentric inversion (inverted segments include centromere). In a
paracentric inversion, due to single crossover, the odd number of crossing over in
inverted region forms dicentric (two centromeres) chromosome and acentric
(no centromere) chromosome. This results in two chromatids: one carrying inversion
and the other remaining normal. This formation can be observed during anaphase I in
the form of a fragment and bridge (Fig. 5.39). Deficiency and duplication are due to
crossing over between inverted segments. However, in pericentric inversion, the
formation of pachytene is very similar to paracentric but differs during crossing over
and configuration at the meiosis stage. Deficiency and duplication are present in two
of the four chromatids. Due to this, gametes involving chromosomes do not function
and result in gametic or zygotic lethality. (Fig. 5.40).

5.10.4 Translocation

A section of one chromosome is transferred to a non-homologous chromosome

which is defined as translocation. If there is an exchange of a segment or part
290 D. Patwardhan et al.

Fig. 5.39 Paracentric inversion and inversion during metaphase

Fig. 5.40 Paracentric inversion, acentric fragment and dicentric bridge at anaphase I and result of
double cross
5 Study of Chromosome 291

Fig. 5.41 Drosophila sex

chromosome showing
translocation

between non-homologous chromosomes, then it is known as reciprocal transloca-

tion. In this, there is no loss or addition of chromosomes. It was first observed in
Drosophila having an unusual behaviour of a second chromosome gene (pale). The
presence of a pale gene in the homozygous condition is lethal. Bridges studied that
the presence of one gene on the third chromosome can suppress the pale gene which
is also lethal in the homologous chromosome. Stern in the year 1926 studied
translocation between some allele on the ‘Y’ chromosome to the ‘X’ (Fig. 5.41).
A chromosome having a single break and gets transferred onto the other chromo-
some end is known as simple translocation. Chromosome involves three breaks so
that any two-break section of a chromosome can get inserted within the break
developed in a chromosome that is non-homologous, and this shows shift or
intercalary translocation. However, in reciprocal translocation, if there is a single
break in homologous chromosome, an interchange of a chromosome segment
between them develops (Fig. 5.42).
In homozygous condition, translocation forms an equal number of homologous
pairs the same as the normal homozygous if centromere is not lost, but in heterozy-
gous translocation, the result of pairing during meiosis is different (Fig. 5.43a).
Reciprocal translocation results in four chromosome sets at each pachytene stage,
and the formation of chiasmata between the chromosome forms quadrivalent gets
disjoined in three different patterns of segregation in I meiotic division (Fig. 5.43b):
alternate segregation, where non-homologous alternate chromosomes move to the
same pole in zigzag/alternative pattern so that translocated and non-translocated
chromosomes are present in different gametes with no duplication or deficiency;
adjacent I segregation, where adjacent non-homologous chromosome moves to the
same pole and each gamete contains non-translocated and translocated chromosome,
and each gamete shows duplication and deficiency; and adjacent II segregation,
which involves the movement of the adjacent centromeres to the same pole but these
are now said to be homologous containing translocated and non-translocated
chromosomes showing supplication and deficiency producing unbalanced gene
components: unbalanced gamete formation in adjacent I and II segregation. The
out-turn of these segregations inhibits the independent assortment between genes
and non-homologous chromosomes due to the absence of deficiency and duplication
(neither of single mutant phenotypes) in offspring.
292 D. Patwardhan et al.

Fig. 5.42 Various forms of

translocation showing simple,
shift and reciprocal
translocation

5.11 Fragile Site: Chromosomal Breakage

Fragile sites are constrictions, gaps or breaks on the chromosome in metaphase that
develop when cells are subjected to perturbation of the deoxyribonucleic acid (DNA)
replication stage (Fig. 5.44). The fragile site can be seen in all human chromosomes
and named based on the band patterns (e.g. fra(X) (q27.3) was the first fragile site
found on the X chromosome). Fragile sites are categorised as rare or common. Rare
fragile site is present in very small portion of the total population having a maximal
5 Study of Chromosome 293

Fig. 5.43 (a) Translocation condition in homozygous and heterozygous forms. (b) Three different
segregation patterns in translocation heterozygotes

frequency (1/20); the common fragile site is commonly present in every individual.
There are around 89 common and 30 rare fragile sites that have been identified.
Rare fragile site FRAXA which is associated with fragile X syndrome is the
common defect of hereditary mental disability. It is caused by the mutation of a
single gene (FMR-1 fragile mental retardation), located on the X chromosome.
Normally, gene consists of a sequence CGG which is repeated 30 times in a row.
However, in fragile X syndrome, these three sequences are repeated around
300 times creating a microscopic gap on the length of the chromosome X. FMR-1
gene instructs the production of FMRP (fragile X mental retardation 1 protein) which
is involved in the normal development of the brain (development of synapse,
specialised connections between nerve cells). Mutation of the FMR-1 (gets switched
off) results in blocking the synthesis of the protein by preventing normal brain
development.
Fragile genes like FRA-3B, FHIT, WWO-X and FRA-16D are large tumour
suppressor genes. Deletion at breakpoints within the fragile site (Fig. 5.45) is
associated with many types of cancers (breast, lung and gastric cancers).
294 D. Patwardhan et al.

Fig. 5.44 Karyogram showing fragile site in chromosome 16q21

Fig. 5.45 Illustrative representation of normal and fragile X chromosomes

5 Study of Chromosome 295

MicroRNA genes are commonly present at the fragile site which is involved in
the alteration of the chromosome. Additionally, diseases like hepatitis B (HBV) and
HPV-16 (human papillomavirus-16) are found to interact most likely around fragile
sites which are described as the crucial significance for the development of cancer
tumours. Various forms of syndromes, e.g. Jacobsen syndrome, breakage near or at
FRA-11B (a part of the long arm of chromosome 11 is lost), and Seckel syndrome, a
rare genetic disease due to presence of low levels of ATR, show a higher level of
instability of chromosome at fragile site.

Box 5.1
Anaphase is the stage where condensation of the mitotic chromosome is
important for the segregation of sister chromatids. Condensing and topoisom-
erase II (ATP hydrolysing enzymes) combination plays an important role in
the reorganisation of chromatin involving the active assembling of
chromosomes. Condensation of mitotic chromosomes involves a process of
interphase chromatin conversion into rod-shaped structures by three different
steps—condensation, individualisation and resolution. These are different
from the condensation of heterochromatin and apoptotic condensation. The
basic unit of chromatin folding is the nucleosomes as oligomers of histones on
the DNA fibre that generate six- to sevenfold compaction by the coiling or
folding of 30 nm fibre into a zigzag, solenoid or crossed fibre. To attain around
10,000–20,000 linear compactions of mitotic chromosome, 200–500
compactions of around 30 nm fibre during the mitotic condition are necessarily
a must. Chromosome scaffold which is formed by DNA or protein (the
backbone of the chromosome) determines the rod-shaped structure of the
chromosome. In many models, two neighbouring elements bind to a scaffold
even if they are separated by up to 100 kb of DNA forming a loop with the
intervening DNA. The scaffold attachment region is a DNA sequence that
functions as a cis-acting sequence in chromosome assembly. This DNA
sequence is AT-rich which acts as a binding site of DNA topoisomerase II
(a chromosome scaffold component).
Altering folding patterns of a mitotic chromosome is determined using
variant dyes. SMA subunit and topoisomerase II condensing complex are
the major biochemical compositions of the mitotic chromosome. These are
known as topo II-IIα (CAP_B), and five subunit complexes are condensin
(CAP, C, -D2, -E, G and H), chromokinesin (CAP-kip1/D) and chromatin
remodelling ATPase (ISWI CAP-F). Mitotic chromosome fractions are rich in
ATPases. Utilising the energy of ATP hydrolysis and energy utilised for
microtubule-dependent movement of chromosomes induces the g, global
and local conformational changes of chromosomes. Topo II
(ATP-dependent) enzyme that crosses the strand of DNA by cleaving one
strand of DNA via breaking passes the second strand of DNA and releases it.

(continued)
296 D. Patwardhan et al.

Box 5.1 (continued)

Invertebrate cells involve the process of removing catenated DNA between
various chromosomes and convert each chromosome into a single mobile unit.
Topo II association with the condensing complex is essential for the compac-
tion of the chromosome. Cytologically identifiable pairs of sister chromatids
are formed by the decatenation activity of topo II in prometaphase and are
completely separated in anaphase. Throughout the cell cycle, the amount of
topo II on chromosomes varies which is maximum in prometaphase and
minimum after telophase. A large protein complex, condensin, is one of the
profuse structural components of mitotic chromosomes.
SMC2 and SMC4 are the two core subunits of this complex, which by an
antiparallel coiled-coil interaction gets folded intramolecularly, and at their
hinge domains, they dimerise to form a V-shaped structure. An ATP binding
catalytic domain is formed at each end of the SMC dimer by the folding of the
SMC subunit which allows the association of N-terminal and C-terminal
sequences. CAP-D2, -G and -H are the three non-SMC subunits that bind to
one or both of the catalytic ends. Supercoiling assay and knotting assay are the
two in vitro assays devised to show that condensin is capable of inducing
super-helical tension in an ATP hydrolysis-dependent manner into DNA. The
super-helical tension model, chiral looping model and protein-protein
interactions that facilitate rod-shaped chromosome formation are the hypothet-
ical models for the condensin contribution to the organisation of the chromo-
some. As observed in eukaryotic organisms so far, with the initiation of
chromosome condensation, phosphorylation of histone H3 takes place at
serine 10 in the prophase of mitosis.
Aurora B, a mitotic kinase, acts as a major physiological kinase that is
responsible for phosphorylation of histone H3; this might contribute to the
sister chromatids’ resolution by possibly enhancing the chromatin fibres’
structural flexibility or by increasing the electrostatic repulsion between the
chromatids. Furthermore, work is necessary to understand the exact role of
phosphorylation of histone H3 in mitotic chromosome dynamics. As we know,
mitotic chromosome condensation is an active process that requires many ATP
hydrolysing enzymes like topo II and the condensin complex; determination of
how these two components contribute to the mitotic chromosome assembly by
their combined action is an important challenge and requires further research
and insights.
5 Study of Chromosome 297

5.12 Summary

• Chromosomes are strands of DNA that are wound around histone proteins and are
coiled and supercoiled to package the DNA in a compact form. Chromosomes
have telomeres at their ends which stabilise the chromosomes. The chromosome
also consists of a centromere where kinetochore complex forms and spindle fibres
get attached during mitosis and meiosis. The sister strands formed after duplica-
tion of a chromosome are also held together at the centromere.
• Humans have 22 pairs of autosomes and 1 pair of sex chromosome. Sex of the
individual depends on the composition of the sex chromosome. In humans, the Y
chromosome contains genes which allow the development of male genitals and
suppress the formation of female genitals during embryonic development. The
presence of Y chromosome therefore determines maleness and absence of it leads
to female development.
• Sutton and Boveri’s independent observations in chromosomal inheritance and
embryonic development led to the suggestion of chromosomal theory of inheri-
tance around 1902. It stated that chromosomes are carrier of genetic material and
are the units involved in Mendelian inheritance. Thomas Morgan and Calvin
Bridge’s work on fruit flies provided undeniable proof for the chromosomal basis
of heredity.
• Mutations in genes present on sex chromosomes lead to sex-linked disorders.
These are called sex linked because the inheritance of these disorders does not
follow Mendelian inheritance and instead occurs preferentially in one of the
sexes. X-linked recessive disorders are more prevalent in males than in females
and show a criss-cross pattern of inheritance.

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Genetic Study of Bacteria
and Bacteriophage 6
Nidhi Sharma

6.1 Bacterial Genetics

Bacterial genomes are made up of a circular chromosome that possesses a single-

stranded DNA (ssDNA) molecule of several million base pairs in total length. A
classic example is E. coli genome that is widely organized in approximately 4.6
million base pairs of DNAs. No wonder that some bacteria contain multiple
chromosomes, for example, Vibrio cholerae that causes cholera has two circular
chromosomes and Rhizobium meliloti has three chromosomes. Apart from this
composition, there are few bacteria that do not carry circular DNA and instead
carry a linear chromosome. In addition to its own genome, the bacterial genome also
possesses an additional structure known as “plasmid”—a small circular DNA
molecule present in many numbers (usually known as copy number). Plasmids
accommodate genes that are not essential at the functional point of view for bacteria
but that might have been involved as an important factor for the life cycle and growth
of their bacterial hosts. Some plasmids are involved in the mating process between
bacteria (provide a channel for the exchange of genetic material during mating,
which will be discussed in the next section). Some plasmid leads to an important role
in the generation of antibiotic resistance among the population. Most plasmids are
circular and of several thousand base pairs in length and, however, some of small
size; about a hundred base pairs in the length have been found. Each plasmid
structure carries at least an origin of replication (ori), a start point for DNA replica-
tion. The main function of ori is to let the plasmid replicate independently without
involving a bacterial chromosome. Episomes are plasmids that are capable of freely
replicating and integrating into the bacterial chromosomes. Episomes are
categorized into several types based on importance and functionality. The F (fertil-
ity) factor of E. coli is one type of episome found robustly. F factor basically

N. Sharma (*)
La Sapienza University of Rome, Rome, Italy

# The Author(s), under exclusive license to Springer Nature Singapore Pte 299
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_6
300 N. Sharma

regulates the mating and gene exchange between E. coli cells (which will be
discussed in the next section). In this part, we have focused on the bacterial mutant
gene mutation process that contributes to genetic variation in the bacterial popula-
tion. We begin with an overview of the chemical nature of the mutation in bacterial
genetics and its effect at both molecular and organismal levels.

6.1.1 Bacterial Mutant Genetics

In microbiology, studying mutants is an important part. The study of mutants

requires a vast knowledge of detection methods; geneticists should be able to find
them out quickly even when they are rare and should be able to isolate them
efficiently from wild type or least interested other mutants. What are the mutants?
The bacterial population is the most important tool in genetics and biotechnology,
and scientists have observed so many unusual new bacterial strains and are still
observing to date. Every time new strain discovery is an outcome of occurrence of
mutation ever so often. A slight variation in the genome of bacterial strains within a
colony results in the rise of new features in the colonies and appears as different from
its wild type and known as mutants.
Geneticist increases the likelihood of obtaining mutants by using mutagens to
increase the rate of mutation from the usual one mutant per 107 to 1011 cells to about
one per 103 to 106 cells. To study bacterial mutants, we should know the selection,
isolation, and detection method that is described in the following section.

6.1.1.1 Mutant Detection

When it comes to detecting and collecting a mutant of a particular organism, one
should know the wild-type characteristic of that organism to recognize the difference
between a mutant and a wild type, and thus detection of mutants has been evolved
along with the time as the necessity in the field of bacterial genetics. Mutation in
prokaryotes is easy to detect even if it is a recessive mutation because it has been
seen immediately and onward. A very simple example of albino mutants, which is a
type of mutant of pigmented bacterium, can be visualized by observing the color
differences of colonies. This mutation simply requires color detection. On the other
hand, there are other detection methods available that are more complex. Replica
plating method is one of them and is used for the detection of auxotrophic mutants,
which has been explained in Fig. 6.1. This method distinguishes a mutant from the
wild-type strain. Auxotrophic mutants are able to grow in the deficiency of particular
biosynthetic end product that is necessary for an efficient growth of cells.

6.1.1.2 Mutant Selection

Specific incubation conditions can be a tool for the selection of specific mutants that
can only grow under such conditions in which wild type will not be able to grow.
Thus, this is an effective method for mutant selection. What exactly mutants do to a
wild-type strain and what makes them grow in a specific condition? A selection
method will either develop a reversion mutation (a mutation that restores the wild-
6 Genetic Study of Bacteria and Bacteriophage 301

Fig. 6.1 Isolation method for bacterial mutants from the culture through replica plating method.
Replica method for separation of bacterial mutants from the culture. Replica plating is popular for
the isolation and detection of lysine auxotrophs. Auxotrophic mutants can be easily generated by
using a mutagen. Both wild type and auxotroph are cultured together and plated on the complete
medium. The culture containing wild type and auxotroph is plated on a complete medium.
Immediate to colony formation, a soft velvet cloth is pressed on the culture plate, and bacterial
colonies are picked by this piece of cloth. This traced colonies are transferred to another culture
plate of minimal media (lacking Lysin) and in the same orientation as the master plate is Location of
302 N. Sharma

type phenomenon if a wild-type strain is already mutated in the forward direction) or

develop a resistance to the environmental stress. For example, if the purpose is to
isolate a revertant (a strain that regains the former capability) from lac Z
auxotroph,
then the method would be simple. Let us assume that a huge population of lac Z
auxotrophs is plated on the minimal media lacking the lactose, incubated, and
examined for selection. In this case, only cells that have mutated which can regain
the ability to grow on lactose will grow on the minimal media (Fig. 6.2). Out of this
experiment, we can suggest that millions of strains can be plated, grown, and isolated
on a single Petri dish, but those revertants that have the ability to manufacture the
nutrient source lacking in the medium will grow selectively. Thus, this method can
be a selective technique to test for mutation by a mere scanning of a single Petri dish.
This method has successfully accounted for screening many environmental
substances containing mutagenicity.
The selection method for mutants due to environmental stress is more likely
similar to the previous approach that has been used for auxotrophs. It is known that
wild-type strains may be susceptible to any environmental stress like virus (phage)
attack or antibiotic treatment. Thus, plating a phage-sensitive wild-type bacterium on
the minimal media produced colonies that are resistant and have become phage-
mutant. This type of selection method is useful for many other environmental stress
conditions, that is, virus attack, temperature, antibiotics, etc.
In addition to the abovementioned selection method, several bacterial strains
utilize few specific substrates to survive. Not all bacteria use all the nutrient
substrates present in the growth medium. Many bacteria just use a few primary
carbon sources rather than the complete ones. In this case, bacteria can isolate by
plating on the Petri dish containing an alternate carbon source. If colonies appear on
the plate, this strain will be considered a mutant for that specific substrate being
utilized in the culture for its survival.
The mutant selection method has eventually become useful for understanding the
complexity of genes and biochemistry of a particular bacterial strain. Particularly,
this technique is a significant tool for understanding the role of carcinogens in
developing mutagenicity.

6.1.2 Spontaneous Mutation in Bacteria

Spontaneous mutation allows a DNA to be mutated without any exogenous factor or

selective pressure such as UV rays or chemicals; instead, endogenous factors are the
primary sources, that is, DNA damage, error in DNA replication machinery, less
effective DNA repair machinery, and so on.

Fig. 6.1 (continued) auxotroph bacteria which must not grow on second plate and can compare to
the master plate to spot the lysin mutant. This bacterium can be picked from the master plate and can
further grow as a single culture of lysine auxotroph
6 Genetic Study of Bacteria and Bacteriophage 303

Fig. 6.2 Production and

selection method for
auxotrophic revertants
(mutants). In this example, lac
Z revertants are selected after
plating a lac Z
auxotroph
because the agar contains a
minimal medium that does not
support auxotroph growth

Foster and her colleagues in 2012 have demonstrated the mutation rate in three
different strains of very primitive and common bacteria E. coli by using the whole-
genome sequencing (WGS) approach. They explained in their work that the muta-
tion rate of 1–2  10
3 mutation per generation and per genome is natural for this
bacterium which has not been induced by any external factors. Experimentally, they
observed that at any given defined medium or growth condition, the mutation rate of
any specific gene remains constant. Interestingly, if a small inoculum that may
contain few mutants will be transferred to a culture medium, then the proportion
of mutants in that growing culture is positively correlated with the progressively
increased bacterial population. From the historical point of view, spontaneous
mutation was first time spotted by “Salvador Luria and Max Delbrück” in 1943. In
their experiment, they have found that when the E. coli was plated on the nutrient
medium in the presence of T1 phage, it appears as a phage-resistant mutant before
phage display on the plate. It means the E. coli observed on the plate were resistant to
the phage attack, which might be the result of a spontaneous mutation. This
discovery brought us the understanding of genetic diversity in the bacterial genome
as a result of spontaneous mutation. Lately, this understanding has widened by
including another piece of information based on spontaneous mutation which
304 N. Sharma

suggested that the occurrence of genetic changes due to spontaneous mutations is

essential for developing antibiotic-resistant strains, host evasion, and acclimatization
to the new environment, thus contributing to the bacterial evolution. Interestingly, a
recent study from Tomasetti, C et al. has revealed that random mutagenesis in
somatic cells of some tissue causes an error in DNA replication which transformed
the normal cells into cancerous cells compared to other cells from different tissue at
the same time. This study has underlined the role of spontaneous mutation in cancer
risk; however, this study has been the reason for debate as well.
How can mutations in bacteria occur spontaneously? What is the method to
detect them? The fluctuation test is one of the experimental techniques widely
used for its detection. As we have discussed the Luria and Delbrück experiments
where they have explained that if a mutation occurs spontaneously, then the inci-
dence of the mutation at a different time in a different culture would be higher which
led to the variation in resistant colonies throughout the cultures. This concept is
known as the “fluctuation hypothesis.” In this experiment, small inocula were grown
in the culture medium, and the number of phage-resistant E. coli from this culture
was measured and compared with other grown multiple E. coli colonies. According
to the spontaneous hypothesis, we can understand two conditions:

1. If the phage resistance mutation occurs after exposure to the phage, then the
number of phage-resistant mutants and mutation rate should be similar between
the colonies of both sets of conditions.
2. On the other hand, if the mutation is spontaneous and occurs before the exposure
to the phage, then the variability in the mutation for all the independently grown
cultures would be highest because the difference in the size of the bacterial
population which received the mutation at first will contribute to the overall
observed variability till the end of generations. The data indicated that the
mutations to phage resistance in E. coli occurred spontaneously with a constant
probability per cell division.

Let’s think about what could be those reasons governing spontaneous mutation?
Spontaneous mutation does not need any exposure to external agents besides a mere
error in DNA replication, or base substitution could bring the results as a spontane-
ous mutation. One of the reasons being considered is a malfunction in the machinery
of DNA polymerase III (an enzyme associated with replication) during DNA
synthesis. Adding, mispairing, or omitting a nucleotide on a parent DNA strand
will appear as a mutated granddaughter DNA strand and thus lead to mutation in the
continuity. In addition, some mobile genetic materials like transposons are also
considered as the source for spontaneous mutation in the bacteria where these
elements are present other than the nuclear chromosome.
Replication error appears when the nitrogenous base of a template nucleotide
exists in a rare form such as tautomeric form. Tautomerization is a chemical process
in which natural forms of nucleotide bases are (keto(C¼O) and amino (C¼NH2))
converted into two rare structural isomers (imino (C¼NH) and enol (C¼OH)). These
isomers of nucleotide base pairs make nonconventional hydrogen bonding and can
6 Genetic Study of Bacteria and Bacteriophage 305

Fig. 6.3 Schematic diagram of base pairs undergoing tautomerization. Normally the keto form of
the base formed a normal hydrogen bond-like A–T and C–G, but the enol tautomer instead produces
A–C and G–T base pairs. The upper lane shows the normal pattern of A–T and C–G pairing, while
the lower lane shows the rare bonding between (1) imino form of adenine and cytosine and (2) enol
form of guanine and thymine

readily change to each other (Fig. 6.3). Thus, tautomeric shifts change the
characteristics of the hydrogen bond among the four bases. In turn, this shift allows
purine to bind with purine and pyrimidine to pyrimidine instead of pyrimidine–
purine binding and eventually generates an alteration in the nucleotide sequence of
the daughter strand after the first round of replication.
Such mutation based on tautomerization, known as transition mutation, is
relatively common to be found. On the other hand, transversion mutation is
another type of mutation in which purine may substitute to a pyrimidine and vice
versa, but this mutation is not frequent due to stearic hindrance in the pairing of
purine to purine and pyrimidine to pyrimidine.
Replication error also occurs when a purine and pyrimidine base leads to apurinic
(loss of purine from the nucleotide sequence) or apyrimidinic (loss of pyrimidine
306 N. Sharma

base from the nucleotide sequence) sites in the sequence. Spontaneous loss of purine
or pyrimidine base due to hydrolytic cleavage of n-glycosylic bonds with sugar
moiety forms lesions, and thus polymerase enzyme is unable to synthesize comple-
mentary nucleotide on this site which leads to mutation.
Redox attack on guanine base results in the conversion of guanine to
8-hydroxydeoxyguanosine (8-OhdG). This modified base ultimately pairs with
adenine instead of cytosine and produces G->T transversion in the end, during the
replication.
Although most geneticists believe that spontaneous mutation is a random process
without any induction through external agents, this theory lately in 1988 has been
modified by John Cairns whose theory is an extended version of Luria and
Delbrück’s concept of spontaneous mutation. He stated that mutation does not
happen only spontaneously in growing cells but preferably happens in
nonproliferating cells by giving lethal conditions. In his experiments, he
demonstrated that when mutant E. coli strain was subjected to grow on a complete
medium, it was unable to utilize the lactose as a whole carbon source and interest-
ingly if lactose were continuously added to the culture medium as an only carbon
source, E.coli would more rigorously avoid the lactose to take as a carbon source. In
this case, John found that lactose seemed to induce the mutation which allows the
E. coli to choose the sugar over lactose for a carbon source. This study took us to
interpret that this type of mutation is “adaptive mutation” where bacteria are
supposed to choose the mutation that occurs frequently so that they can adapt to a
better surrounding for their survival.

6.2 Viral Genetics

Every organism such as plants, animal, fungi, and bacteria are susceptible to viral
infection. A virus is a simple replicating machinery that consists of a core nucleic
acid and is protected by a surrounding protein coat usually known as a capsid. Virus
can be categorized based on their shape and size and nucleic acid whether it is a
double-stranded DNA or single-stranded DNA or sometimes a single-
stranded RNA.
Virus has a particular class that infected their specific host, for example, a virus
infected bacteria is called “bacteriophage,” and this phage will not infect plants or
animals directly but will be carried away with bacteria itself to further infect animals
or plants. Phages have been involved in genetic research since the late 1940s. It has
become an essential and advanced research tool in the area of genetic research since
it contains a small and handy genome, reproduces rapidly, and produces a large
number of progenies. The study of the phage genetic system has been employed long
ago because this microorganism plays an important role in human society. In this
section, we will focus on many unique aspects of phage genetics, that is, study of
structure and life cycle of bacteriophage, detection methods for phage infection,
application in genetic research, and so on.
6 Genetic Study of Bacteria and Bacteriophage 307

The Discovery of phage included many efforts and stories that have been made by
several scientists. First time, it has been spotted something vague in the river of India
by Ernest Hanbury Hankin, a British bacteriologist in 1896. He simply reported that
there is something in the water that had antibacterial properties and killed cholera,
but he did not specify his finding. Later in 1915 another British bacteriologist
Frederick Twort discovered that a very small molecule or an unknown thing kills
bacteria in the bacterial culture; he had published his finding, but unfortunately, this
work had been interrupted due to WWI. Later in 1917, Felix d’Herelle discovered a
killing agent for bacteria at Pasteur Institute, France. He actually observed that when
he added a filtrate that was collected from sewage to the bacterial culture of
dysentery, in a few minutes, the culture colonies disappeared, and he named this
filtrate as “invisible antimicrobial agent,” and later this work was published. In the
row of findings, a first-time phage study was developed in the Elvia institute, Tbilisi,
Georgia, in 1923 to study this invisible thing and to develop phage therapy. In 1969,
Max Delbrück, Alfred Hershey, and Salvador Luria discovered the replication of
virus and virus genetics for which they were awarded the Nobel Prize in Physiology
or Medicine.

6.2.1 Structure of Bacteriophage T4

T4 is one of the most extensively studied bacteriophages among all other phages
such as T1–T7. T4 bacteriophage is specific to E. coli and has been demonstrated as
a model for phage study by Delbruck and coworkers in 1944. In the modern time of
genetic engineering, the study of phage included advanced tools and techniques
particularly to understand its structure at the atomic level. Early discoveries on this
bacteriophage have included the prediction of phage image using electron micros-
copy (EM) obtained by Brenner et al. in 1959. This work extended to an extensive
study on phage head symmetry, tail, and baseplates through EM. For the first time, a
complete T4 genome was sequenced in 2003. In the continuity of this work, high-
resolution cryo-electron microscopy (cryo-EM) image was obtained which revealed
a dome-shaped baseplate structure in the infectious virus. In subsequent years, star-
shaped baseplate and prolate head structure of post-infection T4 were published.
Thereafter, many other techniques like complementation assay (to study recombina-
tion in bacteriophage), cross-linking analysis (protein–protein interaction study),
X-ray crystallography, and cryo-EM provide high-resolution atomic-level structure
model for T4 phage. Studies from the structure model reveal the structural
similarities among phage protein and bacterial protein which suggested the common
evolutionary ancestry or coevolution with the bacterial host. Structural similarities
among phages as well as of T4 components with bacterial proteins demonstrate
common evolutionary ancestry or coevolution with bacterial hosts.
Bacteriophage T4 belongs to the family Myoviridae that infects E. coli. A basic
T4 structure includes a head (capsid), tail, and baseplate. Phage has a rigid tail that is
composed of many layers; the inner layer of the tail is surrounded by a contractile
sheath that helps a phage during infection. Phage Myoviridae family like T4 contains
308 N. Sharma

a massive baseplate at the end of the tail with long attached fibers that guide the
phage to find the receptor in the host cell and mediate the initial contact. A contractile
tail is helpful in penetrating the bacterial outer membrane before the DNA delivery
during the infection. Long attached fibers are made of six short tail fibers that are
folded underneath the baseplate and unfold on the recognition of the host as a host
sensor. The baseplate is a puncture device for phage located at the end of the long tail
and the last element located at the end of the tail.
Capsid of T4 phage is assembled with three main components: (1) gp23 (48.7 kD)
forms hexagonal capsid lattice, (2) gp24 forms pentamers of the vertices, and
(3) gp20 forms a unique dodecameric entrance as a portal vertex that makes a
gateway for DNA packaging and exit during the infection. Genetic material of T4
is a linear dsDNA of 168kbp which has 289 open reading frame (ORF).

6.2.1.1 DNA Packaging

DNA is translocated in the capsid through the central channel of portal protein on
one vertex of the capsid. The portal complex at vertex works as a docking point for
terminase; a viral ATPase complex that is bound to the end of the portal vortex
provides an active packaging motor complex that assists the entry of viral DNA into
the capsid. The packaging of DNA is evenly regulated till the threshold amount of
DNA is entered into the capsid. Interestingly Smith et al. in 2001 have reported in
their work that dense packaging of DNA after some point will raise the pressure
inside the capsid approximately equal to 6Mpa. Termination of DNA packaging,
after enough DNA has been densely packed into the capsid, is mediated by bolting of
the active portal complex, which prevents the phage genome leakage. Portal com-
plex on vertex alone is not enough in the packaging machinery; therefore, in
addition, head completion proteins sometimes also called adaptor proteins are
required to bind with the complex dodecameric portal and form the connecter at
the end of the capsid.
If we take a closer look at the symmetry of capsid and tail, a mature or fully
assembled capsid in a phage follows four to fivefold symmetry or so-called icosahe-
dral symmetry. From the structure point of view, each multiple structural unit in the
capsid has been displayed as a regular lattice with two, three, and five rotational
symmetries. Portal protein shows dodecameric oligomerization pattern while tails
show overall six- or threefold rotational symmetries, and protein repeats in this
symmetry are shown in a helical pattern.
Brenner et al. in 1959 successfully illustrated phage image through electron
microscopy (EM).
EM image of mature phage has been described in Fig. 6.4:

1. 1150 Å-long and 850 Å-wide icosahedron head encompassing genomic DNA
2. 925 Å-long and 240 Å-diameter contractile tail attached to the one end of the head
through portal vertex
3. 270 Å-high and 520 Å-diameter hexagonal baseplate
4. 1450 Å-long six long tail fibers attached to baseplate
6 Genetic Study of Bacteria and Bacteriophage 309

In the past decade, advanced techniques such as single-molecule tweezers and

fluorescence studies revealed a few amazing facts about bacteriophage; T4 motor
packages DNA at the rate of 2000 bp/s which is confirmed as the fastest packaging
motor till date. Another fact that FRET-FCS studies on phage suggested that DNA in
phage gets condensed during the translocation process.

6.2.2 Life Cycle of Bacteriophage T4

A phage life begins and ends during the infection period and has been characterized
into two phases: lytic cycle and lysogenic cycle. Lytic cycle is a virulent phase since
it infects a cell, destroys the cell, lyses the cell, and replicates and produces more
phage particles. A lysogenic phage is a temperate phase because it infects the cell
and incorporates dsDNA to the host cell, and no progeny will be produced. In
lysogenic phase, foreign DNA such as viral DNA is incorporated into the bacterial
chromosome and replicates multiple times as the host cells divide. However lyso-
genic phase also undergoes a lytic phase in some circumstances not capable of
undergoing the lysogenic life cycle and therefore directly undergoes the lytic cycle.
Other than lytic and lysogenic, a phage also has been found in pseudolysogeny
life. It is an unfavorable and unusual life cycle that occurs when a phage grows in
unfavorable growth conditions that enable a phage to survive by preserving the
phage genome until the host growth condition becomes advantageous again.
Lytic phase has been categorized as follows:

Fig. 6.4 Assembled mature

bacteriophage T4. The
assembly of T4 can be divided
into three independent
subassemblies: the head, the
tail, and the long tail fibers.
The tail binds to the head
followed by the attachment of
the fiber in the end. Six long
tail fibers are then attached to
form a viable T4 virion
310 N. Sharma

1. Attachment to the bacterial cell and phage infection

2. Passage of DNA through the bacterial cell wall
3. Infected bacterium converted to phage factory
4. Production of phage DNA and protein
5. Assembly of phage
6. Release of phage particle

6.2.3 The Plaque Assay

Plaque assay has been developed to the necessity of phage detection in several
medical conditions. Plaque assay has shown the ability of host cell to transform into
plaque lawns if any bacterial colonies are being infected within the phage. Spot-on
lawn assay has been identified as one of the simplified versions of the plaque assay
which aims to identify potential viral plaques or virus infection in the growing
bacterial culture. To this aim, approximately 1 μL aliquots of virus suspension
would be enough to be applied to fresh lawns of diverse microbial host strains.
For example, if the suspension of a phage (i.e., T4) is applied to the susceptible
bacterial host (E. coli), then phage infects the bacterial cell, replicates independently,
and promotes the lysis of the cell due to the occurrence of lytic phase and kills the
bacteria in the end. This lysed bacterial cell indicates the formation of a clearing zone
on the bacterial lawn known as plaques. In some circumstances where the lytic cycle
is absent, bacterial colonies grow confluently. Any single plaque determines the
number of phages that have infected the single bacteria and further keep infecting
bacteria in the vicinity and develop the new plaque in the vicinity which together
forms a big plaque, enough to be seen with naked eyes. Notably, plaque does not
continue indefinitely, and the size of plaques is totally dependent on the type of
phage, host cell, and condition of grown culture. Measuring the frequency of plaque
formation or detecting the number of plaques formed on the bacterial lawn can be
calculated by the dilution method. If phage suspension can be diluted in a serial
dilution method, thus appropriate dilution factor can be used for calculation of
plaque formation unit by using the following equation:

Average Number of Plaques

Plaque forming unitðPFUÞ =
Dilution factor  volume of diluted virus added

6.2.4 Lysogeny

Lysogeny is another life cycle or reproductive pathway of phage other than the lytic
cycle. The very first evidence of lysogenic behavior of phage is confirmed by two
subsequent experiments in 1920 and 1940 as follows:
6 Genetic Study of Bacteria and Bacteriophage 311

1. In the 1920s, some remarkable results were obtained in the study of phage in the
E. coli culture. Earlier microbial geneticists have performed an experiment in
which they mixed two microbial strains: lysogenic-resistant and non-lysogenic-
resistant strains. Lately, they have found that non-lysogenic-resistant strain was
lysed in the culture. The geneticist explained that this phenomenon happened
because resistant strain somehow causes lysis of nonresistant strain and thus
resistant cell known as lysogeny or lysogens (a causative factor for cell to
lysis). It is further important to understand that sometimes nonlysogenic bacteria
might get infected with phage-derived lysogenic strain, and very few infected
cells were not lysed but rather itself became lysogen.
2. However, in 1940, André Lwoff performed another experiment in which he
studied the lysogenic bacteria Bacillus megaterium and he followed its behavior
and cell divisions in the culture throughout. Once he established the culture, he
separated each daughter progeny after each cell division. From this daughter
progeny, he has put back one cell in a new culture while he followed the other cell
throughout the cell division. In this experiment, he followed 19 cultures that
represent ten consecutive generations. He also separated culture medium and
found no phage existing in the free medium through which he confirmed that
lysogenic behavior of the bacteria is followed by each cell division or each
reproduction in the absence of any external phage in the medium.
On the other hand, he spreads this separated phage-free medium, on the lawn of
non-lysogenic bacteria, and astonishingly, he observed a spontaneous plaque
formation in the culture. This observation he explained in his proposed hypothe-
sis. Lysogenic behavior of phage passes from generation to generation, and this
gives rise to pure noninfective strain but somehow this noninfective factor
converted into infective phage although no free phages presence in the medium.
This event is an exceptional case. In the term of microbiology, Lwoff named this
factor as “prophage” which can change the noninfective factor to infective factor
by chance or in an inductive manner.

Now we know why non-lysogenic-resistant cells have been found to be infected

and lysed in the culture because prophage, which is a feature of lysogenic bacteria,
eventually produced infective phage into the medium that causes the cells to lyse.
A common lysogenic cycle can be described in few steps (Fig. 6.5):

1. Linear phage DNA first injected to the host cell.

2. Initially phage mRNA will synthesize for short period, followed by synthesis of
(a) repressor protein that inhibits the synthesis of the enzyme needed for lytic
cycle and (b) site-specific recombination enzyme. Later, phage mRNA synthesis
is turned off by repressor protein.
3. Phage DNA molecule incorporated into the bacterial chromosome.
4. As bacteria replicate and grow in the medium, phage DNA also multiplies and
produces more phage progeny as part of the bacterial chromosome.
312 N. Sharma

Chromosome
Circularization replication; Cell
Integration Division
of Phage DNA

Many Lysogens
Transcription Produced
Injection
repression

Fig. 6.5 A lysogeny phase after immediate to virus infection in the host cell. Infection of phage
initiates circularization of phage DNA at first, followed by integration with bacterial chromosome
and replication. Cell division reproduces more numbers of bacterial cells containing integrated
phage DNA which accomplishes the lysogenic cycle of any phage

6.2.5 T4 Phage Modulates Bacterial Genetics

Other than its characteristic to invade bacterial hosts, T4 phage infection has been the
subject of much scientific research to how phage has an impact on the bacterial
genome. Such phenomenon can take place by either generalized transduction or
introduction of phage-encoded protein whose expression results in changes of their
host phenotype and activity. Phages have acquired these genes from their host and
continued to evolve and change within its own genome. These extra genes can be
named as “accessory genes” that can govern the biology of their bacterial host and
find the tune in the way in which bacteria interact with their environments. Such
observation has been possible by our ability to sequence phage genomes, and this
information will serve as a start point for further study to determine how phage
infection can contribute to their bacterial host’s physiology endurance and evalua-
tion. In many ways, phage affects the bacterial genome or phenotype, and few
examples are given below:

6.2.5.1 Human Gut Microbiome Interaction

Human gut microbiome is another example of phage–bacteria interactions which
consist of densely colonizing microorganisms including T4 phage and interacting
with mammalian hosts. Metabolome data from several research on the role of phage
in the gut microbiome suggested that T4 phage remains as a modulator of bacterial
colonization in the human gut and supports the benefits out of this interaction to
human health.

6.2.5.2 Host Communication

T4 phage further manipulates bacteria by accessing quorum sensing pathways which
have effects on one or multiple behaviors of bacteria as shown in (Fig. 6.6). Genomic
study of phage reveals that phage DNA sequence contains homologs of response
regulator that is involved in quorum-sensing pathway. Also, it has been noticed that
induction and release of temperate phages have been observed following the N-acyl
homoserine lactone exposure (LuxS system of quorum-sensing pathway).
6 Genetic Study of Bacteria and Bacteriophage 313

Fig. 6.6 T4 phage modulates bacterial genetics in several ways. After insertion of phage into a
bacterial DNA, it further regulates several signaling in the bacterial cell which are essential for
bacterial life cycle, stress responses, replication, metabolism, and so on

6.2.5.3 Host Replication

Phage uses bacterial machinery to manipulate its own replication and host replica-
tion system, and phages manipulating host replication can be seen in the dimorphic
bacterium Caulobacter crescentus. Moreover, another example of phages
influencing their host’s replicative process is the phage-encoded homologs of
MazG. MazG is a regulator of cell death in E. coli, and its expression influences
bacterial replication in nutrient-limited environments. Interestingly, homologs of
MazG have been found in phages infecting several diverse bacterial species, includ-
ing several cyanophages, Burkholderia cenocepacia phages, and Mycobacterium
phage L5.

6.2.5.4 Host Metabolism and Energy

Many phages that invade bacterial cells also control its metabolic regulation, such as
carbon, nitrogen, and phosphate metabolism, and its utilization. Cyanophage is one
example of such kind of modulation by phage in the host cell.

6.2.6 CRISPR/Cas9 Bacteria in Genetic Engineering

In the past decades, an endogenous machinery of bacteria/archaea has been used as a

powerful tool in genome editing (called gene editing) technology which allows
genetic material to be added, removed, or altered at particular locations in the
314 N. Sharma

genome. To this purpose, several techniques have been developed, and the very
recent one is known as CRISPR/Cas9. CRISPR-Cas9 stands for clustered regularly
interspaced short palindromic repeats and CRISPR-associated protein 9. Interest-
ingly, CRISPR/Cas9 was adapted from a naturally occurring genome editing
machinery in bacteria. Just like us, bacterial cells can be invaded by viruses, and
in response to defense against the virus, the bacterial CRISPR immune system can
thwart the attack by destroying the genome of the invading virus.
Interspersed between the short DNA repeats of bacterial CRISPRs are similarly
short variable sequences called spacers that are derived from viral DNA that have
attacked bacteria previously. However, this spacer helps bacteria to recognize the
viral genome of its attacks again, and CRISPR (Fig. 6.7) defense system will cut up
any viral DNA matching the spacer sequence. Thus, these spacers are termed
“genetic memory.”
Genetic manipulation such as gene insertion or gene overexpression is very well
established; however, inhibition or abrogation of a particular gene is quite challeng-
ing until CRISPR/Cas9 came into existence in the last decade. CRISPR/Cas9 system
has been categorized into types I, II, and III, of which type II is the most successful
and widely used in genome editing since it requires one enzyme and one RNA to
function as a DNA endonuclease. Moreover, the RNA components of the CRISPR/
Cas9 system can be used separately by fusing the crRNA (mature CRISPR RNA) to

Fig. 6.7 CRISPR-mediated gene editing: CRISPR are regions in the bacterial genome that help
participate in the defense against invading viral genome. These regions are composed of short DNA
repeats (black diamond) and spacer (colored boxes). When a new virus infects a bacterium, a new
spacer is generated by the viral genome and incorporated among existing spacers of CRISPR.
CRISPR is transcribed and processed into short CRISSPR RNA molecule. This CRISPR RNA
guides bacterial molecular machinery to a matching target sequence in the invading virus. The
molecular machinery cuts and destroys the invading viral genome
6 Genetic Study of Bacteria and Bacteriophage 315

the tracrRNA (trans-activating CRISPR RNA) generating a single guide RNA that
recruits the Cas9 nuclease to specific genomic locations via standard Watson–Crick
base pairing and facilitates double-strand break. The creation of site-specific double-
strand breaks by the CRISPR/Cas9 complex then triggers genome editing through
two different mechanisms: (1) repair through homologous recombination and
(2) nonhomologous end joining. Notably, both pathways lead to functional inactiva-
tion of targeted genes with high efficiency, and thus CRISPR/Cas9 methodology has
rapidly become the state-of-the-art technique for genetic manipulation of mamma-
lian cells and genetically modified mice and has the potential to be used in a diverse
range of gene therapy approaches in the future. Generation of the knockout mouse
model for many disease studies has been possible by using adenovirus (AVV)-
associated CRISPR/Cas9 system in recent years.

6.2.7 Application of CRISPR/Cas9

CRISPR/Cas9 is a simple and rapid tool that enables the efficient modification of
endogenous genes in various species and cell types. A number of clinical trials using
CRISPR/Cas9 system for genome editing are underway, and the first clinical trial
involving CRISPR/Cas9-mediated gene modification has started in October 2016 at
West China Hospital, Chengdu. CRISPR/Cas9 complex is nowadays an easy tool for
many therapeutic approaches such as for immunotherapy in lung cancer, HIV, beta-
thalassemia, Duchenne muscular dystrophy, hepatitis B virus (HBV) infection, and
so on. CRISPR/Cas9 system is used in the current scenario and its applications are
listed in Table 6.1.

6.3 Conjugation

6.3.1 Discovery of Conjugation

For many years it was thought that bacterial reproduction is only done by simple
binary fission that splits a bacterial cell into two identical daughter cells excluding
the exchange or recombination of genetic material. The very first evidence of
exchanging the genetic material within the bacterial population was “conjuga-
tion”—a method of DNA transfer mediated by direct cell-to-cell contact. This result
became part of the knowledge from a subsequent series of experiments conducted by
Joshua Lederberg and Edward Tatum in 1946 (reported in Nature and the Journal of
Bacteriology (JB) in 1946 and 1947). In this experiment, two auxotrophic strains
were first selected and mixed which is further followed by incubation, culturing into
a nutrient medium for many long hours, and plated on the minimal medium. Later,
they observed a recombinant prototrophic colony on the minimal medium which has
an incorporated recombination chromosome in each cell. Thus, this experiment
suggested that the chromosome of two auxotrophs can associate with each other
and undergo the recombination process (Fig. 6.8).
316 N. Sharma

Table 6.1 CRISPR/Cas9 implementation and its applications in medical science

CRISPR/Cas9 use Application
1. CRISPR/CAS9 delivery to edit the mutation in Gene editing in mouse type I
fumarylacetoacetate hydrolase (genetic disorder) tyrosinemia model in vivo
2. Gene correction by repairing the mutation in the CFTR Therapeutic
gene for cystic fibrosis (genetic disorder)
3. Duchenne muscular dystrophy (genetic disorder) Therapeutic
4. Removing one or more exons from the mutated Therapeutic
transcript by CRISPR/Cas9 system allowed for the
production of truncated but still functional dystrophin
protein in a mouse model of muscular dystrophy
5. Generating autocatalytic mutations to generate Generating homozygous animal
homozygous loss-of-function mutations model
6. Gene deletion in mouse model to generate particular Knock mouse model
disease study model
7. Injection of CRISPR/Cas9 components into zygote/ Genome editing in human
early stage embryo to modify genetic structure permanently

However, Lederberg and Tatum failed to prove the concept of “physical contact
between cells” which is the major requirement for gene transfer. But in the following
years, 1950, Bernard Davis has demonstrated this gene transfer in “U tube” experi-
ment. Bernard Davis constructed a U tube that contains two pieces of curved tube
fused together at the base to form a “U” shape separated with a piece of fritted glass
filter fixed between halves.
This filter does not allow the passage of bacteria; rather it allows the passage of
the medium. During the incubation time, the medium was pumped back and forth
through the filter to make sure that the medium is thoroughly switching between
halves. After 4 h of incubation, bacteria were plated on the minimal media condition.
And interestingly, Davis observed that when the auxotrophs were separated and cells
were not in contact, the conjugation does not occur which means gene transfer needs
direct contact (Fig. 6.9).
The next question rises to know what component or factor promotes conjugation.
F factor (fertility factor)-associated gene transfer is the most common type of
conjugation in bacteria that will be discussed in the next topic.

6.3.2 Discovery of Fertility Factor (F)

After being given the experimental evidence by Lederberg and Davis for conjuga-
tion, William Hayes in 1953 came up with the idea that genetic transfer occurred
only in one direction in the abovementioned crosses. This is the reason that it has
never been found that gene transfer in E. coli could be in a reciprocal manner. Thus,
one cell must act as a donor, and the other cell must act as the recipient. This
unidirectional gene transfer seemed to be compared with the original sexual differ-
ence between participants, according to which donor cell should be known as “male”
6 Genetic Study of Bacteria and Bacteriophage 317

Fig. 6.8 Experimental setup by Lederberg and Tatum. Tube 1 contains a single auxotroph
population that has met
bio
thr+ leu+ thi+, meaning this bacteria contains the functional gene
only for thr, leu, and thi (amino acid) while methionine and biotin genes were absent and that this
bacteria cannot grow on the minimal media which is lacking all the essential amino acid and biotin
needed for any bacterial growth. In tube 3, another auxotroph population was present containing
met+ bio+, thi
, leu
, thi
which is an opposite composition in tube 1. In tube 2, both populations
were mixed and incubated for 4 h. Later when this population was plated on minimal media from
these three tubes, the population from tube 1 and tube 3 was unable to grow on minimal media,
while mixed population from tube 2 was successfully grown on the minimal media, which shows
that transfer of gene between these two population has occurred somehow which has given the new
mutant colonies containing met+ bio+, thr+, leu+, thi+ genes. This experimental evidence has
proven the concept of gene transfer in the bacterial population

and recipient cell should be known as “female.” Although such gene transfer is only
possible in eukaryotic organisms but not in bacteria and hence conjugation is not a
type of sexual reproduction at all. In bacterial gene transfer, one cell that has to
transfer the gene behaves similar to a donor, and the other cell which is supposed to
receive the donor’s genetic material and change its own genetic makeup behaves
similar to a recipient cell, while sexual reproduction has equally contributed to
donor’s and recipient’s genetic information. Lately, it was discovered that gene
transfer in E. coli through conjugation is eventually driven by one of the circular
DNA plasmids known as fertility factor or “F factor” which is sometimes also called
318 N. Sharma

Fig. 6.9 Bernard Davis U tube experimental setup to prove that bacterial mating or physical
contact is a must for gene transfer through conjugation. When auxotrophic strain A and auxotrophic
strain B were plated on the minimal media and incubated for few hours, no growth was been
observed on the media which confirms that no gene was transferred between the bacteria when they
were separated through the filter paper

as sex factor. F factor is found in some species but not in all bacterial cells. First, we
need to understand the characteristics of the F factor prior to following its role in
conjugation.
The size of F factor varies from few kb to 100 kb in the form of duplex DNA
keeping two distinct replication origin regions (Fig. 6.9). Among these two, the
bigger one is denoted as ori V or vegetative replication region which is a point that
supports the F factor to replicate autonomously in a particular situation when the
plasmid is not being transferred such as cell division of F plasmid; this origin is
bidirectional, whereas ori T is unidirectional and responsible for replication and
transfer of F factor to the recipient cell. F factor shows the similar copy number of a
plasmid as bacterial chromosome shows, and therefore one bacterium has one or two
copies per bacterial chromosome.
The conjugation process is regulated by sex pili or F pili, a thin rod-like structure
that appears as an extension of the cell wall. A protein subunit of pili is pilus coded
by gene tra which polymerizes into pili. Bacteria carrying F plasmid (male or donor)
attach to the recipient bacterial (female or recipient) cells for conjugative transfer. An
F-positive bacterium has 23 pili on the surface and a tra operon encoding
6 Genetic Study of Bacteria and Bacteriophage 319

Fig. 6.10 A physical map of F plasmid (100 kb). This circular DNA is further divided into (1) ori
V which is responsible for autonomous replication, (2) ori T which is the origin of replication and
transfer of F plasmid, (3) tra operon which encodes the functional factor required for conjugation,
and (4) IS3, IS2, and Tn 1000 which are transposable elements. The thin arrow indicates the
direction of replication

30 functional genes that promote the transfer of the F plasmid (Fig. 6.10). Other than
this, F plasmid has three transposable elements incorporated in the structure, in
which two copies are of insertion sequence—IS2 and IS3—and one is transposons
Tn1000 (sometimes known as γδ).
During the experiment, a variant of the F factor has been discovered accidentally
by Hayes. He has observed that a variant from its original donor did not reproduce a
recombinant after crossing with the recipient strain. This observation reflects that
this donor cell apparently had lost the ability of gene transfer and had converted into
recipient-like strain known as “sterile donor.”
320 N. Sharma

Through this analysis, Hayes realized that fertility characteristics (ability to

donate) of E. coli could be silent and recover easily. Thus, he suggested that the
donor ability of F factor shows a hereditary phenomenon.

6.3.3 F+ and F2 Bacteria

As we have discussed in the previous section, gene transfer is done through the
fertility factor (F factor) in a donor cell, designated as F+ bacteria, whereas bacteria
that is a recipient and lacking F factor is designated as F
bacteria.
The F factor includes an origin of replication and genes required for conjugations
as discussed previously (see Fig. 6.10). F+ bacteria produce sex pili (singular known
as pilus) that facilitate a physical contact between F+ and F
to pull them together
(Fig. 6.11). The most important fact about conjugation is that this type of gene
transfer can only take place cells that contain F and cells that lack the F factor. Detail
mechanism is highlighted in Figs. 6.3 and 6.4.

6.3.4 Hfr Bacteria

Hfr (high-frequency recombination) bacteria simply became in existence immedi-

ately after F+  F
mating. It has been suggested that this is the second type of F
bacteria that maintains a higher frequency of conjugation than the F+ bacteria. At
first, Lederberg and Tatum have observed that conjugation between F+ and F

allows the transfer of genetic material in F plasmid but does not account for the
transfer of chromosomal genes. Hfr-mediated conjugation transfers the donor chro-
mosomal gene with a great efficiency into the recipient cell but does not change the

Fig. 6.11 Gene transfer mechanism in F+ and F
cells. (a) F+ is a donor cell that will transfer the F
factor to the F
(recipient cell). (b) A conjugation tube or a bridge begins to happen between these
two cells. (c) Single-stranded DNA is generated by nick at the origin and separates it from the
double-stranded circular DNA. (d) This 50 nicked single-stranded DNA is transferred across the
cells and enters in the recipient cell where single-stranded DNA begins to replicate and convert the
F
into F+ cell. (Benjamin. A. Pierce., Genetics: A conceptual approach)
6 Genetic Study of Bacteria and Bacteriophage 321

Fig. 6.12 Construction of Hfr cell from F+ cell. Integration of F factor to the bacterial chromosome
in F+ cells which converted the F+ cell into Hfr strain containing both the features of plasmid and the
bacterial chromosome. (Benjamin. A. Pierce., Genetics: A conceptual approach)

F
into F+, and this is because of a partial transfer of chromosome into F
which
does not change the cell into F+ unless the entire chromosome has been transferred.
If the F factor is integrated into the chromosome and this chromosome has to be
transferred, the chromosome will require 100 min in case of E. coli, but unfortu-
nately, the conjugation breaks before the process is finished. At last, the F factor is
not completely transferred to the recipient cell, and it remains F2.
Thus, Hfr strains contain F integrated chromosome, and F+ cell further can form
sex pili and conjugate with F
cell (Fig. 6.12).
In the mating between Hfr and F
cells (Fig. 6.13), integrated F first nicked at
the one end on one strand. This nicked end moves toward F
cell similar to the
conjugation between F+ and F
cells. Since the F factor is integrated with the
bacterial chromosome, nick transfer further allows the transfer of chromosomal
fraction into the recipient cell. The amount of transferred chromosomes depends
on the duration of conjugation between the two cells and how long they are
connected.
Once the nicked and single-stranded DNA is transferred to the recipient cell (F
),
it starts to replicate, and sooner the crossing/recombination between the donor and
the recipient chromosome will take place. When the crossing over takes place in the
recipient cell, degradation of the donor chromosome occurs instantly. The recombi-
nant recipient chromosome remains intact in the cell and starts to replicate and pass
to generations. It is already mentioned in the mating between Hfr and F
cells that
F
will not become F+ or Hfr unless the entire F factor (F-integrated bacterial
chromosome) will receive an F
-recipient cell. This event seems to be a rare case
or time-consuming because most of the conjugations last for only a short time and
break cells apart any time before the chromosome could have been transferred.
322 N. Sharma

Fig. 6.13 Mating between Hfr cell and F
cell. Mating takes place between Hfr and F
cells which
have been described in steps (a) to (e). This process is time taking so it occurs rarely in the nature

6.3.5 Mapping of Bacterial Chromosome

Before inventing genome sequencing, microbiologists had only one genetic

approach to elucidate the organization of genes in bacterial chromosomes and that
is linkage analysis. Genetic mapping using linkage analysis is a tedious task for
them. To date, three methods for mapping have been identified: (1) interrupted
conjugation, (2) transformation, and (3) transduction. All of them have important
similarities and differences which are discussed in this section.
In bacterial genetics, to map the relative location of bacterial genes, Hfr conjuga-
tion method has been used more often. In Hfr conjugation, when Hfr and F
are
mixed in one culture, Hfr strain begins the chromosome transfer immediately.
Transfer of chromosome from donor to recipient is not synchronous but occurs at
a constant rate and over a period of minutes. The mapping is done by using an
interrupted conjugation. Within an interrupted conjugation, a bridge between donor
and recipient is broken, and thus matting between Hfr and F
is interrupted at
various intervals. This technique is organized after some time when the conjugation
begins in a mixture and is interrupted by vigorous agitation in a blender (Fig. 6.9).
Through this method, the order of gene transfer and time intervals can be
interpreted because timing is a direct reflection of the gene order on the bacterial
chromosome (Fig. 6.14). In the given graph, X-axis of the curve demonstrates the
time point at which the gene began to enter the recipient cell. The demonstrated
curve is a circular chromosome map where the distance between every gene is equals
the minutes lapsed until the gene is passed to the recipient cell. In the graph, the gene
that is more distant from F factor (origin of replication) shows lower plateau
compared to gene that is in the vicinity of F factor. In the given example of
E. coli, trp is the distant gene from the F factor, and thus there is even a greater
chance that the conjugation bridge will beak spontaneously before the trp gene is
transferred to the cell. To generate the map, it is important to know that the transfer
of genes always starts within the point of origin that is the F and thus the orientation
and position of F on the chromosome determine the direction of the starting point of
6 Genetic Study of Bacteria and Bacteriophage 323

Fig. 6.14 Mapping of the bacterial chromosome by using Hfr and F
mating system. Hfr and F

interrupted conjugation experiment at time intervals. (a) Schematic diagram in the left panel shows
that a linear transfer of genes has been paused and a discontinued conjugation bridge is taken into
the consideration of the sequence of gene transfer from donor to the recipient cell. (b) In the right
panel, the graph shows a relationship between time intervals at which a particular gene has been
transferred into the recipient cell obtained by an interrupted conjugation experiment. From the
graph, we can see that the gene order is lac–tsx–gal–trp

gene transfer. In the given example, the point of origin (F factor) is just immediately
before the lac gene in the chromosome. Since the genome of E. coli is relatively
larger, mapping is quite lengthy through Hfr strain. Therefore, the easy way to do so
is to let the several Hfr strains integrate with F plasmid at different locations, and all
the fractions of the map obtained through these different locations must be
superimposed to create the entire map of E. coli. The overall map is adjusted to
100 min in the case of E. coli. In this sense, the term “minutes” not literally indicates
the measurement of time but the distance between the genes on the map.
Gene mapping through transformation follows a few steps including the separa-
tion of DNA from the donor strain, fragmented and integrated to the recipient strain.
Loci that are widely separated on the fragmented DNA from the donor chromosome
and always carried by two different fragments, then the frequency of
cotransformation is different from the single transformants of per103 recipient
(a normal transformation occurs at the rate of one cell per 103 recipient). If the two
loci are very close to each other and are carried by one fragment, the rate of
cotransformation must be similar to a single transformation rate. Thus, the
cotransformation will provide the information of the order of genes on the donor
chromosome and will guide to map the genome.
Mapping through transduction is quite similar to transformation and also depends
on the gene transfer but between two different bacterial traits. Gene transfer occurs
through the bacteriophage. Similar to the transformation, small fragmented DNA
will be cotransformed by phage from donor to the recipient strain. Rates of
cotransformation in transduction will help to calculate the relative distance between
genes and to create a genomic map.
In conclusion, all the three modes of gene transfer—interrupted conjugation,
transformation, and transduction—are based on the same basic strategies that are
324 N. Sharma

used for mapping. The way of DNA transfer is slightly differing such as through the
physical contact between bacteria in interrupted conjugation with interrupted conju-
gation, small naked fragmented DNA in transformation, and fragmented DNA
through bacteriophage in transduction.
Using these techniques, researchers mapped about 2200 genes of E. coli K12 and
compared this with the actual nucleotide sequence of the genome (i.e., physical map
of the genome). Genome sequencing has revealed about 4300 possible genes. Thus,
genetic analysis is defined over half of the potential genes. The genetic map
approximates the physical map, but they do not correspond perfectly. This is because
the genetic map is derived from genetic linkage frequencies that do not correlate
exactly with the number of nucleotides that separate the two genes. Roughly
speaking, 1 min of the E. coli genetic map corresponds to 40 kilobase of DNA
sequence.

6.3.6 F+ 3 F2 Mating

It was demonstrated by William Hayes in 1952 that the gene transfer observed by
Lederberg and Tatum was one-directional and had performed between polar cells.
Therefore, it is predictable that there must be cells having characteristics of a donor
(F+ or fertile) and the recipient (F
or infertile). This type of gene transfer is
nonreversible.
An extra chromosome such as F factor in the F+ strain encoded sex pili which is
an essential need for plasmid transfer. Major role of sex pilus is to establish a
physical cell–cell contact between the F+ and F
mating. Figure 6.11 shows the
mechanism through which bacteria can transfer its plasmids such as F to the recipient
cells. Once F+ and F
cells come into the vicinity, the F plasmid of F+ strain directs
the pili synthesis followed by its projection toward recipient cell to make contact and
pull the recipient cell closer (Fig. 6.15). This protruding pilus makes a pore on the
recipient cell, and thus F plasmid passes through this pore into the recipient cell. It is
notable that during this transfer DNA does not transfer in a double-stranded form
while it carries only one strand of F DNA, which initiates replication of the
complementary strand in the conjugation tube (basically bridge like structure)
which connects both the donor and the recipient cell. This replication concluded in
two copies of F DNA, one remaining in donor and one appearing in the recipient cell
as shown in (Fig. 6.15).
Replication of the F factor is accommodated by rolling mechanism and replica-
tion initiated by the help of a protein complex known as “relaxosome.” This
relaxosome first recognizes ori T (Fig. 6.10) site and nicks one strand from this
point. Relax enzyme is a part of this relexosome and remain attached at the 50 end of
the nicked strand. During the replication of the F plasmid, replicated strand displaced
and attached relax enzyme move along through the type IV secretion system to the
recipient cell. Because pilus is embedded into secretion system, it has been
suggested that the DNA moves through a lumen in the pilus.
6 Genetic Study of Bacteria and Bacteriophage 325

a b
Donor
Bacterial
chromosome

Conjugation
bridge
Plasmid
Pilus

Recipient

Fig. 6.15 F+ and F
mating system. Conjugation between F+ and F
initiates with pilus formation
and pulls them together and during conjugation, shown in (a). This pilus formation and cell-to-cell
contact are further followed by the formation of a bridge or a pore (essential passageway) between
two cells. Single-stranded DNA passes into the recipient cell and becomes double strand by rolling
replication mechanism as shown in (b)

6.3.7 F Plasmid

Plasmid has the capacity to replicate independently and to integrate into the bacterial
chromosome. F plasmid is one of the important factors also known as fertility factor
that integrates with bacterial chromosome to generate Hfr cells. Homologous recom-
bination site present on either bacterial chromosome or F factor allows repairing and
release of F plasmid. Occasionally integrated F plasmid exists from the bacterial
chromosome by reverse recombination process. F factor is responsible for mating
and gene exchange between bacteria and so the conjugation process is a totally F
plasmid-dependent process. Most importantly, the F plasmid contains an origin site
for replication and other genes required for conjugation and sex pili formation to
make contact with the recipient cell (F
cell). DNA is always transferred from F+ to
F
. Thus, F plasmid is essential and needed for conjugation in bacteria. One more
important thing about F plasmid is the integration of F plasmid into a bacterial
mutant (known as F0 ) that is unable to replicate known as “integrative suppression.”
For its own replication, F plasmid uses many E coli replication proteins, but it does
not use the dnaA protein usually required for bacterial chromosome replication. In
case if dna A is mutated in bacteria and temperature is elevated to the 42 (on which
dna A is inactivated), initiation of replication in bacterial chromosome will not be
possible, but F plasmid will replicate since it has its own origin of replication. In
dnaA mutant strain where the F plasmid is integrated into the chromosome, the
replication of the chromosome is still possible and independent at high temperatures.
However, replication does not occur at the origin of the chromosome and instead
occurs at oriV, origin site on the F plasmid. In this way, integration of F plasmid
suppresses the importance of dnaA as a phenotype by replacing it with F plasmid-
derived replication.
Note that it is also tricky to select the F integrated positive strains, and therefore
integrative suppression can be a possible way to select those that have integrated F
326 N. Sharma

plasmid. In a given example of mating between F0 Lac+/trps and Lac
(dnaA)/trpr

would result in the colonies to be selected on high temperature and lactose/trypto-
phan selective medium. Colonies were successfully survived on the medium and
high temperature should have contained an integrated F0 Lac+.

6.3.8 R Plasmid

A bacterial chromosome often regulates antibiotic sensitivity or resistance. Alter-

ation in the pathogenicity of bacteria is thought to occur due to another plasmid that
allows the bacteria to grow in the host cell escaping the toxic effects of antibacterial
drugs. Antibiotic resistance is a net result of a group of genes located on the R
plasmid. Drug-resistant R plasmid has evolved in the past 60 years. The Discovery
of antibiotic resistance came into existence after the incident in a Japanese hospital,
where the first time an antibiotic resistance has been observed in Shigella; a bacteria
causes dysentery, which later on with multiple treatments of antibiotics (penicillin,
tetracycline, sulfanilamide, streptomycin, and chloramphenicol, etc.) has become
resistant to drugs. Similar to the F plasmid, R plasmid contains circular small DNA
that has divided into two fragments; one fragment contains a group of gene respon-
sible for replication and copy number, transfer of gene, and sometimes resistance to
the tetracycline, and another fragment includes the number of other resistance gene
or factor (thus known as R factor or R plasmid). R plasmid serves several genes to act
on antibiotic resistance and establish antibiotic resistance in the strains. R plasmid
carrying the marker for resistance like chloramphenicol (Cam), streptomycin (Str).
Stone, A. B. has mentioned in his study that R plasmid is a major factor that is
responsible for the epidemic spread of multiple drug resistance throughout the
bacterial population. For example, research shows that plasmid carrying genes for
resistance to multiple antibiotics might have been transmitted from a cow udder
infected with a hand towel. The practice of milking an infected cow followed by a
farmer cleaning his hand with a towel was the reason for the antibiotic resistance
passage from bovine to the human–microbe reservoir. This incident shows a severe
problem in antibiotic therapy since plasmid transmission is not restricted to particu-
lar species or genera, and it may invade the unrelated species or population.

6.4 Transformation

Transformation is another method for gene transfer in bacteria that facilitates a DNA
transfer without any donor bacteria or physical contact, rather a naked foreign DNA
exogenously being uptakes by the recipient cell. This method suggested that bacte-
rial genetic information is somehow transferable within the bacterial population
where physical contact unlike conjugation is not needed. This discovery belongs
to the time before when DNA structure was discovered.
6 Genetic Study of Bacteria and Bacteriophage 327

6.4.1 Discovery of Transformation

Transformation is the second most abundant mode of gene transfer after conjugation.
In the transformation, a naked DNA or partial DNA fragments are uptake by the
recipient cell from the environment and incorporated into the recipient’s chromo-
some as a result of recombination.
Transformation introduced a gene transfer method that allows the transfer of one
genotype into another by the exchange of exogenous DNA. First-time transforma-
tion was confirmed in Streptococcus pneumoniae in 1928 by Frederick Griffith.
Following this, in 1944, Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty
explained that the “transforming principle” was none other than bacterial DNA in
this method. This experiment was the first explanation that reveals the phenomenon
like DNA can be transferrable.
Fredrik Griffith’s experiment is shown in Fig. 6.16, where he used two distin-
guished Streptococcus pneumoniae strain; one is virulent and has lethal effect on
most laboratory animals, and other one is nonvirulent and is not lethal for animals.
Virulent strain designated as (S) and enclosed by polysaccharide cover (capsule like
structure) and gives a smooth appearance when it grows on the medium so easily can
be detectable on culture. Nonvirulent strain designated as (R) strain which is lacking
any cover or capsule shows rough appearance when it grows on medium and thus
can be detectable on medium. Griffith first boiled some virulent strains to kill them
and injected these heat-killed strains into the mice, and he observed that these mice
survived, and the cells do not show any lethal effect on the mice. In the next round, a
mixture of heat-killed virulent cells and live nonvirulent cells were injected into the
mice and mice did die. He isolated the live cells from the dead cells and grew them
on the medium, gave the smooth colonies, and showed virulent characteristics on
subsequent injection to the other mice. These results made him realize that heat-
killed virulent (S) cells somehow converted the nonvirulent live cells (R) into live
virulent cells (S) which was the reason for the death of mice (Fig. 6.16).

Fig. 6.16 Transformation of the nonvirulent live cell (R) into the live virulent cell (S). An
experimental setup explains that chemical substances produced from heat-killed cells someway
transferred into live nonvirulent cells and transformed it into live virulent cells (S). This substance
can be any biomolecule whose competency is to be transformed is chemically unknown
328 N. Sharma

But unfortunately, Griffith was not able to reason why this had happened and
what made the nonvirulent live cells behave like virulent live cells?
Next to this achievement, a question was awaiting that what was the chemical
composition of dead donor cells had caused this transformation? Was this protein or
any other element? Since it was clear that this substance that had changed the
genotype of the recipient cell must be something transmissible or heritable. A
group of Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty solved this
doubt when they proposed a study to destroy all the chemical substances of the dead
cells and see the transforming capacity of this dead extract from the cell. They found
that this mixture still could develop the ability of transformation. However, heat-
killed virulent cell contains polysaccharide coat while live nonvirulent does not
contain any coating; hence, it is assumed that the transforming agent could be this
polysaccharide coat. Destroying polysaccharide coats does not diminish the
transforming activity. Proteins, fats, and RNAs show similar results not to be
transforming agents.
An interesting result obtained when the mixture was treated with DNase, it lost its
transforming ability which strongly suggested that DNA was the genetic material
that has transformed from one bacteria (dead virulent) to other bacteria (live
nonvirulent), named transformation (Fig. 6.17).
It is noteworthy that natural transformation is a random process between two
bacterium and DNA transfers from the donor bacterium to Hfr + bacterium. Any
portion of the entire genome may be able to transfer.

6.4.2 Transformation Process

In the previous section, studies on transformation in S. pneumoniae have been

intensively explained and now it has to be understood how this process takes
place? An entire transformation involves (1) the uptake of foreign DNA from the
surrounding and (2) its incorporation in the recipient bacterial chromosome or
plasmid. Naturally occurring transformation takes DNA from the dead bacterium
which might have been released to the environment after fragmentation of the DNA
due to the death of the bacterium. Thus, the marine and soil microbiome utilizes this
mode of transfer of genetic material at a massive level. A cell that uptakes the DNA
is known as a competent cell that is the target for exogenous double-stranded DNA.
This DNA was later cleaved and processed by endonuclease into double-stranded
chunks of 5–15 kb size. The entire process of transformation is energy dependent.
During the DNA uptake process, one strand of DNA is hydrolyzed by an envelope
associated with exonuclease while the other strand is coupled with small proteins
and move along with the plasma membrane. Thus, only one strand of DNA is
available for interaction with recipient DNA. Fragmented single-stranded DNA
aligns with the homologous region on the recipient genome and is integrated
successfully.
However, transformation in Hemophilus influenzae, a gram-negative bacteria,
unlike S. pneumoniae is different in many aspects of the transformation process. It
6 Genetic Study of Bacteria and Bacteriophage 329

Fig. 6.17 Experimental evidence for the transformation of DNA in mice between different strains.
S strain was recovered from the live cells, all the chemical components were destroyed step wise,
and the extract mixture was reinjected with the R strain into the mice. As the result, (1) R strain
converted into S strain when no component destroyed, (2) R strain converted to S stain when
polysaccharide destroyed, (3) R strain converted to the S strain when lipid destroyed, (4) R strain
converted into S strain when protein destroyed, and (5) R strain does not convert into S strain while
DNA destroyed. Thus, these results confirm that when all the other components were destroyed but
not DNA, R strain was not able to be transformed into virulent death causing lethal S strain, and
destroying the DNA makes the R strain incapable to transformed into s strain and mice left alive

does not produce competence factor to make cells competent and it takes up DNA
from closely related S. pneumoniae; thus, the case of H. influenzae transformation is
a selective method while in the case of S. pneumoniae is less particular about the
source of DNA.
It has been demonstrated that the specificity of Hemophilus influenzae transfor-
mation came from the 11 base pair sequence, 50 AAGTGCG-GTCA30 , which exists
in a repeat over 1400 times in DNA, and this repeat must bind with competent cells.
Nonetheless, transformation is not restricted to only a few selective bacterium DNA
rather a DNA source for a competent call can be anything in an appropriate condition
(Fig. 6.18).
However, recombination or crossover is common in bacteria but efficiently DNA
uptake is limited. Even it has been seen that a species is capable of transformation,
but a very small fragment of DNA is transformed in a growing population of
bacteria. For a long time, bacterial geneticists were in the search of developing a
novel technique to increase the transformation frequency that may enhance the DNA
uptake into the cell. Hence, transformation supplemented with an artificial add-on
330 N. Sharma

Fig. 6.18 Gene can be transferred between bacteria through transformation. Transformation in the
bacterial cell initiates with DNA uptake and its integration to the bacterial chromosome that
procures a recombinant DNA that contains both bacterial and exogenous DNA intact in the
daughter bacterial cell

such as calcium chloride in the medium, heat shock, or electric field makes the cell
membrane more porous and permeable to uptake DNA more efficiently. Increasing
the DNA concentration will also work as an enhancer for transformation efficiency.
These enriched techniques for the transformation of foreign DNA into any cell are
vastly in practice for molecular biology studies in laboratories.

6.4.3 Transformation Linked Genes

Likewise, conjugation transformation has been used to map the bacterial gene,
especially for those species that do not undergo conjugation or transduction.
Mapping in such strains is only possible when they are entirely different genetic
strains or traits that have to mate through the transformation. For example, a recipient
strain might be auxotrophic for three nutrients p
q
r
(in the figure), mate with
donor cell, and prototrophic for alleles p+ q+ r+ (in the figure). DNA from the donor
strain is treated and fragmented to increase competency. Fragmented donor DNA is
added to the medium of the culture of recipient strain (competent cells). Eventually,
fragmented donor DNA enters the recipient cell and immediately undergoes recom-
bination. The recombination process must be followed by a homologous sequence
on the recipient bacterial chromosome where the donor DNA is attached and intact
throughout the procedure. Recipient cells that positively have received the genetic
material from the donor cells through transformation are called “transformed.”
How to understand that how many genes and what frequency they have
transformed? To this end, we first need to observe the rate at which two or more
genes are transferred together (usually termed as cotransformed). The recombination
rate of these genes is the basic need for the measurement of transformation fre-
quency. Gene can be mapped by observing the rate at which two or more genes are
transferred together (cotransformed) in transformation. We assume that genes that
are physically close to each other on the same DNA after fragmentation are more
likely to be transformed contiguously into the competent cell. For example, in
Fig. 6.19, gene p and q on the DNA of donor strain are physically linked so that
6 Genetic Study of Bacteria and Bacteriophage 331

Fig. 6.19 Transformation and linkage for mapping the bacterial genome. Gene p and q are close
enough to be transformed together, gene q and r are also close enough to transform together and
therefore genotype observed as (1) p+ q+ r
and (2) p
q+ r+ are cotransformed. Note that p+ q+ r+
and p+ q
r+ are rare genotypes because p+ and r+ are two distant genes transformed together, and
thus, the rate of cotransformation is inversely proportional to the distance between the genes

they would preferably transform together. However, genes that are far apart are
unlikely to be present on the same DNA fragment and rarely will be transferred
together. In Fig. 6.19, we can observe that gene p and r are separated from each other
and no fragments are produced containing p+ r+ on the same DNA and therefore we
have not observed cotransformed for p+ q
r+ which is the rarest event.
Thus, after performing the transformation, transformed colonies must have been
obtained on selective media and performed the genotyping of each strain. Let us
assume that if gene p and q are frequently cotransformed and gene q and r frequently
cotransformed, then gene q must be in between p and r and the gene order on the
DNA must be p q r.
332 N. Sharma

6.5 Transduction

We have already spotted a light on bacterial gene transfer in the previous section.
Transduction is the third type of gene transfer after conjugation and transformation.
So, let us take a closer look at the third way of gene transfer in which gene is
transferred between bacteria through the phage/bacteriophage. Transduction is a
type of horizontal gene transfer that occurs naturally via phage infection.
Based on structure, the virus is simple, often composed of just a nucleic acid, and
the genome is always protected by a protein coat known as a capsid. Phage does not
replicate autonomously; instead, they first infect the cell, take control of host
machinery, use it, and force the host cell to produce multiple copies of phage
particles. Most phages initiate their replication immediately after infection. When
a phage begins the replication, it reaches a certain number of copies or let us say a
threshold number of copies that cause bursting of cells and produces many new
phage progenies to further infect new bacterial host, such phages known as a virulent
and the process is called “Lytic cycle.” Few bacteriophages do not kill the bacteria
immediately after infection and instead insert their genome into the bacterial genome
without affecting it. The inserted phage called prophage. These bacteriophages
passively replicated as the host cells genome does and thus this bacteriophage is
known as temperate bacteriophage and the relationship between phage and its host
cell is called lysogeny (Fig. 6.20). Temperate phages can remain inactive as an insert
for many generations in their hosts. However, they are prone to some conditions like
UV irradiation which can induce lytic cycle temperate phages.
Transduction is categorized into two: (1) generalized transduction in which any
gene may transferred and (2) specialized transduction where only few genes are
possibly transferred. How this transduction has been identified by researchers? It is
discussed in the next section.

Fig. 6.20 Transduction comprised of lytic cycle and lysogenic cycle. A typical transduction
process has both lytic and lysogenic cycles which either follows one each after or remains separated
as shown in the image
6 Genetic Study of Bacteria and Bacteriophage 333

6.5.1 Discovery of Transduction

Joshua Lederberg and Norton Zinder in 1951 were testing the recombination in
Salmonella typhimurim by using the same techniques as Lederberg had found in
E. coli. For their experiment, they used two distinguished strains of Salmonella; one
was auxotroph for phe
, trp
, tyr
and the other was auxotroph for met
, his
. They
mixed these two strains in one culture and plated on the minimal media, then similar
to the E.coli experiment, they did not observe any wild-type strain When these two
strains were plated on minimal medium, no wild-type cells were observed; however,
at a low frequency of about 1/105, recombination was observed. In the discovery of
transduction, researchers referred to the U tube experiment with few modifications.
They put the porous filter instead of the fritted filter paper in the conjugation
experiment to prevent cell–cell contact. Later they observed that the agent responsi-
ble for the recombination is the size of the phage of P22 which is a known temperate
phage of Salmonella. Furthermore, many studies together suggest that the vector for
recombination is a P22 phage. But there was uncertainty among the researchers
whether this filterable recombination agent is a phage or something else. Therefore, a
comparison of the properties of this agent with phage, where it shows the similarity
in the size, sensitivity to the antiserum, immunity to the hydrolytic enzyme, and so
on has confirmed its virus alike characteristics.
As a result, Lederberg and Zinder have confirmed this new type of gene transfer
through the virus and named it transduction instead of conjugation.
However, in the lytic cycle, sometimes phages interact with host DNA and
integrate their own DNA that is then transferred to another host bacteria and insert
its contents into bacterial DNA. Both temperate and virulent phages follow the
transduction method to transfer the genes.

6.5.2 Generalized Transduction

Besides understanding the transduction further, it came with the next question that
how transducing phages are reproduced after infection? To address this question, in
1965, K. Ikeda and J. Tomizawa had discussed the experiment on temperate phage
P1 in E. coli. They highlighted in their experiment that when P1 infected the E. coli
and lysed the donor cell where bacterial chromosome was broken up into small
fragments; however, some of these pieces were captured mistakenly by phage
particles and assembled in the head instead of phage DNA. Eventually, this has
become the source of transducing phage.
During the infection, phage capsid (coat proteins) determines phage’s ability to
recognize and attack the host bacteria and transfer its content to the host cell. But
now in the case of transducing phage, the transferrable material is the donor’s
chromosome which the phage had assembled during the transduction. Interestingly,
transduction through the transducing phage could rise the situation of merodiploid
(a partial diploid bacteria) since it transferred the donor’s chromosome which
recombined with the recipient chromosome and now the recipient cell will have
334 N. Sharma

two bacterial chromosomes which is a merodiploid situation (Fig. 6.21). This type of
transduction allows the passage of any kind of host (bacterial) markers to other
bacteria and is thus known as generalized transduction where any gene can possibly
be transferred to another bacteria.
Phages P1 and P22 belong to the group that shows features of generalized
transduction. Looking at their cycle, P1 phage is usually integrated to the host
chromosome while P22 remains free in the cytoplasm.

6.5.3 Specialized Transduction

Unlike generalized transduction, specialized transduction will allow a few particular

genes (few host markers) to be transduced as shown in Fig. 6.22.
λ phage is very well known for specialized transduction. λ prophage (shown as
the red circle in the figure) integrated between gal and bio region of host chromo-
some (shown blue linear strand in figure) as shown in Fig. 6.23. Hence, λ can
transduce only the gal and bio genes. Let us understand the mechanism of λ
transduction.
Recombination between λ phage and host chromosome is catalyzed by a specific
enzyme system. This enzyme guides λ phage to integrate at the same site as on
chromosome which is denoted in the diagram with att (λ attachment site). This site
also decides the lytic cycle and ensures that prophage excise at the correct point to
produce a normal circular λ chromosome (see the figure, top left—a normal exci-
sion). However, errors in excision can occur as abnormal excision (see the figure top
right abnormal excision). Under this type of excision, the prophage gene during the
lytic cycle becomes circular but contains a nearby gene of the host chromosome and
leaves behind some phage gene intact to the host chromosome (Fig. 6.23).
If we see the figure, we can assume that λ has a nearby gene gal on one side while
bio on the other side. Resulting phage λ is now referred to as defective particle
because it left gene behind on the host chromosome and carry either partial gal
known as λdgal (λ-defective gal) or partial bio or λdbio. The resulting particles are
defective due to the genes left behind and others transferred from the chromosome
are referred to as λdgal (λ-defective gal) or λ dbio. This defective phage gene can be
packaged into head and infect another host bacterium. In the second round of
infection, λdgal gene will integrate to the λ attachment site on the second host
chromosome (Fig. 6.23). This way, gal gene from the first host will transduce to
the second host. It should be noticed that this type of transduction carries a few
limited genes very near the original integrated prophage, so very few genes are able
to transfer and are called specialized transduction.

6.5.4 Lambda (l) Genetics

Lambda phage is the most extensively studied bacteriophage among all the other
phages. Lambda phage is an important model system for latent infection of
6 Genetic Study of Bacteria and Bacteriophage 335

Fig. 6.21 Generalized transduction in bacteria. Generalized transduction is processed with infec-
tion of phage to the host cell and releases its genetic material into the cytoplasm. Once the phage
infects the host cell, simultaneously host DNA hydrolyzed and synthesized partial phage DNA and
proteins. Assemble phage contains a small fraction of the host bacterial chromosome. This phage
further transduces and infects other host cells where the crossing over between phage DNA and
bacterial chromosome takes place. In the above example, crossing over between bacterial
336 N. Sharma

mammalian cells by a retrovirus, and this model system has been widely used for
cloning purposes.
Lambda is the prototype of a group of phages that is a well-characterized virus
with both lytic and lysogenic alternatives to its life cycle.
DNA inside the phage is linear but it circularizes on the infection to E. Coli
chromosome (Fig. 6.24). At each end are complementary 12 bp long overhangs
known as cos sequences (cohesive ends). Once inside the E. coli host cell, these pair
up and the cohesive ends are ligated together by host enzymes forming the circular
version of the lambda genome. Lambda can only be compatible for packaging of
genome size of 37–52 Kb and also small fragments of extra DNA can be packaged
into lambda genome without hindrance. Although accommodate longer insert, some
of the lambda genomes must be removed. In the lambda genome, the left-hand
region has essential genes for structural proteins while the right-hand region consists
of genes responsible for replication and lysis. Cro has been believed to play an active
role in switching lysogenic cells to the lytic state following induction. However such
lambda replacement vectors cannot integrate to host genome and form lysogeny by
themselves. The Middle region is necessary for integration and recombination
(Fig. 6.22).
This lambda phage has made many undiscovered questions easy for a scientist to
address and to develop advanced techniques such as how to sequence DNA and
discovered essential enzyme for RNA synthesis. Also, studies on Lambda phage led
to the discovery of (1) basic molecular biology principles of how gene transcription
is halted with rho-dependent termination manner, (2) first transcription factor, and
(3) gene regulation including “Operon” concept.

6.5.5 Nature of Transduction

Nature of transduction reflects here the question to understand the nature of pro-
phage or prophage–host interaction. As sooner as the prophage is induced immedi-
ately after the infection, as more it will produce the prophage and the genome will be
restored in the prophage. Before its discovery, it is necessary to understand whether a
phage is a mere small invisible particle or a plasmid that lives in the bacterial
cytoplasm or a part of the bacterial chromosome. From the past, it has been observed
that a temperate phage lambda (λ) promotes the lysogenic cycle in its particular host
bacterium E.coli used by Lederberg and Tatum (mentioned earlier). So far, the
studies on lysogenic cycle of phage λ have introduced the λ phage as a first
preference to refer for lysogenic and well-characterized known phage. If we look
at the crosses between F+and F
cells obtain interesting results such as F
 F+ (λ)
gives recombinant lysogenic recipient while F+ (λ)  F
results in nonlysogenic

Fig. 6.21 (continued) chromosome (his
Lys
) and phage DNA (his+) results in his+ Lys+ positive
recipient host cell. They again cross over
6 Genetic Study of Bacteria and Bacteriophage 337

Fig. 6.22 Specialized transduction in host bacteria. In specialized transduction, prophage that
contains some bacterial gene disintegrates on the specific induction. The disintegration of prophage
produces a new circular chromosome in the same host cell. Replication in the host cells took place
immediately and was followed by the assembly of new phage that released and infected other host
338 N. Sharma

recombinants. These results were of more importance when Hfr strain had discov-
ered and was used for crosses. If crosses happened between Hfr  F
(λ), lysogenic
F
exconjugants readily recovered with Hfr genes.
If a cross occurs between Hfr (lysogenic or lambda containing strain) with F

(non-lysogenic or nonimmune recipient), entry of lambda prophage into
non-lysogenic cell will immediately trigger the prophage into the lytic cycle. This
process is known as Zygotic induction. On the other hand, if the cross between Hfr
(λ)  F
(λ) occurs, the resultant recombinants are readily recovered, and no
prophage lytic cycle occurs. From this observation we can say that cytoplasm of
F
cell must have interchanged between two states (depends on whether recipient
contains λ prophage). So when the recipient cell is a nonimmune cell, the entry of
prophage will induce the lytic cycle. This cytoplasmic state is specifying the fact that
prophage represses the multiplication of the virus and therefore when lambda infects
the nonlysogenic cell, these prophage repressing factors are diluted immediately
after the infection and thus virus will multiply and reproduce the progenies. But what
if a virus specifies the repressing factor and why the virus does not shut off the
replication of itself?
The answer is, it does because a fraction of infected cells can become lysogenic
(prophage). But there is a race between lambda gene signal for reproduction and
repressor specify signal to shut down the replication. In this way, a phage-directed
cytoplasmic repressor model explains the immunity of lysogenic bacteria. In con-
clusion, a superinfected phage will immediately encounter the repressor and become
inactivated.

6.6 Infectious (Bacterial/Viral) Disease

Coronavirus (CoVs) is a large family of single-stranded RNA virus that can infect a
wide variety of animals including humans, causing respiratory, enteric fever,
hepatic, and neurological disease. In human coronavirus mainly causes respiratory
tract infection. Till date, six coronaviruses have been identified including (1) alpha-
CoVs HCoV-NL63, (2) HCoV-229E, (3) beta-CoVs HCoV-OC43, (4) HCoV-
HKU1, (5) severe acute respiratory syndrome-CoV (SARS-CoV), and (6) Middle
East respiratory syndrome-CoV (MERS-CoV). However, a novel coronavirus,
SARS-CoV-2 (COVID-19) lately in 2019, has been added to the list of existing
coronavirus. Although human coronavirus has been identified decades ago, their
clinical and epidemic importance was not recognized until the outbreak of SARS
(2002) and MERS (2012–2017). In the next section, SARS, MERS, and COVID19
will be discussed in detail.

Fig. 6.22 (continued) cells. Multiple crossing overs between prophage and bacterial chromosome
result in (1) bacterial chromosome containing only donor DNA and (2) bacterial chromosome
containing both viral DNA and donor DNA
6 Genetic Study of Bacteria and Bacteriophage 339

Fig. 6.23 The mechanism of transduction for phage lambda and E. coli. Integrated phage lies
between gal and bio genes. When a normal excise occurs (top left) new phage is complete and does
not contain any bacterial gene. While rare excise occurs (top right), either the gal or the bio genes
are picked up by bacterial genes and some are lost. As a result, a defective lambda phage that
contains a bacterial gene can transfer to the new host cell

6.6.1 SARS-CoV

SARS CoV virus was identified in 2003. SARS-CoV is a zoonotic disease and was
thought to be an animal virus from an uncertain animal reservoir, like bats and civet
cats, and found to be the first to infect humans in the Guangdong province of south
China in 2002 (Fig. 6.25). However, these animals were only incidental hosts, as
there was no evidence for the circulation of SARS-CoV-like viruses in palm civets in
the wild or breeding facilities. Studies reported that bats are the reservoir of a wide
variety of coronavirus including SARS-CoV-like and MERS-CoV-like virus.
340 N. Sharma

Fig. 6.24 Lambda replacement cloning vector. Lambda phage is easy to grow and therefore it has
been modified to accept foreign DNA inserts. Both left and right ends are overhangs with cohesive
regions known as COS and regulate the circularization of the DNA. The green region has genes that
are nonessential for lambda growth and packaging but can be replaced by the foreign DNA insert
(up to 23 kb) during the cloning. The yellow region codes for proteins essential for head and tail
packaging

Fig. 6.25 Insights into SARS and MERS infection Cycle: SARS-CoV crossed the species barrier
into masked palm civets and other animals in live animal market in China responsible for SARS-
CoV infection occurred in late 2002. Later in 2012, the cross-infection in dromedary Camel was
identified as MERS-CoV infection in the Middle East. SARS-CoV and MERS-CoV spread between
humans mainly through nosocomial transmission, which results in the infection of healthcare
workers and patients at a higher frequency than infection of their relatives

Transmission of this disease was primarily spread from person to person and
appeared to occur in the second week of illness where excretion of virus in respira-
tory secretion and stool was on the peak. Lately, most of the cases of human-to-
human transmission were due to negligence in the healthcare setting, absence of
adequate infection control, and precautions. Symptoms of SARS include influenza-
like fever, malaise, myalgia, headache, diarrhea, and shivering. Cough was initially
6 Genetic Study of Bacteria and Bacteriophage 341

dry, and shortness of breath and diarrhea are most prominent in first or second week
of infection. Mostly severe cases developed rapidly, progressing to respiratory
distress and required intensive care. Unlike COVID-19, SARS transmission counted
as an epidemic since the geographic distribution was limited such as Toronto in
Canada, Hong Kong, China, Chinese Taipei, Singapore, and Hanoi in Viet Nam,
thus SARS infection.

6.6.2 MERS-CoV

Ten years after the emergence of SARS, in June 2012 a man in Saudi Arabia died
with acute pneumonia and renal failure, which lately had identified infected with a
novel coronavirus named Middle East respiratory syndrome coronavirus (MERS-
CoV). MERS was also identified outside of Arabian Peninsula such as Jordan and
United Kingdom, as a result of traveling; often, these imported MERS cases resulted
in nosocomial transmission (Transmission usually occurs via healthcare workers,
patients, hospital equipment, or interventional procedures). In the case of MERS,
serological tests from dromedary camels from Oman and Qatar camel farms con-
firmed its transmission from camel to human first in Arab peninsula and later in the
Middle East, Eastern Africa, and Northern Africa. Like SARS, MERSS causes acute
respiratory syndrome as well which is associated with the upregulation of
proinflammatory cytokines and chemokines. Immune response to SARS and
MERS during the infection plays a key role in its spread since SARS-CoV and
MERS-CoV use several strategies to avoid the innate immune response.

6.6.3 COVID-19

The 2019 novel coronavirus, also known as COVID-19 or SARS-CoV-2, is a novel

human coronavirus that has emerged at the end of December 2019 in Wuhan, China.
It is currently spreading all over the world in the form of a pandemic. Coronavirus
disease (COVID 19) is a clinical syndrome associated with SARS-CoV-2 infection
and is characterized by severe respiratory syndrome.
SARS-CoV-2 belongs to the same Betacoronavirus genus as the other
coronaviruses that are responsible for severe acute respiratory syndrome (SARS-
CoV) and the Middle East respiratory syndrome (MERS-CoV). Phylogenic analysis
of novel coronavirus suggested that SARS-CoV-2 belongs together with SARS-CoV
and Bat SARS-like coronavirus which is a different clade from MERS-CoV but
more phylogenetically related to Bat SARS-like coronavirus (isolated from horse-
shoe Bats between 2015 and 2018) than SARS-CoV. Genomic comparison between
SAARS-CoV-2 and SARS has shown that there are almost 380 amino acid
substitutions and mainly contained in structural protein, and 27 mutations were
found in viral spike protein (S) which is responsible for receptor binding and cell
entry. It is assumed that due to this mutation, SARS-CoV-2 is less pathogenic than
342 N. Sharma

Table 6.2 Overview of SARS-CoV-2, SARS-CoV, and MERS-CoV

Case
Phylogenetic Animal Intermediate fatality
Strain origin reservoir host Receptor rate
SARS- Clade I, Bats Controversial Angiotensin-converting 2.3%
CoV-2 cluster IIa enzyme 2 (ACE2)
SARS- Clade I, Bats Palm civets Angiotensin-converting 9.5%
CoV cluster IIb enzyme 2 (ACE2)
MERS- Clade II Bats Camels Dipeptidyl peptidase 34.4%
CoV 4 (DPP4)

the SARS-CoV; however, further studies are still going on to understand its pathol-
ogy and its contribution to other diseases like cancer (Table 6.2).
Similar to SARS-CoV, a mutation in the receptor-binding domain (RBD) of S
protein in SARS-CoV-2, which directly interacts with human cell receptor—angio-
tensin converting enzyme 2 (ACE-2)—is thought to be the cause for its pathogenic-
ity. Interestingly data on affinity analysis confirms that SARS-CoV-2 binds to
ACE-2 more efficiently than SARS-CoV strain from 2003 although less efficiently
than the 2002 strain.
ACE-2 is an ectoenzyme (an enzyme that has the catalytic site outside the plasma
membrane and is mostly found in the endothelial cell) occurring in many tissues
including, the lower respiratory tract, kidney, heart, and gastrointestinal tract. In
vitro studies on SARS-CoV-2 show that inoculation of 2019nCoV (SARS-CoV-2)
on the surface layer of human airway epithelial cells causes cytopathy effects and
cessation of cilia movement. SARS-CoV induces downregulation of ACE-2 in lung
epithelium, but SARS-CoV-2 shows higher affinity to ACE-2 and results in more
severe lung infection than the SARS-CoV.
From the clinical aspects, virus loads are higher at the time of symptoms onset
and are higher in the nose than the throat specimens which is why it is suggested to
collect the patient specimens from the nose. In patients affected by COVID-19, viral
loads progressively decrease within days following a different pattern from SARS, in
which the highest shedding is recorded after 10 days of symptoms onset. Therefore,
it has been suggested that SARS-CoV-2 can easily spread within the community
than SARS even when mild or no symptoms (asymptomatic) are present. To date,
there are no vaccines and drugs developed which can directly target COVID-19, but
many clinical trials for vaccines are underway in different parts of the world, namely,
Serum India technology (India), Oxford University (UK), Moscow’s Gamaleya
Institute (Russia), and AstraZeneca (USA).
Antiviral drugs including ribavirin, lopinavir, ritonavir, and remdesivir in combi-
nation with other drugs like chloroquine, cyclophilin chlorpromazine, loperamide,
and cyclosporine A has been reported effective in some cases. In addition, antibody
therapies and plasma therapies have been the leading proposed treatment in the case
of MERS. Recently, plasma therapy has shown a potential advantage in COVID-19
treatment. Many countries such as India, United Kingdom, and United States are one
6 Genetic Study of Bacteria and Bacteriophage 343

of those who are successfully running a trial for plasma therapy and antibody
therapies for COVID-19.

Box 6.1 Scientific Concept: Genetic Exchange Between Escherichia coli

Strains in the Mouse Intestine—Jones R T et al.
Bacterial genome usually possesses the phenomenon of gene flow between
species. Gene flow largely contributes to the evolution of bacterial species;
however, the significance of gene flow is still unknown. In vitro conjugal
transfer of genes in E. coli K-12 and some other species has been widely
studied and also revealed that plasmids harbored by bacteria from the natural
environment are continuously increasing. Such plasmids may introduce the
determination of colicin production, multiple drug resistance, surface antigen,
enterotoxin production, suppressor and mutagenic factors, hemolysin produc-
tion, and so on. Some of these plasmids are enabled to conjugally transfer to
the recipient cells and some of these has been identified as a fertility factor that
supports the transfer of chromosomal gene at low frequency. Considering the
abovementioned prospective gene flow among bacterial species in nature or in
ecological niches, it has been observed that the study of gene transfer in some
particular niche has become a difficult task due to the microbial complexity of
the niche where these species are likely to occur. One of the common niches is
the mammalian intestinal tract where enormous microorganism species live
together and are associated within the tract facilitating the genetic transfer. In
the given niche, Enterobacteriaceae is the common inhabitants where they
possess the genetic transfer very often. So far, many studies have been focused
to understand the in vivo transfer of genetic material between the E. coli K12
(Fig. 6.26) and the formation of recombinants. K12 strain is genetically
compatible to investigate the in vivo gene transfer and to avoid the microbial
contaminant, and germ-free mice have been used.
Recombination frequencies between three donor strains from in vitro liquid
mating, which have been selected further for in vivo studies and the F

recipient 820 are shown in Table 6.3.Cross between F+  F
gave the
recombination frequencies of 5  10
5 for any maker while the cross between
Hfr  F
gives high frequencies for proximally transferred markers and
decreased for markers situated at increasing distance from the origin of the
chromosome transfer.
Recombination frequency for pur E marker (1.2  10
1) is slightly lower
than that for the more distantly transferred pro C (2.4  10
1). It is because of
integration of markers located next to the origin of chromosome transfer (i.e.,
pur E locus in Hfr strain OR74) is always lower than more distantly transferred
markers.
Thus, proximally transferred markers showed high frequencies of inheri-
tance, which further decreased as the distance from the origin of the

(continued)
344 N. Sharma

Box 6.1 (continued)

chromosome transfer increased. Thus, these data show that the donor or
recipient strain for in vivo experiments behaved in the predictable manner as
they do in in vitro mating.
Therefore, gene transfer between microbial species is quite difficult to
understand since the ecosystem is more complex. By creating a simplified
ecosystem of microbial species through contaminating the intestine tract of
mice with genetically well-characterized bacterial strains is one of the easy
ways to study the gene transfer between two species. In this study, Hfr strain
OR74 was chosen and maintained in the intestine of mice as its in vitro stable.
Forty days after inoculation, F
and F+ strain/clone was isolated in vivo. The
transition of Hfr , F+ , F
is unpredictable in in vivo and in vitro and will
not remain the same during the gene transfer. The reason might be the
influence of chemical, selection, and environmental factors. The study of
recombinants in the natural environment based on selection and survival rate
plays an important role to understand the relationship between its in vivo host
and gene transfer pattern. It would be interesting to set up the experiments
in vivo to compare the mechanism of gene transfer and formation of
recombinants in animals similar to its natural habitats. The complexity of
gene transfer among the microbial ecosystem in nature can be possible to
study by performing in vivo experiments on well-characterized bacteria and
produce new germ-free host animals.

Box 6.2 Scientific Concept: Role of Pili in Bacterial Conjugation—Ou, J. T

et al.
Bacterial conjugation has been discovered in 1946 by Laderberg and Tatum
where they have proved that cell–cell contact is required for the transfer of
bacterial chromosomes from a donor to a recipient. Later, in 1958 Anderson
et al. have provided the electron micrograph images of conjugation bridge
formation between mating pairs which confirmed the hypothesis of Laderberg
and Tatum. To understand the role of F-pili to F-factor and male-specific
phages, Brinton proposed that F-pili not only serves in pair formation but is
also important for tube formation through which bacterial chromosomes are
transferred. Evidence from various studies and experiments suggested that
F-pili connect the male and female and then bring them a close wall to wall
contact by retracting the F-pili into male or female cells. To obtain clear
evidence, microbiologists further studied the role of F-pili by isolating mating
pairs by using a micromanipulator (Fig. 6.27). They compared the production
of recombinants from loosely connected pairs with the ones produced by

(continued)
6 Genetic Study of Bacteria and Bacteriophage 345

Box 6.2 (continued)

mates who were in intimate connect. Results from this study suggest that F-pili
accelerates the transfer of bacterial chromosome, but close contact is one of the
strong phenomena that facilitates an efficient transfer of bacterial
chromosomes.
Micromanipulator is associated with the micropipette that separates
microdroplets of medium containing single bacteria to be studied under high
magnification. This microdroplet is larger enough to facilitate the bacterial cell
division, and further this daughter bacterial cell can be taken from the drop and
transferred to the other microdroplets.
During the experimental setup, the primary goal was to identify the mating
pairs in the oil chamber of the micromanipulator where two mixed cultures
were seeded. Morphological features observed under the microscope such as
short and round denotes the male mate and long and thin denotes the female
mate. Motility factor also supports the evidence to their identification such as
male could be motile and could be seen pulling the nonmotile female through
the medium. Mating pairs that have been chosen for this study have been
described in Table 6.4.
From the given sources earlier, mating between F+ (lac+, pro+ leu+ strr øIIr,
MS2s) and F
p678 (lac
pro+ leu
strr øIIs, MS2r) were done by mixing
these two strains and placed them on an oil chamber and both loose and close
contacted mating pairs were identified with the help of visualization under a
microscope. The clones after transfer and cell division were tested whether
they are resistant or sensitive to the phage øII or MS2. If they are resistant to
øII and sensitive to mS2, this indicates that they were received the F factor. To
analyze the close contact and loose contact between F+and F
p678, Table 6.3
further indicates that close mating pairs generate more viable F+p678 clones
with high frequency than the loose mating pairs. However, four viable F+p678
clones from loose mate pairs suggest that F-pili might indeed aid as the means
for transfer (Table 6.5).
During the experimental set up, it takes at least 6 min to isolate the first
mating pairs after mixing male and female cells. Previous studies suggested
that chromosomal bacterial transfer takes maximum 3–5 min and if we assume
that the F factor takes a similar time like chromosome (3  104 nucleotide
pairs/min) and it gives 7.5  104 Nucleotide pairs so it is predictable that time
for transfer the F factor would be approximately 2 min. Therefore, one thing is
clear that the F factor consumes a minimum 5 min to complete the transfer.
However, it is difficult to exclude the fact that the F factor had already been
transferred during unobserved close contacts before mating pair were isolated.
To this end, experiments were further extended to mating between Hfr X F

where bacterial chromosomal transfer could not take place at least 8 m in after
the mixing the cultures.

(continued)
346 N. Sharma

Box 6.2 (continued)

Evidence together suggest that initial contact between mating pair of cells
appears to be a thin thread that is difficult to observe under light microscope
and the simplest assumption is that this thin thread is F-pili that can be seen
under electron microscope as a connecting bridge between male and female
bacteria. It has also been proved that the mating pairs that were separated
during mating from each other produce F-exconjugants that lately forms
recombinant clones.
In the conclusion, such studies provide us with three important information;
F pili play a significant role in the mating between two E. coli bacteria by
providing an external organelle for attachment of mating pairs.
F-pili is a medium for the transfer of bacterial chromosomes and F factor.
F-pili beings two mating pairs in proximity to create close contact between
them and help to build the conjugation bridge more efficiently.

Fig. 6.26 Image shows Linkage map of E. coli K-12. The arrow indicates the origin, direction, and
gradient of chromosome transfer of Hfr strain OR74. The F0 strain ORF-210 used in these studies
had the same origin and direction of chromosome transfer as Hfr OR74 when F0 was integrated into
the chromosome. The relevant fermentation and auxotrophic markers of F-recipient strain 820 are
shown (Jones and Curtiss (1970))
6 Genetic Study of Bacteria and Bacteriophage 347

Table 6.3 Recombination frequencies obtained from in vitro liquid

mating between the Hfr, F0 and F+ strains chosen for further in vivo
studies and F
recipient 820a strain

Fig. 6.27 Image shows the

arrangement of the equipment
for micromanipulation.
(a) Glove box, (b) hinged
opening for glove box,
(c) opening for arm insertion,
(d) opening for microscope,
(e) leveling platform, (f) fan,
(g) heat strip,
(h) thermoregulator, (i) 10-mL
hypodermic syringe, (j) rubber
tubing, (k) micropipette, (l) oil
chamber, (m) micropipette
receiver, (n) transformer,
(o) controlling level

6.7 Summary

• Bacteria and virus genomes show a potential scope for genetic studies. Bacterial
and virus genome is small and haploid in nature. Bacterial genome is a simple
circular ds DNA, while virus genome varies as ssDNA, dsDNA or ssRNA.
• Mutation is a very common feature in bacterial DNA and mutant selection;
detection has been developed through various techniques including, replica
348 N. Sharma

Table 6.4 Escherichia coli K-12 strains were used for recombination experiments

Table 6.5 Transfer of F factor between loose and close pair mate in the cross F+ X F
p678

a
Value of P (χ 2 test) gives the probability that there is no difference between close- and loose-
mating pairs in the production of recombinants

plate method, phage display, and plaque assay. The spontaneous mutation is one
of the potential sources and have been reported as the first experimental evidence
for microbial evolution which is a random process.
• Plasmids are extrachromosomal nongenetic DNA molecules that coexist with
bacterial chromosomes. They rapidly and independently replicate in the bacterial
cell. Episome is a plasmid that can either live freely or integrate into the bacterial
chromosome.
• Plasmids are involved in DNA transfer in the bacterial population by conjugation,
transformation, and transduction.
• Conjugation referred a physical interaction between two bacterial cells to
exchange the DNA through the bridge, known as a conjugation tube. F factor
(fertility factor) is responsible for DNA transfer. F plasmid containing cell only
can transfer the DNA, known as F+cell while cells lacking F factor meant to
6 Genetic Study of Bacteria and Bacteriophage 349

receive the DNA, know F
cell. Hfr cell contains F factor integrated with
bacterial chromosome and shows the high frequency for DNA transfer.
• Mating between F+ and F
or Hfr and F
determines the rate of gene transfer
from Hfr to F
in terms of the time unit. The rate of gene transfer represents the
order of the gene on the chromosome.
• In transformation, bacteria uptake exogenous DNA from the environment without
any physical contact between cells. Cotransformation is a rate at which linked
genes transferred and the frequency of cotransformation defines the physical
distance between a gene on the chromosome.
• The virus is auto replicating machinery with DNA or RNA presence in either
circular or linear forms. Most common virus being studied in genetics is bacteri-
ophage—a virus that infects the bacterial cell. Bacterial genome can be trans-
ferred through a phage known as transduction. Likewise, conjugation
transformation, transduction also provides the information to map the gene
order on the bacterial chromosome about the rate of cotransduction reveals the
gene order on the bacterial chromosome.
• CRISPR/Cas9 is an advanced technique using strong bacterial adaptive immune
machinery to adapt in genetic engineering to edit genes of interest in any kind of
eukaryotic or prokaryotic cellular system.
• In generalized transduction, any gene can be transferred from one bacterium to
another bacteria. In specialized transduction, a gene linked to the site of phage
integration can only transfer from one bacterium to another bacteria.
• A phage life has been differentiated in the lysogeny phase and lytic phase. In the
lytic phase, phage causes bacterial cell lysis over the infection and does not
integrate with bacterial chromosomes. In lysogeny phase, phage DNA integrates
with bacterial chromosome and remains dormant for generations.
• Coronavirus is single-stranded RNA virus that mainly causes acute respiratory
syndrome in humans. Corona to date has been identified as SARS (2002), MERS
(2012), and novel coronavirus (COVID-19) in 2019.

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 Part II
Molecular Genetics I: Analysis of Gene
Replication of DNA
7
Tanushree Banerjee

We observe around us a lot of variation amongst individuals, like height, skin colour,
facial contour, eye colour, etc. All these variations are encoded in the language of
four nucleotide bases present in the nucleic acid called deoxyribonucleic acid or
DNA. The enormous information present in the nucleic acids also needs to be carried
forwards from one generation to the next. That needs a highly precise and accurate
method to replicate the genetic material.
Imagine when we had no idea about the existence of nucleic acids or the fact that
DNA is the genetic material. It is interesting to think about how people ventured into
this large unknown, step by step, to unravel the mystery of life. In this chapter we
will learn about the classical studies which showed that DNA is the genetic material.
We will also learn about how the genetic information is coded and organized and
what are the precision mechanisms which have evolved to ensure error-free propa-
gation of genetic information.

7.1 Classical Experiments: DNA as Genetic Material

Even before nucleic acids were discovered, people had figured out that there has to
be some substance called genetic material which is causing the variation in
individuals, and it is the same material which is being transferred to the progeny
from the parent. Therefore, genetic material should have certain basic characteristics
like the following:

T. Banerjee (*)
Molecular Neuroscience Research Laboratory, Dr. D. Y. Patil Biotechnology and Bioinformatics
Institute, Dr. D. Y. Patil Vidyapeeth, Pune, India
e-mail: tanushree.banerjee@dpu.edu.in

# The Author(s), under exclusive license to Springer Nature Singapore Pte 353
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_7
354 T. Banerjee

• Variation: It should be capable of shuffling the information for creating

variation.
• Stability: It should be stable enough so as not to create intolerable variation.
• Replication: It should be able to replicate so that information can be passed on
from the mother cell to the daughter cell.
• Transmission: It should be able to carry the information from one generation to
the next.

During Crimean War in 1869, a Swiss biochemist Friedrich Miescher isolated a

substance from the pus found in the bandages of war victims. Upon analyses he
observed that the material contained carbon, hydrogen, oxygen, nitrogen and phos-
phorus. Upon searching he observed that the material is present in the nucleus of
cells, so he called it nuclein. It was only in the 1900s that threadlike structures called
chromosomes were discovered and over the next 40 years they were found to be
made of protein and nucleic acids.

7.1.1 Transformation: Early Study

In 1928 Frederick Griffith, a British medical officer, was working with Streptococ-
cus pneumoniae. He had two strains of the bacterium, smooth virulent strain and
rough nonvirulent strain. Smooth strain had a polysaccharide coat which made it
virulent and shiny. Rough strains could not produce the polysaccharide coat due to a
mutation and due to which it was avirulent.
There are several types of S strains like IIS and IIIS. IIS mutates to form IIR, and
IIIS mutates to form IIIR, but IIS never forms IIIR and likewise IIIS never forms IIR.
R stains can also mutate back to S strains of the same type.
Griffith infected mice with various strains of S. pneumoniae. When infected with
living IIR, mice lived, but when infected with living IIIS, mice died. IIIS bacteria
could also be isolated from the dead mice. However, when mice were infected with
heat-killed IIIS strains, mice lived. This showed that bacteria need to be alive and
have polysaccharide coat to kill the mice.
Next, Griffith infected the mice with live IIR and heat-killed IIIS bacteria and the
mice died. Griffith concluded that avirulent IIR was somehow transformed by
virulent IIIS bacteria upon coming in contact with dead IIIS bacteria. He named
this process transformation (Fig. 7.1). Griffith identified the material of the dead
IIIS which transformed the live IIR as transforming principle. However, he could
not establish the biochemical nature of the transforming principle and initially
believed it to be a protein, but he was later proved wrong.
His experiments established that the transforming principle should be something
which is stable enough to make the shiny capsule; it should be capable of replication
so that it can be transmitted to the progeny cells. Therefore, Griffith’s transforming
principle had the properties of genetic material. The biochemical nature of this
genetic material was later established by Avery, MacLeod and McCarty’s
experiment.
7 Replication of DNA 355

Fig. 7.1 Griffith’s transformation experiment. Live bacterial colonies containing cells with
capsules (type IIIS) when injected in mice, they died. When avirulent live bacterial cells without
capsules (type IIR) were injected in mice, mice survived. When mice were infected with heat-killed
IIIS bacteria, they survived. When live avirulent IIR and heat-killed virulent IIIS bacteria were
injected together, the mice died (Adapted from: Klug et al. 2012)
356 T. Banerjee

7.1.2 Avery, MacLeod and McCarty’s Experiment

In the 1930s, American biologist Oswald T. Avery, along with his colleagues Colin
M. MacLeod and Maclyn McCarty, started working towards identifying the
Griffith’s transforming principle. The cells were heat killed at 65
C. They lysed
the S-type bacteria, and extracts were prepared using saline solution of sodium
desoxycholate. The crude extract was precipitated using ethyl alcohol. To identify
the transforming principle from the crude cell extract, it was necessary to carry out
stepwise extraction and removal of one biochemical component at each step. The
extract was then deproteinized using chloroform, and polysaccharide coat was
removed by enzymatic digestion. Each of these extracts was then analysed using
qualitative chemical tests like Dische diphenylamine reaction for deoxyribonucleic
acid, orcinol test for ribonucleic acid and so on. Extracts from each of these steps was
then incubated with R-type bacterial culture and plated on nutrient agar medium. The
extract which contained DNA was able to transform the R-type bacteria to have
glistening polysaccharide coat. This proved that DNA is the genetic material
(Fig. 7.2) (Avery et al. 1944).
To ensure that the DNA extract was not contaminated with other biochemical
components like protein and RNA, proteinases and RNAses were added. Even when
proteins and RNA were enzymatically destroyed, the transforming ability of the
extract was not lost. However, when DNAses were added, the transforming ability
was lost. This further confirmed that DNA is the transforming principle and the
genetic material.

7.1.3 Hershey and Chase Experiment

In 1952, Alfred Hershey and Martha Chase confirmed that DNA is the genetic
material. They used T2 bacteriophage which infects E.coli. Viruses can only repli-
cate inside the host. Bacteriophages need bacterial host for their propagation. Since,
bacteria are being used as host for making progeny bacteriophage, the infecting
bacteriophage must be transferred with its genetic material to the host bacterial cell.
T2 phages have simple organization having only DNA and protein. Upon infection,
they insert their genetic material while leaving the outer coat as ghost phage
adsorbed on the surface of the bacterium.
Hershey and Chase used different radioactive isotopes for labelling the protein
and DNA of the bacteriophage. S35 was used to label protein, and P32 was used to
label DNA. T2 phages were grown in separate media containing either S35 or P32.
Bacteriophages were allowed to grow in those media for 4 h which was enough to
incorporate the radioactive isotopes. Later, those two separately labelled phages
were made to infect two separate cultures of E. coli. The phage coats were separated
from the bacteria by agitation using a high-speed blender followed by centrifugation.
The E. coli culture which was infected with S35-labelled bacteriophages showed
radioactivity in the culture medium and not inside the bacteria. The bacterial culture
7 Replication of DNA 357

Fig. 7.2 Avery, MacLeod and McCarty’s experiment showing DNA as the transforming principle.
Virulent IIIS cells were heat killed, and carbohydrates, lipids and proteins were extracted from
them. The extract was subsequently treated one by one in a stepwise manner with protease,
ribonuclease and deoxyribonuclease. Avirulent live IIR bacterial cells were then incubated with
these extracts which were treated with protease, ribonuclease and deoxyribonuclease. IIR cells were
transformed to IIIS cells when treated with extracts incubated with protease and RNase. Transfor-
mation ability was lost when the IIR cells were incubated with deoxyribonuclease-treated
extract (Adapted from: Klug et al. 2012)

which was infected by P32-labelled phages showed activity in the bacterial cells
(Fig. 7.3). This again proved that genetic material is DNA and not protein.

7.1.4 Transfection Study

Transfection is the process of introducing purified nucleic acid into eukaryotic cell.
Although the pioneering experiments which proved DNA as the genetic material
involved transformation of nucleic acid into bacterial cells and bacteriophages,
currently transfection is widely used as a part of molecular cloning technique. It
358 T. Banerjee

Fig. 7.3 Demonstration of DNA being the genetic material instead of protein by Hershey and
Chase. T2 bacteriophages were added to media containing S35 and P32 radiolabel, in which E. coli
was already growing. S35 was used to label protein, and P32 was used to label DNA. Progeny phages
became labelled with S35 and P32. Labelled phages were made to infect unlabelled bacteria. When
phage ghosts were separated from the infected bacteria, P32-labelled bacteria and unlabelled phage
ghosts were obtained. However, S35-labelled bacteria were not obtained (Adapted from: Klug et al.
2012)

means ‘infection by transformation of genetic material’. Transfection can be brought

about by various techniques like electroporation, liposome mediated, nanoparticles,
magnetofection, gene gun, etc. Viruses like lentivirus and adenovirus are also used
as vectors for bringing about transfection.
7 Replication of DNA 359

7.2 Molecular Evidence: DNA as Genetic Material

When Friedrich Miescher isolated genetic material in 1869, he realized that this
substance is present in the nucleus, and hence he called it nuclein. However, it took
decades of further research to understand the distribution and organization of DNA.

7.2.1 Indirect Evidence: DNA Distribution

Around the 1880s, Walther Flemming observed threadlike structures present in the
nucleus and named it as chromatin or stainable material. Using innovative
microscopy, he observed threadlike bodies that formed during cell division and
named it as ‘mitosen’. He correctly deduced the movement of chromosomes during
mitosis. Towards the end of the nineteenth century, advancements in microscopy
techniques helped cytogenetics to progress further. Theodor Boveri took
Flemming’s observation to the next level by linking chromosomes as the hereditary
material. He used roundworm Ascaris megalocephala embryo as a model organism
for observing cell cleavage, chromosome distribution and reorganization during cell
division. Walter Sutton in 1902 published the images of individual chromosomes
undergoing various stages of meiosis. He identified 11 pairs of chromosomes, which
had different sizes in the testes of Brachystola magna. However, it was in the early
twentieth century that the work of Thomas Hunt Morgan could establish a correla-
tion between inheritance of genetic traits to the behaviour of chromosomes, thereby
proving ‘chromosome theory of heredity’.
In twentieth century, chromosomes were stained with various dyes and were
found to have a banding pattern. Quinacrine mustard (QM) was one such dye which
was initially used to stain the chromosomes. It alkylates bases preferentially guanine
and also intercalates between the DNA strands. The metaphase stage in human
chromosomes from blood cultures, stained with QM, showed a distinct banding
pattern of fluorescence. Chromosomes 3, 13–15 and Y were observed to be most
intensely stained and were further analysed by ultrafluorimeter (Fig. 7.4).

Fig. 7.4 Banding pattern of

chromosomes in metaphase.
Metaphase stage of human
male chromosome from
leukocyte culture stained with
quinacrine mustard.
Fluorescence image,  2000.
(Image adapted from: T
Caspersson et al.
Experimental Cell Research,
60, 1970, 315–319)
360 T. Banerjee

Later techniques like in situ hybridization methods were developed to analyse the
chromosome distribution. The first generation of in situ hybridization methods used
radioactive nucleic acid probes that produced autoradiographs when a small piece of
photographic film was placed on top of a chromosome spread on a microscope slide.
Decay of 32P radioactive label in the probe exposed the photographic film, which
was then developed in the same way as an autoradiograph. In chromosome
autoradiographs, dark regions corresponded to the chromosome locations of a
DNA sequence hybridized by the probe.

7.2.2 Indirect Evidence: DNA Mutagenesis

Further evidence that proved DNA to be the genetic material was the identification of
variants amongst the wild-type organisms. In 1941, George Wells Beadle and
Edward Lawrie Tatum’s article ‘Genetic Control of Biochemical Reactions in
Neurospora’ explained the mechanism by which genes control biochemical reactions
and how those chemical reactions control the development of organism (Beadle and
Tatum 1941). Initially, Beadle and Tatum observed variations in the eye colour of
Drosophila and later switched to Neurospora crassa. It was easier to study Neuros-
pora as it requires a simple growth medium, has short life cycle, and during
reproduction produces ascospores which are easy to isolate for genetic and biochem-
ical analysis. They exposed the fungus to X-rays and allowed random mutagenesis.
Exposed fungus was then grown on minimal growth medium. Most of the mutants
died on the minimal growth medium. Then they supplemented the growth medium
with essential amino acids or vitamins to make complete medium. Mutants were able
to grow on the complete medium. Those mutants were called as auxotrophs. Most of
the time mutants required one essential nutrient for growth. This showed that during
random mutagenesis only one metabolic pathway got affected. Each mutation
affected the activity of a single enzyme. This led to ‘one gene-one enzyme’
hypothesis.
Another assay based on DNA mutagenesis was developed in the 1970s by
Professor Bruce Ames. This assay tests the ability of a chemical compound or
mixture to induce mutations in DNA. In this assay, mutant strains of the bacteria
Salmonella typhimurium (S. typhimurium) were used. These haploid bacteria had
some mutations in the gene encoding histidine synthesizing enzyme; their genotype
is given as his. Since histidine is essential for making proteins, the mutant bacteria
lacking histidine synthesizing enzymes will die. If the growth media is supplemented
with histidine, these mutants can survive. Sometimes the his mutants revert to his+
state spontaneously by acquiring additional mutations. These new mutations are
known as secondary mutations. Occurrence of secondary mutation causing reversal
of phenotype is rare. Presence of certain chemicals can lead to an increase in the
frequency of occurrence of secondary mutations and cause the his mutants to
become his+ again. Hence, obtaining revertant of S. typhimurium in media
supplemented with trace amounts of histidine and chemicals proves the mutagenic
ability of the added chemicals.
7 Replication of DNA 361

7.2.3 Direct Evidence: rDNA Study

In the 1970s, the advancement of science and advent of recombinant DNA technol-
ogy provided direct evidences for DNA being the genetic material. In 1973, Herbert
Boyer, University of California, and Stanley Cohen, Stanford University, used
E. coli restriction enzyme to insert foreign DNA into bacterial plasmid. Restriction
enzyme EcoRI, discovered by Boyer, cut DNA in a way that it creates staggered
ends. Another DNA molecule cut by the same enzyme will have the same staggered
ends. One DNA molecule cut by the enzyme EcoRI can latch onto another DNA
molecule cut by the same enzyme as they possess the same staggered ends and are
complementary to each other. Stanley Cohen discovered the process by which
plasmid DNA could be isolated and inserted back into other bacteria. Plasmids are
self-replicating DNA molecules present in bacteria apart from its chromosomal
DNA. Plasmid pSC101, isolated by Cohen which was known to impart resistance
to tetracycline, was cleaved by EcoRI. The linearized plasmid was mixed with
another DNA molecule cut by the same enzyme. DNA from both sources joined
together to form new loops. The bacteria E. coli was then transformed with recom-
binant DNA molecule and plated on tetracycline plates. Only the bacteria which
carried the recombinant plasmid having tetracycline resistance gene could grow on
the plate. Hence, the first study of interspecies molecular cloning provided concrete
evidence that DNA imparted the tetracycline resistance phenotype and hence DNA
is the genetic material.

7.2.4 RNA Serves as Genetic Material

Ribonucleic acid (RNA) is the other form of nucleic acid apart from DNA. If both
these are nucleic acids capable of storing genetic information, then why has DNA
evolved as the preferred genetic material over RNA? In the 1960s, it was shown that
mRNA is capable of storing information and ribosomal RNA and transfer RNA are
capable of translating the genetic material into proteins. Around two decades later, it
was also shown that RNA has catalytic activity, and those RNAs are known as
ribozymes. Considering the wide variety of functions, RNA seems to be a better
genetic material than DNA. Based on these, theories were proposed which suggested
that RNA was the first genetic material. Since it is single stranded unlike DNA, it is
highly unstable and could be damaged easily. Therefore, DNA evolved as the
preferred genetic material.

7.2.4.1 RNA as the Genetic Material in Viruses

Tobacco mosaic virus (TMV) was one of the early examples to have RNA as the
genetic material. It just contains protein and RNA without any DNA. Gierer and
Schramm, in 1956, showed that RNA alone is enough to infect tobacco plants.
Hence, RNA was shown to be the genetic material.
In viruses, RNA could exist as a single-stranded or even as a double-stranded
molecule. Based on the RNA, the viruses are classified as single-stranded RNA virus
362 T. Banerjee

or double-stranded RNA viruses. Single-stranded RNA viruses could be further

classified as positive- and negative-strand viruses. In positive-strand virus, the
RNA directly codes for protein acting like mRNA. In negative-strand virus, the
RNA is first transcribed into complementary positive strand by RNA-dependent
RNA polymerase which then codes for the proteins.
There is one more class of viruses containing single-stranded RNA but have a
DNA intermediate. These are known as retroviruses. An enzyme coded by the viral
genetic material, reverse transcriptase, converts the viral RNA into complementary
DNA which is then replicated into double-stranded DNA. This double-stranded
DNA is then integrated into host DNA by the enzyme integrase to code for viral
proteins along with the host proteins. An important member of this family is human
immunodeficiency virus (HIV).
Similar to retroviruses, there are mobile DNA elements, known as
retrotransposons, which are found in eukaryotes. They also encode for reverse
transcriptase enzyme. They are first transcribed into RNA and with the help of
reverse transcriptase are converted back to DNA which then integrates with the
chromosomal DNA at various regions. Since they can change their position by
integrating at different regions, they are called as jumping genes or mobile
elements.

7.3 Structure of DNA Helix

DNA is composed of four nucleotide bases which are linked to each other by
phosphodiester bond into a polynucleotide chain. Bases of one polynucleotide
chain can base pair with the bases of another polynucleotide chain to form double-
stranded DNA duplex. The two polynucleotide chains are in opposite orientation
with respect to each other.

7.3.1 Base Composition Study

Each nucleotide has three basic components: nitrogenous base, a cyclic five carbon
sugar and a phosphate group attached to the 50 -carbon of the sugar. We will discuss
about each component in detail:
Nitrogenous base: The nitrogenous bases could be of two types, single ringed
called pyrimidines and double ringed called purines. Cytosine and thymine are
pyrimidine bases, and adenine and guanine are purine bases. These nitrogen bases
are attached to the 10 -carbon atom of the sugar by an N-glycosidic bond (Fig. 7.5). A
base linked to the sugar is called nucleoside. In DNA, the bases are linked to
deoxyribose sugar and hence forms deoxyribonucleoside, viz. adenine forms adeno-
sine, guanine forms guanosine, cytosine forms cytidine and thymine forms thymi-
dine (Fig. 7.6). In RNA, the bases are linked to ribose sugar and hence are
ribonucleoside. Uracil is a base found in RNA instead of thymine. Upon being
linked to ribose, it is called uridine.
7 Replication of DNA 363

Fig. 7.5 Structure of bases. (a) Chemical structure of nitrogenous bases in RNA and DNA. (b)
Chemical structure of pentose sugars in DNA and RNA (Adapted from: Klug et al. 2012)

Five-carbon sugar: The sugar present in nucleic acids have five carbons and have
cyclic conformation. In RNA, hydroxyl groups are present in 20 and 30 position. In
DNA the 20 hydroxyl is absent, hence is called 20 deoxyribose sugar.
Phosphate: The phosphate group is attached to the 50 -carbon of the sugar by a
phosphoester linkage. When the phosphate group is attached to the nucleoside, it
then becomes nucleotide. The nucleotides are covalently linked by a second
phosphoester bond that joins the 50 -phosphate of one nucleotide to the 30 -OH
group of the adjacent nucleotide. This bond between the phosphate group of 5-
0
-carbon and hydroxyl group of 30 carbon is called phosphodiester bond.
Nucleotides present in the polynucleotide chain have one phosphate and hence are
called deoxynucleotide monophosphate (dNMP) where N is any nitrogenous base.
However, nucleotides present freely have three phosphates and are called
deoxynucleotide triphosphate (dNTP) (Fig. 7.7). Two of the three phosphates are
removed during the formation of phosphodiester bond. The successive linkage of
nucleotides by phosphodiester linkage results in a polynucleotide chain which has
free 30 -OH at one end and free 50 -phosphate at the other end (Fig. 7.8).
364 T. Banerjee

Fig. 7.6 Nomenclature of nucleosides and nucleotides. Structure and nomenclature of nucleosides
and nucleotides of RNA and DNA (Adapted from: Klug et al. 2012)

Fig. 7.7 Structure of nucleoside diphosphates and triphosphates. Structure of deoxythymidine diphos-
phate (dTDP) and adenosine triphosphate (ATP) (Adapted from: Klug et al. 2012)
7 Replication of DNA 365

Fig. 7.8 Depiction of phosphodiester bond. (a) Phosphodiester bond between C-30 and C50 of the
adjacent nucleotides. (b) Short hand notation for a polynucleotide chain (Adapted from: Klug et al.
2012)

7.3.2 X-Ray Diffraction Study

In 1953, Rosalind Franklin and Raymond Gosling solved the X-ray crystal structure
of DNA.
From their crystallographic data, Franklin deduced that DNA existed in two
forms, which are in equilibrium. They reasoned that the presence of two forms of
DNA resulted in unclear diffraction pattern. These forms are A form, the dehydrated
form of DNA, and B form, the hydrated form of DNA. Franklin could isolate the two
forms of DNA by techniques like ‘manipulation of the critical hydration of her
specimens’. A and B forms were then separately crystallized, and those crystal
structures became the basis for Watson and Crick’s double helical DNA model.
The diffraction pattern obtained by Franklin and Wilkins hinted of a two-stranded
helical form. Patterns were observed to be consistent; hence they deciphered that
helix diameter must be constant. The helical turn of DNA correlates to the horizontal
lines in the picture which measures to 34 Å. They also calculated the gap between the
366 T. Banerjee

Table 7.1 Base composition of DNAs from various sources

Base composition Base ratio Combined base ratio
Organism A T G C A/T G/C A + G/C + T A + T/C + G
Human 30.9 29.4 19.9 19.8 1.05 1.00 1.04 1.52
E. coli 24.7 23.6 26.0 25.7 1.04 1.01 1.03 0.93
T7 26.0 26.0 24.0 24.0 1.00 1.00 1.00 1.08
bacteriophage
Source: Chargaff (1950)

base pairs to be 3.4 Å, which led them to conclude that there are ten nucleotide bases
per turn. Franklins X-ray diffraction also showed that sugar phosphate backbone was
on the outside. Chargaff did chemical analysis on the molar content of the bases in
DNA molecule in various organisms and concluded [A] ¼ [T] and [G] ¼ [C], and
total molar content of purines is equal to that of pyrimidines [A + G] ¼ [C + T]
(Table 7.1).

7.3.3 Watson and Crick’s Model

Watson and Crick’s model of DNA double helix was based on the X-ray diffraction
pattern analysis of Franklin and Wilkins (Fig. 7.9). Watson and Crick combined the
physical and chemical data and determined that the two strands are coiled around
each other. The 3D structure of DNA is the B form of DNA. B form exists when
there is plenty of water in the medium. Watson Crick’s model based on Franklin’s
X-ray crystallography study has the following features:

1. DNA molecule consists of two polynucleotide chains which are wound around
each other forming a right-handed (clockwise) helix.
2. The two chains are antiparallel, which means that the free 30 -OH group of one
strand is opposite to the free 50 -phosphate group of the other strand and vice
versa.
3. Sugar phosphate backbone is on the outer side of the helix with the bases
occupying the inner side. The bases are perpendicular to the sugar phosphate
backbone and are stacked on top of each other. The B form of DNA is narrow and
elongated. Helical conformation creates major and minor groove along the axis.
4. The bases of one polynucleotide strand are hydrogen bonded to the opposite
bases present in the other polynucleotide strand. Based on Chargaff’s rule, they
predicted that A is bonded with T by two hydrogen bonds and C is bonded with G
by three hydrogen bonds. AT and GC are called complementary base pairing, and
as per the space filling model, these are the only two permissible base pairings
which can exist in the helix as per Chargaff’s rule.
5. There are approximately 10 bp per 360
rotation of the helix. Each base pair is
twisted 36
to the adjacent base. Each base pair is 0.34 nm apart, so ten base pairs
in each turn encompasses 3.4 nm. The diameter of the helix is 2 nm.
7 Replication of DNA 367

Fig. 7.9 X-ray diffraction pattern of DNA. (a) X-ray diffraction photograph obtained by Rosalind
Franklin. The DNA fibres which were used for diffraction were of the B form. Diffraction pattern
showed strong arcs on the periphery. These arcs represented the periodicity of nitrogenous bases,
which are 3.4 Å apart. The inner cross pattern of spots indicated that DNA is helical in structure. (b)
Watson and Crick DNA double helix model showing base pair interactions, major and minor
groove, pitch and the diameter (Adapted from: Brooker 2018)
368 T. Banerjee

Table 7.2 Differences between A, B and Z forms of DNA

Parameters B-DNA A-DNA Z-DNA
Conformation B-DNA is the Watson- Ribbon-like helix with a Z-DNA is a long and
Crick form of the shorter, broader and thin duplex structure
double helix. The two more open core compared to other forms
strands of the duplex of DNA. Zigzag (hence
are antiparallel and the name) pattern in the
plectonemically coiled phosphodiester
backbone is due to
alternating purine-
pyrimidine sequence
Helix sense Right-handed helix Right-handed helix Left-handed helix
Number of 10 bp per turn 11 bp per turn 12 bp per turn
bases per turn
Helix pitch 34
helix pitch with 28
helix pitch with rise 45
helix pitch with rise
rise of 0.34 nm per of 0.256 nm per base of 0.37 nm per base
base
Helix 2 nm helix diameter 2.3 nm helix diameter 1.8 nm helix diameter
diameter
Base pair Base pairs are almost Base pairs are tilted and Base pairs accommodate
orientation centred over the displaced laterally away distortion
helical axis from the central axis and
closer to the major
groove
Sugar pucker Sugars have C20 Sugars have C30 Guanine base has C30
endoconformation endoconformation endoconformation sugar,
and alternate cytosine
has normal C20
endoconformation
Glycosyl Anti Anti Pyrimidine-anti
angle Purine-syn

6. Keeping in account spatial constraint induced by the complementary base pairing

of AT and GC, the two sugar phosphate backbones are not equidistant from each
other along the helical axis. At certain point they are closer to the axis and at other
points they drift apart from the axis. This unequal spacing results in formation of
grooves; the wider grooves are called major groove, and narrower groove is
called minor groove (Table 7.2).

If the water present in the medium is less, the DNA forms another right-handed
helix known as A form of DNA. A form is shorter and wider than the B-DNA, and
bases are tilted away from the main axis. A form of DNA is found in spores of some
bacteria and some protein-DNA complexes (Fig. 7.10).
Another form of DNA found in nature is the Z-DNA. Unlike the A and B forms, it
is a left-handed helix. Sugar phosphate backbone follows the zigzag path moving
back and forth hence is called Z-DNA. Z-DNA has been shown to contain multiple
stretches of alternating C and G nucleotides. Z-DNA-specific antibodies bind to
regions of the DNA which are undergoing transcription. Hence, it is probable that
7 Replication of DNA 369

Fig. 7.10 Comparison of different forms of DNA: (a) Molecular structures deduced from X-ray
crystallographic image of short fragments of B-DNA and Z-DNA. (b) Space filling model depicting
that B-DNA has distinct minor and major grooves. In Z-DNA, due to the zigzag pattern, minor and
major grooves are not well distinct. Black zigzag lines in the Z-DNA are connecting the phosphate
groups of the DNA backbone (Adapted from: Brooker 2018)

Z-DNA may have role in gene expression. Other secondary structure of DNA like
H-DNA also exists. In this form, a part of the double helix unwinds, and the single-
stranded nucleotide chain then pairs with double helical region to form three
stranded helices.

7.3.3.1 Triplex DNA

In 1957, Alexander Rich, David Davies and Gary Felsenfeld observed that DNA can
attain triple helical forms in the in vitro conditions. Three synthetic strands of DNA
were shown to form a triple helical structure. After 30 years, it was observed that
naturally occurring DNA double helix can also base pair with a synthetic DNA
especially at the major groove creating a DNA triple helix. The base pairing between
the double helix and synthetic DNA followed the normal rules of base pairing as
depicted in the Fig. 7.11. This observation was of particular interest to the scientific
community as it gave a handle to regulate DNA transcription. Short synthetic DNA
strands could be synthesized complementary to the DNA of interest and allowed to
form DNA triple helix. Such triple helical structures were shown to inhibit the DNA
transcription and hence emerged as an important tool for gene silencing.

7.3.3.2 Alternative Forms of DNA

Several alternative forms of DNA right-handed helix models have been shown to
exist in the in vitro conditions. C-DNA exists at highly dehydrated conditions and
has only 9.3 base pairs per turn. It has a helical diameter of 1.9 nm, and the base pairs
are tilted relative to the axis. The sequences which lack guanine residues can exist in
370 T. Banerjee

Fig. 7.11 DNA triplex: (a) In

the ribbon model of DNA
triple helix, it is shown that the
major groove of the DNA
double helix binds with a
synthetic DNA strand. (b) The
third strand base pairs with the
complementary bases of the
major groove following AT
and GC rule. Cytosines of the
third strand are protonated
hence is positively charged
(Adapted from: Brooker
2018)

two other forms in the in vitro conditions, D and E. D has eight bases per turn, and E
has seven bases per turn. Recently discovered P-DNA (named after Linus Pauling) is
narrower and longer with only 2.62 bases per turn. In this form of DNA, phosphate
groups are towards the inside of molecules, and nitrogenous bases are towards the
surface. Other secondary structures of nucleic acids are formed by guanine-rich
sequences. These are helical structures formed by guanine tetrads. Guanine tetrads
are formed by hydrogen bonding in a square plane. This type of hydrogen bonding is
called as Hoogsteen base pairing. Such guanine tetrads can form from one, two or
four strands of DNA molecules. These guanine tetrads can stack on top of each other
to form G-quadruplex. Such quadruplexes have gene regulatory roles and are found
at certain regions more frequently like in the telomeres (Fig. 7.12).

7.4 Analytical Study on DNA and RNA

7.4.1 Absorption of UV Light

DNA absorbs UV light. Its absorption maxima lie at 260 nm (Fig. 7.13).
7 Replication of DNA 371

Fig. 7.12 G quadruplex: Guanine residues of intra or inter strands form Hoogsteen bond to form a
planer structure which stack on top of each other to form G-quartet or G quadruplex (Adapted from:
https://science.institut-curie.org/research/biology-chemistry-of-radiations-cell-signaling-and-cancer-
axis/cmbc/team-teulade-fichou/probes-targeting-g-quadruplex-nucleic-acids)

Fig. 7.13 Absorption spectra of DNA. Absorption of UV light by DNA vs protein at 260 nm.
Absorption maxima lie at 260 nm for DNA. Another peak is observed at 280 nm

Table 7.3 Concentration Nucleic acid Concentration (μg/mL) per A260 unit
of nucleic acid per A260 unit
dsDNA 50
ssDNA 33
ssRNA 40

As shown in the graph (this figure), DNA absorbs maximum UV light around
260 nm depicted by the broad peak. At 280 nm it absorbs only half of the maximum
UV light (this figure). UV absorption is a property of the bases, and each base
absorbs differently. Therefore, when the bases are not hydrogen bonded and are
exposed to UV, they have higher ability to absorb the light. Different DNA
preparations have slightly different absorption peak depending upon the DNA
composition. DNA concentration can be determined by the equation: 1 OD260
unit ¼ 50 μg/mL (Table 7.3).
372 T. Banerjee

Single nucleotides absorb even more strongly than single-stranded DNA because
the stacking interactions between bases of the DNA strand decrease their exposure to
UV light and hence decrease their ability to absorb UV light.

7.4.2 Sedimentation Velocity Centrifugation

The velocity with which a macromolecule moves is mainly a function of its

molecular weight and its shape. The ratio of molecular velocity to centrifugal
force is called the sedimentation coefficient designated as ‘S’ or Svedberg unit.
Therefore, S ¼ velocity/centrifugal force. The size of biological macromolecules is
often referred to in terms of Svedberg units, like prokaryotic ribosome smaller
subunits are called 30S subunit as its S value is 30. S value depends on molecular
weight and shape. Therefore, changes in ‘S’ value depicts changes in molecular
shape (unfolding of biomolecules) or molecular weight (aggregation or dissociation)
depending upon experimental conditions.
One common type of velocity centrifugation is density gradient centrifugation. In
this technique a liquid medium is used whose density is varied along the length of the
centrifugation tube. Sucrose medium is one of the earliest such media which was
used to create density gradient. Various concentrations of sucrose solutions are made
and layered one by one in the centrifugation tube. The highest concentration has the
highest density and is layered at the bottom. Lowest density solution is layered on
top. The density of the molecules to be sedimented is adjusted so that the molecules
having lower density than the density of the sucrose solution are layered at the top of
the tube. The sample is layered on the surface forming a band or a zone. Hence, this
technique is also known as zonal centrifugation (Fig. 7.14). The density gradient
stabilizes the liquid against mixing that might be caused by mechanical forces. After
layering the sample, the tube is centrifuged by swinging bucket rotor. Once the
centrifugation is complete, a hole is punched at the bottom, and successive layers are
collected drop by drop. The presence of different macromolecules in the sample
solution which was layered on top of the sucrose gradient could be analysed.
Equilibrium centrifugation is another velocity centrifugation technique based on
density gradient. In this the macromolecules which are to be sedimented are
suspended in a solution of CsCl whose concentration is approximately equal to
that of the macromolecules. During centrifugation Cs+ ions start sedimenting and
diffusing throughout the tube. Continuous sedimentation and diffusion create a
linear concentration gradient with maximum density at the bottom and decreasing
towards the top. The macromolecules move in the density gradient and stops at the
position where the density of CsCl solution is equal to that of the molecule. Hence,
the macromolecules form a narrow band in the tube. If there are multiple types of
macromolecules with different densities, they form multiple bands. The densest
macromolecule will form the lowest band. This technique has very high resolution.
The naturally occurring DNA molecule with N14 and density 1.708 g/cm3 can be
separated from N15 which has the density of 1.722 g/cm3.
7 Replication of DNA 373

Fig. 7.14 Zonal centrifugation. (a) Formation of gradient. (b) Layering of sample on top of
gradient. (c) Tube is placed in swinging bucket rotor for sample separation. (d) Collection of
samples by punching hole at the bottom

Although both zonal and equilibrium centrifugation use a liquid medium of

varying density for separation of macromolecules, these two methods are working
on different principles. Therefore, on lengthening the centrifugation time, there
might be mixing of different zones of the sucrose gradient with all the sucrose
molecules settling at the bottom, but the CsCl gradient will always maintain the
linear concentration gradient.

7.4.3 Denaturation and Renaturation of Nucleic Acids

DNA double helix has two polynucleotide chains. These are held together by the
hydrogen bonding forces present between the complementary base pairs. Adenine is
374 T. Banerjee

Fig. 7.15 DNA melting curve. Melting temperature, Tm, is depicted by a dotted line. DNA melting
starts at around 70
C. Between 70 and 90
C, absorption at 260 nm constantly increases as the
denaturation continues. Beyond 90
C, curve reaches a plateau phase when the DNA strands are
completely separated (Adapted from: https://members.tripod.com/arnold_dion/RecDNA/)

attached to thymine by two hydrogen bonds, and guanine is attached to cytosine by

three hydrogen bonds. However, hydrogen bonds have very low bond energy and
can be easily disrupted by heat. When the hydrogen bonds are disrupted, the two
polynucleotide chains move apart from each other and lose the normal helical
structure or the native state. This state is called as denatured state of DNA. The
process of transition from the native state to the denatured state is called
denaturation.
The physical property of DNA changes during the process of denaturation.
Single-stranded DNA absorbs light at 260 nm more strongly than the double-
stranded DNA. When the DNA solution is heated, it is observed that after 70
C,
the absorption at 260 nm increases linearly till 90
C (Fig. 7.15). After that there is
no more increase, but the absorption reaches a plateau phase. This process of
denaturation where the DNA strands separate out from each other and become single
stranded is melting of DNA, and the graph showing the linear increase in absorption
as a function of temperature is called the DNA melting curve. Before the increase in
absorption begins, the DNA is in the native double-stranded state. The linear
increase in the absorption is due to the increase in the number of broken hydrogen
bonds in the base pairs. When all the base pairs are separated, the initial part of the
upper plateau is reached indicating complete denaturation and presence of only
single-stranded DNA. The temperature at which the rise in A260 is half complete is
7 Replication of DNA 375

called melting temperature and is designated as Tm. AT has two hydrogen bonds,
whereas GC has three hydrogen bonds. Therefore, more energy is required for
breaking GC bonds than AT bonds. DNA having more GC content will have higher
Tm than DNA with more of AT content. Hence, Tm varies with base composition.
DNA denaturation can also be brought about by increasing the pH. At high pH,
the charge of several groups engaged in hydrogen bonding is changed, and hence
they can no longer participate in hydrogen bonding interactions. At pH higher than
11.3, all the hydrogen bonds are disrupted, and the DNA is completely denatured. If
the salt concentration is low, the strong negative charges present in the phosphate
groups keep the DNA fully extended and single stranded. If the salt concentration is
high enough to neutralize the negative charge of the phosphate groups, DNA folds
back on itself forming intra-strand hydrogen bonds.
Very high temperature can also disrupt the phosphodiester linkage present in the
sugar phosphate backbone leading to breakage of DNA strand. However,
phosphodiester linkage is resistant to alkaline denaturation. Therefore, alkaline
denaturation is considered to be a better method for denaturation.
When the denatured DNA returns back to its native form upon reducing the
temperature or neutralizing the pH, it is called renaturation or reannealing. The
reannealed DNA is called renatured DNA. Renaturation is a slower process than
denaturation and hence requires optimal conditions like:

1. The concentration of salt must be high enough to neutralize the negative charge of
the phosphate groups so as to remove electrostatic repulsion. Usually 0.15–0.5
molar NaCl is sufficient to attain renaturation.
2. The temperature should be high enough to remove non-specific, intra-strand
hydrogen bonding, yet the temperature cannot be too high that inter-strand
hydrogen bonds are not formed. The optimal temperature for renaturation is
20–25
C below the Tm.

The rate-limiting step of renaturation is precise collision between the comple-

mentary strands so that the correct bases are coming opposite to each other. Since
this is dependent on random motion, renaturation is also a function of concentration.
Renaturation can be ascertained by decrease in absorption at 260 nm.

7.4.4 Hypochromic Effect

As discussed above, DNA absorbs UV light of wavelength 260 nm. The absorption
increases as the DNA dissociates into single strands. The increase in absorption at
260 nm due to DNA denaturation is called hyperchromic shift. During DNA
renaturation, the absorption at 260 nm decreases and hence is called the hypochro-
mic shift. Therefore, it can be interpreted that hyperchromic shift is an indication of
DNA denaturation and hypochromic shift depicts DNA renaturation. Hypochromic
and hyperchromic shifts are indications of thermal stability of reassociated DNA
duplexes in a solution (Fig. 7.16).
376 T. Banerjee

Fig. 7.16 A DNA melting curve depicting the increase in UV absorption versus temperature
(hyperchromic effect) for two different DNA molecules having different GC content (Adapted from:
Klug 2012). Molecule having higher GC content has higher Tm

7.4.5 FISH

Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique to

identify certain DNA sequences. Fluorescent probes are used to hybridize with the
complementary DNA sequence. Fluorescent microscopy is then used to localize the
hybridized probe. FISH can also be used for identification and localization of certain
RNA like mRNA, miRNA, etc. (Fig. 7.17). Cot hybridization is also used to isolate
fractions of DNA for use in FISH experiments. The DNA repeats isolated by Cot
fractionation can be used as blocking agents to reduce background signals during
FISH techniques.

7.4.6 DNA Renaturation Kinetics

When DNA renaturation was proposed to be a function of length and complexity of

DNA and solvent viscosity, following facts were already known:

1. The renaturation reaction is approximately second-order reaction.

2. The renaturation occurs at a maximum rate if the temperature is 20–30
C below
the melting temperature (Tm,) of the DNA.
3. A decrease in the molecular weight results in a reduction in the rate of
renaturation.
7 Replication of DNA 377

Fig. 7.17 Fluorescent in situ hybridization. Localization of telomeric DNA using DNA probe (red)
and rDNA probe (green) on metaphase chromosomes from C. porcellus. (Adapted from Svetlana
A. Romanenko et al. A First-Generation Comparative Chromosome Map between Guinea Pig
(Cavia porcellus) and Humans, Plos One 2015)

4. The rate of renaturation of DNA of complex organisms, such as human, is much

slower than the rate of renaturation of DNA of simpler organisms, such as
bacteria.
5. Ionic strength of the electrolyte like NaCl acts as a factor for controlling rate of
renaturation at concentration below 0.4 M.

The formation of first few base pairs is called the nucleation event. It is the rate-
limiting event for renaturation, and the rest of the reannealing is the zippering up
action which is the faster process. Since there are two rates of the renaturation
reaction, it is a second-order rate kinetics. It was proposed that nucleation starts at
certain preferred sites known as nucleation sites. Let’s denote nucleation site by β.
Let L be the average number of nucleotides per single strand of the denatured DNA
preparation and P be the denatured phosphate concentration. Then βP/2N is the
average concentration of the nucleation site. kn represents the rate constant for
nucleation at any such site. Rate of nucleation is then kn(βP/2N )2. The rate of
nucleation at all sites is kn(βP/2N )2βN. Rate of base pair formation is v ¼ knβ3(L/
4N )P2.
Renaturation is dependent on temperature and concentration of DNA. When the
temperature decreases below Tm, the reaction rate increases, with the maximum rate
378 T. Banerjee

at 15–30
C below Tm. If the temperature decreases further, then the renaturation rate
decreases. Let N be the number of base pairs in non-repeating sequence of a DNA
and L be the average number of nucleotides per single strand of the denatured DNA
preparation; the rate constant is approximately k2 ¼ 3  105L0.5N1. mole1 s1 at
(Tm  25)
C and at [Na+] ¼ 1.0 mole in aqueous solution. This equation also
depicts a second-order rate kinetics. If the GC content of the DNA is high, the
reaction rate increases slightly. The reaction rate at maximum temperature is
inversely proportional to the solvent viscosity. If the viscosity of the medium is
changed by adding components like glycerol or NaClO4, Tm can change
significantly (Wetmur 1968).

7.4.7 Repetitive DNA

Repetitive DNA sequences renature faster than single copy sequences. Their
reassociation rates and thermal stability of renatured duplexes are also significantly
different. These differences could be studied by Cot analysis which yields valuable
information about the types of DNA sequences that are present in genomes and their
organization. Repetitive sequences are known to reassociate much faster than the
single-copy sequences. Hence, depending upon the reassociation time, the amount of
repetitive sequence present in the DNA can be revealed (Fig. 7.18) (Graham 2001).
There are various types of repetitive sequences present in eukaryotes. The fastest
reassociating class is the ‘foldback’ sequence. These are sequences within the single
DNA strand and can fold back upon itself. As there is no diffusion and random
collision factors involved, the snapping back process is really rapid. A second class
of repetitive DNA is satellite DNA sequences. They are tandemly repeated
sequences. Tandem repeats range in length from a few base pairs to a few thousand
base pairs. The third type of reassociating sequences are interspersed repeats. The
number of copies of these sequences may vary from a few to hundreds of thousand

Fig. 7.18 Cot curve of DNA.

Log Cot vs ssDNA represent
the amount of repetitive and
single-copy
sequences (Adapted from:
https://en.wikipedia.org/wiki/
Cot_analysis)
7 Replication of DNA 379

copies. ‘Alu’ is one such repeat present in 500,000 copies. Fourth category is ‘low
copy long repeats’. Red-green colour vision gene belongs to this category. Genetic
changes have occurred over time in all these groups, and they have acquired
sequence divergence. Divergence amongst DNA sequence repeats can be measured
by Cot analysis.
DNA concentration (Co), the time allowed for reassociation (t) and the sequence
organization of the DNA are the factors deciding the extent of reassociation. Since
the extent of reassociation is directly proportional to both Co and t, their arithmetic
product (Cot) denotes the extent of reassociation. A DNA solution whose
reassociation is 50% complete at Cot ¼ 100 reassociates much more rapidly than
the DNA solution whose reassociation is 50% complete at Cot ¼ 10,000. The DNA
of the first solution is said to have much less sequence complexity. The sequence
complexity of a genome is the total length of the different sequences it contains,
measured in nucleotide pairs. For species with no repetitive DNA, the sequence
complexity is equal to the genome size. Thermal stability of a renatured DNA can be
assessed by Cot analysis. If there is DNA mismatch, thermal stability is less, and
hence lesser temperature will be required to separate the strands. The difference in
the dissociation temperature when compared to the dissociation of native DNA can
be used to calculate the percentage of DNA mismatch during reassociation. Greater
is the difference; higher is the percentage of mismatch. The ability of RNA
associating with DNA to form RNA/DNA duplex can be analysed by Cot curve.
In this case it is known as Rot curve.

7.4.8 Electrophoresis of Nucleic Acids

DNA is negatively charged due to the presence of phosphate groups. In an electric

field, the DNA moves from negative to positive electrode (Fig. 7.19). The rate of
movement depends on the charge of the molecule. The mass of the molecule plays
no direct role in electrophoretic migration; however, larger molecules face larger
frictional forces which adversely affect their mobility. Hence, separation of
molecules in the electrophoretic field is dependent on charge to mass ratio. The
most common electrophoresis is gel electrophoresis. Agarose or polyacrylamide gels
are prepared containing small slots for loading the DNA samples. Those slots are
called wells. The speed of migration of DNA through the gel matrix will depend on
the size of the DNA fragment (smaller fragment will run faster) and on its tertiary
structure (supercoiled, relaxed or open circular or linear). Supercoiled DNA is very
compact (less surface area and less frictional resistance) and hence moves fastest.
For linear DNA, rate of migration is inversely proportional to molecular mass. The
distance (D) moved is approximately D ¼ a  b log M (molecular mass), in which
a and b are constants, depending on buffer, gel concentration and temperature.

7.4.8.1 Pulse Field Gel Electrophoresis

Standard gel electrophoresis cannot separate DNA molecules larger than 20 kb.
PFGE can separate as large as 200–3000 kb DNA molecules. During PFGE,
380 T. Banerjee

Fig. 7.19 Electrophoresis of nucleic acids. Unidirectional electric field is applied, and the DNA
being negatively charged moves towards the positive electrode. Charge to mass ratio determines the
mobility of proteins in the gel. The smallest fragment moves fastest, and the larger fragments stay
near the well

orientation of electric field changes periodically (Fig. 7.20). This allows the DNA to
bend like a snake through the gel matrix pores.

7.5 DNA Replication: Semi-conservative

DNA was proposed to be a double helix having antiparallel strands. That means, if
one of the strands is running from 30 to 50 , then the other strand is running 50 to 30 .
Therefore, 30 -OH group of one strand is opposite to the 50 -PO3 group of the other.
Directionality is important for understanding the functions of enzymes involved in
DNA replication.
In the 1950s, three different modes of replication were proposed. First model
proposed conservative mode of replication. As per this hypothesis, the parental
strands replicate to form daughter strands, and after replication, the parental strands
stay together to form one double helix, and the new daughter strands form a new
7 Replication of DNA 381

Fig. 7.20 Pulse field gel electrophoresis of DNA separation. The direction of electric field changes
periodically, and depending on the direction of the electric field, direction of movement of DNA
also changes. DNA moves through the gel like a snake (Adapted from: Sharma-Kuinkel et al. 2016)

double helix. Second model proposed semi-conservative mode of replication. As

per this, the parental strands separate out, and each strand synthesizes the comple-
mentary daughter strand. The double helix is then formed between one parental and
one daughter strand. Dispersive model proposed that the segment of parental strands
is interspersed by newly synthesized daughter strands (Fig. 7.21).
In 1958, Matthew Meselson and Franklin Stahl showed that DNA replication
actually follows semi-conservative model. They grew E. coli in a medium containing
N15 for several generations (Meselson and Stahl 1958). The DNA of those bacteria
was all labelled by heavy isotope of nitrogen having no N14 present in the DNA.
They then grew the bacteria for different time periods in normal medium containing
only N14 and no N15. The rationale behind their methodology was that after every
cycle of DNA replication N14 will be incorporated in the daughter strands. New
strands incorporating N14 will be lighter than the parental strands containing N15 and
hence will give separate bands in the CsCl density gradient. 108 E. coli were lysed
after different time periods and subjected to CsCl density gradient centrifugation at
382 T. Banerjee

Fig. 7.21 Three possible models of DNA replication. The parent strands are shown in purple, and
daughter strands are shown in blue. (a) In conservative model, the parental strands form a separate
duplex, and daughter strands form a separate double strand. (b) In semi-conservative model, one of
the parent strand base pairs with one of the daughter strands. (c) In dispersive model, both parent
and daughter DNA are present in the same strand (Adapted from: Brooker 2018)

around 31,000 rpm. Since the sedimentation and diffusion are opposing forces, they
produce a stable concentration gradient of the caesium chloride. These forces create
a continuous increase of density along the direction of centrifugal force. The
resulting gradient drives the DNA where its density is equal to the density of the
CsCl solution. This is called equilibrium point. DNA stays as a band at this point.
Lighter DNA will form wider band, and heavier DNA will form narrow band. The
reason is lighter DNA is subjected to more diffusion force compared to the heavier
DNA (Fig. 7.22).
Since the parental strands had N15 and with each replication cycle more and more
14
N was getting incorporated, it was expected that there will be multiple DNA
molecules with different densities. Density of the DNA molecules will be reducing
after each cycle. Due to the centrifugal force, the densest band will be at the extreme
end from the axis of centrifugation. As the density decreases, the bands will move
closer to the axis. They aliquoted around 109 E. coli, lysed them, centrifuged at
45,000 rpm and allowed them to reach equilibrium with the CsCl gradient. The DNA
in the tube was then observed by UV light. Important interpretations of the experi-
ment are as follows (Fig. 7.23)

1. The bands are moving closer to the axis of rotation with time. So, the density of
sample is reducing with incorporation of N14.
2. Initially there was only one band, so all the DNA had same molecular weight.
Then at 0.3 and 0.7 cycle, lighter bands appeared on the left. It indicated the
presence of an intermediate density DNA species whose concentration was less
than the initial band. The intensity of the band denoted the concentration of the
DNA species.
7 Replication of DNA 383

Fig. 7.22 Ultraviolet absorption photographs showing DNA bands resulting from density gradient
centrifugation of bacterial lysates. Samples were collected at different time intervals after adding
excess of N14 substrates to a growing N15-labelled culture. Each photograph was taken after 20 h of
centrifugation at 44,770 rpm. The time of sampling is measured from the time of the addition of N14
in units of the generation time. (Adapted from: Meselson M and Stahl F.W. 1958, 44, PNAS,
671–682)
384 T. Banerjee

Fig. 7.23 The conceptual explanation of Meselson and Stahl experiment. Cells were initially
grown in N15-containing media so that the entire DNA is labelled with heavy isotope of nitrogen.
Then N14 was added and incubated for various lengths of time and was then lysed. The lysate is then
loaded on CsCl gradient and centrifuged. DNA in the gradient can be observed under UV
light (Adapted from: Brooker 2018)

3. After one round of replication, a single band appeared which is half heavy as the
initial band. It is consistent with both semi-conservative and dispersive model. In
contrast, as per the conservative model, there should have been two bands, one
heavy and one light. Clearly conservative model was incorrect.
4. To confirm whether it was semi-conservative or dispersive model, Meselson and
Stahl checked further replication cycle. At 1.9 cycle, there were two bands, one
for the mixture of heavy and light bands and the other for the all light DNA. As
per dispersive model, there should have been only one band as each strand of
7 Replication of DNA 385

DNA would have carried 1/4 heavy DNA and 3/4 light DNA. Since single band
was not obtained after two replication cycles, dispersive model was also
discarded, and semi-conservative model was accepted.

7.6 Different Modes of DNA Replication

Till now we have learnt how DNA was shown to be the genetic material, what are the
various techniques to analyse DNA, the structure of DNA and the semi-conservative
mode of its replication. In this section we will learn that different organisms have
different modes of replication. We will begin with the bacterial replication and then
move on to eukaryotic replication.
Bacteria have single origin of replication: The Fig. 7.24 shows the autoradio-
gram of bacterial chromosomal DNA replication. The site at which the DNA
replication begins is called origin of replication. At this site the DNA double strand
separates from each other forming two single strands at that site. Cairns showed by
autoradiography the replication in E.coli. The autoradiographic image appeared like
Greek letter θ (Fig. 7.24). So, it was known as theta mode of replication. As the
single strands are present for only a few base pairs and the rest of the DNA is still in
the double-stranded form, it gives an appearance of two ‘Y’-shaped fork facing each
other. These are called replication forks. As the replication proceeds, the single-
stranded region lengthens, and so the replication forks also increase in size. Hence,
the replication proceeds in both the directions, opposite to each other. Therefore, it is
called bidirectional replication. Eventually the replication forks extending on
opposite directions meet each other to complete the replication. Since it is closed
circular DNA, unwinding of DNA induces positive supercoiling. Most of the
circular DNAs are negatively supercoiled; therefore, initial unwinding is not diffi-
cult; however as replication proceeds, unwinding becomes difficult due to the
formation of positive supercoils. Those are removed by the action of topoisomerase

Fig. 7.24 θ-mode of

replication in E. coli. In
bacteria, as closed circular
DNA proceeds for replication,
the parent strands separate,
and replication fork proceeds
in opposite direction; it takes
the form of Greek letter
θ (Adapted from: Brooker
2018)
386 T. Banerjee

Fig. 7.25 Rolling circle mode of replication. The parent strand is shown in black and daughter
strand in orange (Adapted from: Maloy et al. 1994)

II which creates a nick in one of the strands which swivels around the other strand to
release the positive supercoil.
In some of the viruses, some bacterial plasmids and circular genomes like that of
mitochondria replicate by rolling circle replication. In this mode of replication, one
of the strands of closed circular double-stranded DNA is nicked. A free 30 -OH and
free 50 P are generated. The other strand remains intact and acts as template for the
leading strand. DNA polymerase uses the free 30 -OH of the nicked strand and starts
synthesizing the DNA complementary to the intact circular DNA (Fig. 7.25). There-
fore, the newly synthesized DNA is covalently linked nicked strand. The new
daughter molecules are present as linear DNA. Since the template DNA is present
as a closed circle, the DNA polymerase synthesizes the complementary strand
multiple times producing a linear concatemer of repeated sequences. These
concatemeric DNAs are often essential in phage replication where these are pack-
aged inside the viral particles. Even in bacterial mating, the donor DNA is trans-
ferred to the recipient by rolling circle replication. The parental strand for the lagging
strand is displaced and replicates by usual discontinuous replication method.
Another rolling circle replication variant generates linear DNA from closed
circular double-stranded DNA. It is called looped rolling circle replication. ϕ174
replicates by this mode. A phage protein (the A protein) nicks the viral strand
replication origin and becomes covalently linked to the newly formed 50 P. DNA
pol III recognizes the newly formed 30 -OH group and starts synthesis displacing the
nicked parental strand called the (+) strand. This strand becomes coated with SSB
proteins and cannot act as template. Synthesis continues until the origin is reached.
At this point the A protein binds to the 30 -OH group of the (+) strand and joins the
30 -OH and 50 -P groups of the (+) strand, dissociates and attaches to the newly
synthesized (+) strand (Fig. 7.25) (Khan et al. 1997).
In looped rolling circle replication of phage ϕ174, the A protein nicks a supercoil
terminus of a strand, known as the (+) strand. Rolling circle replication ensues to
generate a daughter strand (orange) and a displaced (+) strand bound by single-
strand binding (SSB) proteins. The displaced strand remains covalently linked to the
A protein and hangs like a loop. Hence, is called looped rolling circle replication.
When the synthesis of daughter (+) is complete, the entire parent (+) strand is
displaced and is then cleaved from the daughter (+) strand. It is thereafter
circularized by the joining activity of the A protein. The DNA molecule with the
7 Replication of DNA 387

Fig. 7.26 Looped rolling circle replication of phage 174. Parent strands are shown in black and
daughter strand in orange. As the two strands separate out, one of the strands is displaced and gets
bound by SSB proteins. The displaced strand remains bound by A protein and hangs like a
loop (Adapted from: Maloy et al. 1994)

new daughter strand is now ready for next round of replication. In this mode, the ()
strand is never cleaved (Fig. 7.26).
In eukaryotes, the chromosomes are longer and linear. Hence, to complete the
process of replication faster, they have multiple origins of replication (Fig. 7.27).
Evidence for multiple origins of replication was provided in 1968 by Joel Huberman
and Arthur Riggs provided by adding a radiolabelled nucleoside
(3H-deoxythymidine) to a culture of actively proliferating cells. For a brief period
of time, labelled thymidine was given, and then large amount of unlabelled thymi-
dine was added. Chromosomes were then isolated and subjected to autoradiography.
It was observed that labelled thymidine was incorporated at certain intervals.
Therefore, it was proven that there are multiple origins of replication. The replication
fork is formed similar to that of prokaryotes and proceeds bidirectionally. There are
multiple replication forks which proceed simultaneously. Once the adjacent replica-
tion forks meet each other, they stop progressing, and replication eventually comes
to an end when all the replications forks stop proceeding.

7.7 Mechanism of DNA Polymerase

DNA polymerase II has many subunits. Out of those, the β-subunit acts as a clamp
(Table 7.5). The two β-subunits of the two DNA polymerase come together to form a
ring. The hole of the ring is wide enough to accommodate one turn of DNA double
strand. The clamp is loaded on the DNA with the help of other subunits which are
388 T. Banerjee

Fig. 7.27 Replication of eukaryotic chromosomes. (a) There are multiple origins of replication,
and all replication forks proceed directionally. When the replication fork merges, the replication is
completed forming two sister chromatids (Adapted from: Brooker 2018). (b) A microscopic image
of multiple origins of replication in a eukaryotic chromosome. Each origin is observed as a
replication bubble. Arrows are marked to identify the bubbles [Adapted from: A. Blumenthal,
H. Kriegstein, and D. Hogness, ‘The units of DNA replication in Drosophila melanogaster
chromosomes’, Cold Spring Harbor Symp. Auant. Biol. 38, 205–223 (1974)]

Table 7.5 Subunits of the DNA polymerase III holoenzyme

Subunit Function Groupings
α 50 –30 polymerization
ε 30 –50 exonuclease
θ Core assembly
γ, δ, δ’ Load enzyme on template (serves as clamp loader) γ complex
χ, ψ
β Sliding clamp structure (processivity factor)
τ Dimerizes core complex

together known as clamp loader. In the presence of β-subunit, the DNA polymerase
has the synthesis rate of 750 nucleotides per second. In the absence of β-subunit, the
synthesis rate is only 20 nucleotides per second as the enzyme falls off the template
DNA after synthesis of only a few bonds. Therefore, the β-subunit is known to
increase the processivity of the enzyme.
The α subunit is the catalytic subunit. In the catalytic site, the incoming nucleo-
tide enters following the base pair rule. The incoming nucleotide has three
phosphates which gets covalently linked to the last nucleotide added to the newly
synthesized strand at its free 30 -OH group. The catalysis leads to removal of two
terminal (β and γ) phosphates of the incoming nucleotide in the form of pyrophos-
phate. The pyrophosphate is later degraded into Pi. The ester bond is formed
between the α-phosphate of the incoming nucleotide and 30 -OH of the last nucleotide
added in the newly synthesized strand. Therefore, the direction of DNA synthesis is
always 50 to 30 .
7 Replication of DNA 389

Table 7.4 Properties of Properties I II III

bacterial DNA
Initiation of chain synthesis   
polymerase I, II and III
50 –30 polymerization + + +
30 –50 exonuclease activity + + +
50 –30 exonuclease activity +  
Molecules of polymerase/cell 400 ? 15

DNA synthesis occurs with high amount of accuracy; only one mismatch occurs
per 100 million bases. That is called as fidelity of the enzyme.
The structure of the active site of DNA polymerase allows only AT and GC
pairing. Any incorrect nucleotide entering the active site leads to helix distortion
Therefore, incorrect nucleotides can rarely occupy the active site. The correct
nucleotide after entering the active site leads to conformational change in the active
site and induced fit mechanism of catalysis ensues. AT and GC base pairs have very
low potential energy and hence are extremely stable compared to incorrect base
pairs. The stability of the bases is another factor leading to high accuracy of DNA
synthesis.
DNA polymerase III has 30 to 50 exonucleolytic activity (Table 7.4). In case there
is any mismatch, the enzyme has 30 exonuclease site which can remove the wrongly
incorporated base. This is called proofreading activity. Due to the proofreading
ability of the enzyme, its fidelity increases further (Table 7.5) (Lamers et al. 2006).

7.8 Process of DNA Replication

Replication is initiated by DnaA protein at the origin: Origin of replication in E.

coli is called OriC. Three types of DNA sequences are found in OriC: AT rich, DnaA
box binding region and GATC methylation sites.
There are five DnaA boxes and three AT-rich regions. GATC methylation sites
precede the AT-rich regions. DnaA protein in the ATP-bound state binds to the five
DnaA boxes, and due to protein binding, DNA strands bend around the proteins
(Fig. 7.28). Protein HU and IHF then bind and help to unwind the DNA at the
AT-rich sites. Since AT has only two hydrogen bonds, it is relatively easy to be
separated compared to GC-rich regions.
Once the DNA strands have separated, DnaA along with DnaC recruits a helicase
to unwind the double helix. The separation of DNA strands to form single strands
requires ATP. The DnaA protein remains bound to the single strand and moves
along the replication fork as the directional replication proceeds (Fig. 7.29,
Table 7.6).
GATC methylation sites are methylated at the adenine residues by the enzyme
DNA adenine methyl transferase or Dam. All the methylation sites need to be
methylated before the replication can begin. Since the newly made daughter strands
are unmethylated, they cannot undergo replication again unless all the methylation
sites are methylated.
390 T. Banerjee

Fig. 7.28 The sequences of OriC in E. coli. The AT-rich sequence is composed of three similar
sequences that are 13 bp long and highlighted in blue and five DnaA boxes that are highlighted in
orange. The GATC methylation sites are underlined (Adapted from: Brooker 2018)

7.8.1 DNA Replication at the Replication Fork

Several enzymes are involved in replication accomplishing different functions. In

this section we will learn about those enzymes.

7.8.2 Unwinding of Double Helix

As we learnt in the earlier section, DNA helicases are required to unwind the DNA. It
requires ATP to separate the two strands. As the strand separation proceeds, positive
supercoils are induced in the region ahead of separated strands. DNA gyrase or
topoisomerase II helps in releasing the positive supercoiling. To ensure that the
separated strands stay in the single-stranded state, single-strand binding proteins
bind to the single strand of DNA.

7.8.3 Synthesis of RNA Primer

The enzyme principally involved in replication is called DNA polymerase. How-

ever, this enzyme cannot start de novo synthesis of DNA strand. It can only extend
7 Replication of DNA 391

Fig. 7.29 Replication in E. coli. Proteins involved in replication at various stages have been
indicated. AT-rich strands separate first, and DnaA protein binds to DnaA region. DNA helicase
binds in the AT-rich region and proceeds in the opposite directions (Adapted from: Brooker 2018)
392 T. Banerjee

Table 7.6 Proteins involved in E.coli DNA replication

Common name Function
DnaA proteins Bind to DnaA box sequence within the origin to initiate DNA
replication
DnaC proteins Helps DnaA in the recruitment of DNA helicase
DNA helicase (DnaB) Separate double-stranded DNA
Topoisomerase II (DNA Removes positive supercoiling in front of the replication fork
gyrase)
Single-strand binding Bind to single-stranded DNA and stabilizes in single-stranded
proteins form
Primase Synthesizes short RNA primers
DNA polymerase III Synthesizes DNA in the leading and lagging strand
DNA polymerase I Removes RNA primers, fills in gap with DNA
DNA ligase Covalently attaches adjacent Okazaki fragment
Tus Binds to ter sequences and prevents the advancement of the
replication fork

the polynucleotide chain to form DNA. Therefore, it requires a short RNA primer of
10–12 nucleotides, which can be extended by DNA polymerase to synthesize the
new strand. Primase enzyme has the capability of synthesizing de novo short RNA
primers. The direction of synthesis of polynucleotide chain synthesis is always from
50 to 30 direction. Therefore, one of the parental strands having 30 to 50 orientation
acts as template for synthesis of new strand from 50 to 30 direction maintaining the
antiparallel orientation. For the synthesis of this strand, only one RNA primer is
required and is called the leading strand. For the other parental strand having
orientation 50 to 30 , the new strand synthesis requires several primers at short
intervals, and those primers are then extended in the 50 to 30 direction, and those
short DNA strands are later ligated together. This mode of synthesis is slower and
hence called the lagging strand.

7.8.4 Synthesis of DNA by DNA Polymerase

In E. coli there are five DNA polymerase enzymes. DNA polymerases I and III are
the major enzymes which are involved in DNA replication, whereas the DNA
polymerases II, IV and V are involved in repair of damaged DNA. DNA polymerase
III is the principal enzyme involved in replication. It has ten subunits. The α subunit
carries out the function of synthesizing phosphodiester linkage between adjacent
nucleotides (Table 7.5). The other nine subunits are involved in significant accessory
roles in polynucleotide chain synthesis (Fig. 7.30, Table 7.5). DNA polymerase I is
involved in removing the RNA primer and filling the gaps between the short
polynucleotide chains (Fig. 7.31).
Different bacterial polymerases have different subunit compositions. However,
the catalytic subunit in all these polymerases resembles the structure of the human
fist (Fig. 7.32). The template traverse through the palm, thumb and fingers is
7 Replication of DNA 393

Fig. 7.30 Proteins involved in bacterial DNA replication. Proteins like topoisomerase II, DNA
helicase, single-strand binding proteins, primase, DNA ligase and DNA pol III binding to the DNA
strands at the replication fork (Adapted from: Brooker 2018)

wrapped around it. DNA polymerase allows attachment of incoming nucleotides

only at the 30 end. It also removes incorrect nucleotides though 30 to 50 exonuclease
activity.

7.8.5 Synthesis of Leading and Lagging Strand

We learnt in the previous section that DNA polymerase lacks the ability of de novo
polynucleotide chain synthesis and can extend polynucleotide only in 50 to 30
direction by adding new nucleotides at the 30 -OH end. Due to these limitations,
both the DNA strands cannot be synthesized at the same rate. One of these becomes
the leading strand, and the other becomes the lagging strand. In the leading strand,
one RNA primer is made by primase, and DNA polymerase III extends it towards the
opening of the replication fork in the 50 to 30 direction.
In the lagging strand, although the direction of synthesis is from 50 to 30 , it occurs
away from the opening of the replication fork. Several RNA primers are made by
primase to facilitate synthesis of DNA fragments in the 50 to 30 direction. These
fragments are 1000–2000 nucleotides long. In humans, these fragments are 100–200
nucleotides long (Fig. 7.33). All these fragments have short RNA primers which are
later removed by DNA pol I. The shorter fragments of DNA are called Okazaki
fragments after their discoverer, Reiji and Tsuneko Okazaki. DNA pol I has 50 to 30
exonuclease activity. It removes the RNA primer from 50 to 30 direction and then fills
the vacant space by synthesizing DNA in the 50 to 30 direction. It uses the 30 end of
the adjacent Okazaki fragment to fill in the space. The adjacent Okazaki fragments
are then ligated together by DNA ligase. DNA ligase in bacteria depends on NAD+
for energy, and in eukaryotes and archaea, it depends on ATP.
394 T. Banerjee

Fig. 7.31 DNA replication prokaryotes. (1) Helicase breaks hydrogen bonds and relaxes
supercoiling. (2) SSB proteins stabilize single strand. (3) DnaG synthesizes RNA primers.
(4) DNA pol III synthesizes daughter strands. (5) Leading and lagging strand synthesis. (6) DNA
pol I removes RNA primers. (7) DNA ligase joins Okazaki fragments (Adapted from: Sanders and
Bowman 2015)
7 Replication of DNA 395

Fig. 7.32 DNA pol III holoenzyme. (a) Two core enzymes attached to tau arms, clamp loader and
a sliding clamp (Adapted from: Sanders and Bowman 2015). (b) Model of pol III side view binding
to DNA (Adapted from: Brooker 2018). The catalytic subunit of DNA polymerase resembles a hand
that is wrapped around the template strand. Thus, the movement of DNA polymerase along the
template strand is similar to a hand that is sliding along a rope (Lamers et al. 2006)

7.8.6 Formation of DNA Replication Complex

Separation of DNA strands and synthesis of RNA primers proceed along the
complex of helicase and primase enzymes. These together make the primosome
complex. This complex helps to lead the replication fork ahead. The primosome then
physically associates with two DNA polymerase holoenzymes to form the
replisome complex. Two DNA polymerases III act to replicate the leading and
lagging strands (Fig. 7.34).
Two DNA polymerases move together as a unit. The lagging strand is looped out
with respect to the DNA polymerase that synthesizes the lagging strand. This looping
makes DNA accessible to the primase for RNA primer synthesis as well as to the
polymerase so that it can synthesize DNA in 50 to 30 direction. Therefore, looping
helps DNA polymerase dimer to move as a unit although there is a difference in the
rate and mechanism of synthesis of the two strands. When the lagging strand poly-
merase reaches the end of Okazaki fragment, it gets released from the template and
jumps to the RNA primer next to it. DNA clamp loader loads the DNA polymerase to
the next RNA primer. The proliferating cell nuclear antigen (PCNA) protein
functions as the sliding clamp in archaeal and eukaryotic replication, encircling the
DNA template strand. Replication factor C (RFC) complex connects the DNA
polymerases to the clamp loader and sliding clamp (Figs. 7.34 and 7.35).
Replication termination: In E. coli, opposite to the OriC sequence, there are
termination sequences. Usually there are two termination sequences. One of the
termination sequences, T1, inhibits the replication fork from left to right, and the
other termination sequence, T2 prevents progression of replication fork from right to
left (Fig. 7.36). Termination sequences are bound by proteins known as termination
utilization sequences (tus). If one of the replication forks is halted by one of the
termination sequences, the other replication fork progression stops when it meets the
halted DNA polymerase. DNA ligase covalently links the two newly synthesized
396 T. Banerjee

Fig. 7.33 Synthesis of leading and lagging strands. Parental strands are shown in purple, newly
synthesized DNA in light blue and primers in light red (Adapted from: Brooker 2018)
7 Replication of DNA 397

Fig. 7.34 A three-dimensional view of DNA replication. DNA helicase and primase associate
together to form a primosome. The primosome associates with two DNA polymerase holoenzymes
to form the replisome (Adapted from: Brooker 2018)

Fig. 7.35 DNA synthesis at a single replication fork. Enzymes and proteins involved in the
process (Adapted from: Klug et al. 2012)

strands and forms two double-stranded closed circular DNA molecules. Sometimes,
two double-stranded circular DNA molecules are intertwined after replication.
Topoisomerase II then introduces a nick in one of the DNA molecules to release
the intertwining.

7.9 Mechanism of DNA Ligase

DNA ligase is an enzyme that can join DNA chains to each other. The most well-
studied ligases are those isolated from E.coli and T4 phage-infected E. coli known as
T4 DNA ligase. Both the enzymes catalyse the synthesis of phosphodiester bonds
between adjacent 30 -hydroxyl and 50 -phosphoryl termini in duplex DNA.
Phosphodiester bond synthesis catalysed by the E. coli ligase is coupled to cleavage
of the pyrophosphate bond of diphosphopyridine nucleotide (DPN). Hydrolysis of
398 T. Banerjee

Fig. 7.36 Termination of DNA replication. Two sites in bacterial chromosome shown in rose-
coloured rectangle and ter sequences designated T1 and T2. The T1 site prevents the advancement
of fork from left to right, and T2 site prevents the advancement from right to left. Binding of Tus
prevents the replication fork from proceeding past the ter sequences (Adapted from: Brooker 2018)

Fig. 7.37 Synthesis of a phosphodiester bond. It is formed between adjacent 30 -hydroxyl and
50 -phosphoryl group in duplex DNA by E. coli DPN and T4 (ATP) DNA ligases. (Adapted from:
Lehman I R, Science, 186, 790–797)

the α-β-pyrophosphate bond of adenosine triphosphate (ATP) provides the energy

for phosphodiester bond synthesis by the ligases.
In the case of the E. coli ligase, enzyme itself links with DPN to form ligase-
adenylate. Next, the adenylyl group is transferred from the enzyme to the DNA. This
creates a new pyrophosphate linkage between the adenosine monophosphate (AMP)
and the 50 -phosphoryl terminus at the nick. Finally, the 50 -phosphate is attacked by
the adjacent 30 -hydroxyl group to form a phosphodiester bond, and AMP is
eliminated. The same sequence of reactions is catalysed by the T4 DNA ligase,
except that in the first step the enzyme reacts with ATP rather than with DPN, and
PPi is released (Fig. 7.37) (Lehman 1974).
7 Replication of DNA 399

7.10 Prokaryotic vs Eukaryotic DNA Replication

In eukaryote, the DNA is packaged more tightly with many proteins like histones to
form linear DNA called chromosomes. The cell cycle is regulated much more tightly
than prokaryotes. Therefore, eukaryotic replication is different from that of
prokaryotes. In this section we will learn about the eukaryotic replication in detail
highlighting its differences from prokaryotic replication.
DNA replication in both prokaryotes and eukaryotes require enzymes like
primase, DNA polymerase, helicase, topoisomerase, single-strand binding proteins
and DNA ligase. However, the molecular structure of these enzymes may be
different in prokaryotes and eukaryotes.
As discussed earlier, eukaryotic chromosomes have multiple origins of replica-
tion. The molecular details of origin of replication in eukaryotes have been studied in
Saccharomyces cerevisiae. The origins are known as autonomously replicating
sequences (ARS). These are about 50 bp in length and are rich in AT sequences.
Certain consensus sequences like ATTTAT(A or G)TTTA are present in ARS.
These are similar to bacteria having AT-rich regions called DnaA box.
In lower eukaryotes like Saccharomyces cerevisiae, origins are determined by
DNA sequences. But in higher eukaryotes, origin may not be determined by DNA
sequences. Chromatin packaging and histone modifications also play significant
roles in deciding origins of replication.
In eukaryotes, a complex of proteins known as origin recognition complex
(ORC) forms to initiate the formation of pre-replication complex (preRC) in G1
phase by promoting the binding of Cdc6, Cdt1 and a group of 6 MCM helicases. The
binding of MCM is called DNA replication licensing. As S phase approaches,
several protein kinases are recruited which removes the Cdc, Cdt1 proteins and
ORC proteins. Once those proteins leave, other replication factors assemble at
origin. MCM proteins moves in 30 to 50 direction and unwinds the double helix.
As prokaryotes have clamp and a clamp loader, eukaryotes have PCDNA as sliding
clamp. Replication factor C connects clamp to the polymerase in eukaryotes as Tau
protein in prokaryotes.

7.10.1 Eukaryotes Have Many Different DNA Polymerases

Eukaryotes have many polymerases. Mammalian cells have over 12 different DNA
polymerases. Four of these, alpha (α), epsilon (ε), delta (δ) and gamma (γ), have
primary function of replicating DNA (Table 7.7). DNA polymerase γ replicates
mitochondrial DNA, and α, ε and δ replicate DNA in nucleus. DNA polymerase α
associates with primase. DNA polymerase ε and δ carry out the processive elonga-
tion of the DNA strands. DNA polymerase α and primase complex initiate the DNA
synthesis at the replication fork and later are exchanged for ε and δ. ε plays a role in
leading strand synthesis, and DNA polymerase δ is involved in lagging strand
synthesis. Remaining DNA polymerases are involved in repair of damaged DNA,
and some newly discovered DNA polymerases are called translesion replication
400 T. Banerjee

Table 7.7 Properties of eukaryotic DNA polymerases

DNA 30 –50
polymerase Subunits exonuclease Function
α 4 No RNA/DNA primers, initiation of DNA
synthesis
δ 4 Yes Lagging strand synthesis, DNA repair,
proofreading
ε 4 Yes Leading strand synthesis, DNA repair
γ 2 Yes Mitochondrial DNA replication and repair
β 1 No Base excision DNA repair
η 1 No Translesion DNA synthesis
ζ 2 No Translesion DNA synthesis
κ 1 No Translesion DNA synthesis
ι 1 No Translesion DNA synthesis
θ 1 No DNA repair
λ 1 No DNA repair
μ 1 No DNA repair
ν 1 No Unknown
Rev1 1 No DNA repair

polymerases. Different types of translesion DNA polymerases are able to replicate

over different kinds of DNA damage. For example, polymerase κ can replicate over
DNA lesions caused by benzo[α]pyrene, an agent found in cigarette smoke, whereas
polymerase η can replicate over thymine dimers, which are caused by UV light.

7.10.2 Removal of RNA Primers

In bacteria, RNA primers are removed by bacterial DNA polymerase I. None of the
eukaryotic DNA polymerases can remove the RNA primer. Another enzyme called
Flap endonuclease removes the RNA primer. DNA polymerase δ continues to
extend the Okazaki fragment and reaches the short RNA primer of the next Okazaki
fragment. This causes the RNA primer to form a short flap which is then removed by
the FLAP endonuclease. If the RNA flap is long, then another enzyme called Dna2/
helicase cuts the flap. It creates a shorter flap which is then removed.

7.10.3 End Replication Problem

The 30 end of DNA cannot be replicated by DNA polymerase because RNA primer
cannot be made upstream of this region. In this situation the DNA length would keep
reducing in every replication cycle. Since bacterial DNA is circular, end replication
problem does not exist in them. The loss of genetic information due to chromosome
shortening is avoided by the presence of tandemly repeated sequences at the end of
the chromosome, called telomeres. Enzyme telomerase synthesizes the tandem
7 Replication of DNA 401

repeats of the telomeres. This enzyme was discovered by Carol Greider and
Elizabeth Blackburn in 1984.
Action of Telomerase: Telomerase contains both RNA and protein component.
Telomerase RNA component (TERC) contains a sequence complementary to the
telomere repeat end. This allows telomere to bind to the 30 overhang of the telomere.
RNA sequence of the telomerase beyond the binding site acts as template for the
synthesis of the end of the telomere DNA by adding six nucleotides (Fig. 7.38).
Telomere lengthening is catalysed by the telomerase reverse transcriptase (TERT).
TERT has two identical protein subunits that catalyse DNA synthesis having RNA
as template. Following polymerization, it translocates to the new end of DNA,
adding six nucleotides again.

7.10.4 Role of Telomeres in Cancer and Ageing

The length of telomere shortens with age. At birth the telomere length initially may
be 8000 base pairs, but in an elderly person, it may be around 1500 bp. This decrease
occurs as the activity of telomerase decreases with age. When telomeres become too
short, cells undergo senescence and stop dividing (Fig. 7.39).
However, the cancer cells can undergo uncontrolled division which may not stop
even if the telomere length is short. In them, the telomerase undergoes mutations
which increase the activity of the enzyme.

7.11 Regulation of DNA Replication

We have learnt so far that DNA replication is semi-conservative, with continuous

and discontinuous strand synthesis mostly occurring directionally. Some circular
genomes replicate unidirectionally. Some of the viruses like M13 replicate by rolling
circle replication.
In eukaryotes, the DNA replication is regulated by the cell cycle. In prokaryotes,
DNA replication depends on cell size, availability of energy and precursor
molecules. In presence of rich carbon sources and simple inorganic nutrients,
doubling time is reduced. DNA synthesis is principally regulated at the initiation
stage. In enriched medium the second round of initiation begins even before the first
round of replication is complete. The rate of initiation is controlled relative to growth
rate. Initiation of primer synthesis by the primase is the major rate controlling
mechanism in prokaryotes. Rate of initiation is controlled by DnaA protein. Expres-
sion of DnaA protein is regulated by auto-feedback loop. There are more DNA
copies per cell of those genes which are closer to OriC than of genes far away from
OriC. It is expected that the genes whose products are needed in large amount are
present close to the OriC. Rapidly growing cells can have multiple copies of the
genome, while cells with a very low growth rate have only one copy.
The control of copy number of multi-copy number plasmid is mediated by the
synthesis of anti-sense RNA of the replication initiator protein Rep. Rep anti-sense
402 T. Banerjee

Fig. 7.38 Replication of chromosome telomeres. Telomere lengthening is catalysed by the

telomerase reverse transcriptase (TERT). TERT has two identical protein subunits that catalyse
DNA synthesis having RNA as template. Following polymerization, it translocates to the new end
of DNA, adding six nucleotides again (Adapted from: https://bio.libretexts.org/Courses/University_
of_California_Davis/BIS_2A%3A_Introductory_Biology_(Britt)/02%3A_Face-2-Face/2.05%3A_
DNA_Replication)
7 Replication of DNA 403

Fig. 7.39 Loss of DNA telomeres. Telomeres shorten with each cell division. Telomerase extends
the telomere sequence along the RNA template (Adapted from: https://www.mechanobio.info/
genome-regulation/what-are-telomeres/)

RNA is copied from the non-transcribed DNA strand and is therefore complemen-
tary to the normal RNA. Anti-sense RNA prevents synthesis of the Rep protein. Rep
protein is required for initiation of DNA synthesis, and its concentration controls the
frequency of initiation. Rep proteins encoded by plasmids bind to additional copies
of binding sites called ‘iterons’, often present upstream of the ori sequences in the
plasmids.
Regulation of eukaryotic genome replication is mainly brought about by cell
cycle. It is very tightly regulated to ascertain that DNA replication occurs only once
before division. In cell cycle, there are G0, G1 S, G2 and M phases. In the S phase,
the DNA replication occurs, and at G1 checkpoint, regulatory proteins ensure that
the cell is prepared for replication. At G2 checkpoint, DNA replication is cross-
checked so that cell can proceed for division. DNA licensing is a process to ensure
that chromatin is competent for DNA replication. A complex of proteins called
origin recognition complex (ORC) bind to the ori sequences. Although ORC is
present throughout the cell cycle, other proteins like MCM (minichromosome
maintenance) are loaded stepwise. The loading of MCM proteins and organization
of ORC is the important regulator for controlling the rate of initiation in eukaryotes.
Length of telomeres and activity of telomerase are also important for regulating
DNA replication. If the length of telomeres decreases beyond a certain limit, the cell
reaches senescence and stops replicating.
404 T. Banerjee

Fig. 7.40 Scheme of amino-labelled DNA probes for formaldehyde fixation. (a) Chemical
reactions of formaldehyde fixation. (b) Scheme of formaldehyde-mediated cross-link between
amino-labelled probe and cellular proteins in vicinity

Fig. 7.41 RNA-DNA FISH of terra (RNA) and telomere (DNA) using an amino-labelled oligo-
nucleotide probe. (a) Either a FITC-labelled PNA probe (green) or an amino-labelled oligonucleo-
tide probe (fluorescently labelled with FAM, green) was used to detect terra in RNA FISH.
Telomere DNA was detected by Cy3-labelled oligonucleotide probes (red) with the sequence of
(GGGTTA). Nuclei were stained by DAPI (blue)
7 Replication of DNA 405

Box 7.1 Scientific Concept: Using Amino-Labelled Nucleotide Probes

for Simultaneous Single Molecule RNA-DNA FISH, Reelina Basu et al.
In the earlier section, we have learnt about the importance of FISH (fluores-
cence in situ hybridization). This technique can be slightly modified to detect
DNA and its corresponding RNA transcript in the cell by RNA-DNA FISH.
The problem with this technique is that the RNA is too fragile to survive the
harsh treatments of DNA FISH. Therefore, Reelina Basu et al. designed a
method to amino label the oligonucleotide probes and simultaneously detect
DNA and its RNA transcript (Basu et al. 2014). They selected a lnc (long
noncoding) RNA terra (telomeric repeat-containing RNA) which is a repeti-
tive sequence present in the DNA telomeres. Thirty-six nucleotide long oligo
probes were synthesized by DNA synthesizer. An amino-modified thymine
(amino-dT) was used to introduce an alkylamino group to the oligonucleotide
probe with a density of at least one amino group every six nucleotides. The
oligonucleotide with dual label of alkylamine and fluorophore was
synthesized.
Terra RNA FISH was done using amino-labelled oligonucleotide probe and
PNA probe (peptide nucleic acid). PNA probes are extremely stable and hence
were chosen as control. RNA signals of terra were directly fixed right after
RNA FISH. Terra signals detected by PNA probe were lost during the harsh
treatments of DNA FISH, but amino-labelled probe signals could be easily
detected even after DNA FISH. Authors could infer that not all telomere DNA
is associated with terra RNA. The terra RNA transcripts are located in close
proximity of telomeres, but some terra signals do not overlap with
telomere DNA.
Synthetic amino-labelled oligo nucleotide probe was sufficient to detect
signals of terra as it is a repetitive sequence and probe has multiple binding
sites. For lnc RNA which exist in single copy, like HOTAIR, NEAT2 and xist,
the amino-labelled probe needs to be created by nick translation so that there
could be more incorporation of labelled amino group. More label
incorporation would give more intense signal for detection of single copy
lncRNA (Figs. 7.40 and 7.41).

Box 7.2 DNA Mutation Motifs in the Genes Associated with Inherited
Diseases, Michal Ruzicka et al.
We learnt that DNA replication is a highly accurate mechanism. The error rate
that is allowed is 1 in 109 or 1010. Those errors are repaired by various
mechanisms like nucleotide excision repair (NER), mismatch repair (MMR),
homologous excision repair, post replication repair, etc. Despite all these

(continued)
406 T. Banerjee

Fig. 7.42 Mutation hotspot and cold spot based on their occurrences. Occurrences of top 20 cold
spots (top) and top 20 hotspots (bottom) in the five studied genes visualized with the number of
detected mutations and substitutions in middle position (Adapted from: Ruzicka 2017)

Box 7.2 (continued)

repair mechanisms, mutations still occur. If the mutation is present in the germ
cells, it gets easily inherited. There are certain sequence motifs which are more
prone to mutation and are called mutation hotspots and other regions which are
less prone to mutation than normal and are called cold spots. In this work the
authors have analysed certain genes which are closely associated with genetic
disorders like the PAH gene (associated with hyperphenylalaninemia), LDLR

(continued)
7 Replication of DNA 407

Box 7.2 (continued)

gene (associated with hypercholesterolemia), CFTR gene (associated with
cystic fibrosis) and F8 and F9 genes (associated with hemophilia A and B,
respectively) reported in Human Gene Mutation Database (HGMD). They
analysed the sequences using advanced bioinformatics tools like molecular
dynamics (MD) simulations implemented in the AMBER program package
and free energy calculations using the adaptive biasing method (ABF)
enhanced by the multiple walker approach (MWA).
They identified sequence motifs which could be divided into two groups:
the hotspots and cold spots. They observed in one of the datasets that 18 out of
20 cold spots contain four or five purine tracts. The identified hotspots were
observed to contain CpG dinucleotide sequence in the middle position of the
motif. In the top 20 identified cold spots, CpG dinucleotide was not present.
Next, they analysed the reason why certain sequences are prevalent in
hotspots and others in cold spots. In a cell, mismatches and small insertion/
deletion are repaired by the MutSα protein in the MMR pathway. MutSα
protein with bound ADP can diffuse freely on DNA and search for
mismatches. Wherever it finds a mismatch, it binds to it and bends the DNA
and repairs the sequence. The bending of DNA was quantified by the free
energy change that is needed to bend a relaxed straight DNA to the bend
conformation that is observed in the MutSα/DNA complex. The free energy
profile showed that the hotspots are less flexible than the cold spots and hence
are not repaired efficiently by MMR (Fig. 7.42) (Ruzicka, M., et al. Plos One.
12: e0182377, 2017).

7.12 Summary

• Molecular transformation factor was shown to be responsible for transformation

of living R bacteria into S form by pioneering experiments of F Griffith. Avery,
MacLeod and McCarty’s transformation experiment proved that DNA is the
genetic material. Hershey and Chase further demonstrated that DNA is passed
to the progeny phages and hence carry the genetic information.
• Walther Flemming observed thread-like bodies forming during cell division and
called it ‘mitosen’. Later it was named as chromatin or stainable material present
in the nucleus. Various dyes were later used for staining chromosomes like
quinacrine mustard.
• DNA replication was shown to be semi-conservative by Meselson and Stahl using
transient N15 labelling and then growing in excess of N14 followed by density
centrifugation in CsCl gradient.
• Bacteria have closed circular DNA which replicates bidirectionally from single
origin of replication. Around the origin of replication, the DNA becomes single
408 T. Banerjee

stranded for a few bases. Cairn showed by autoradiography that during DNA
replication in E.coli it looks like Greek letter θ and hence is known as θ mode of
replication.
• Viruses have rolling circle mode of replication forming concatemeric DNA. In
this mode of replication, one of the strands of closed circular double-stranded
DNA is nicked. A free 30 -OH and free 50 P is generated, and the free 30 -OH is used
for extending the new DNA stand. The other strand remains intact and acts as
template for the leading strand.
• DNA contains deoxyribose sugar, phosphate group and nitrogenous base. RNA
contains ribose sugar, phosphate group and nitrogenous base. RNA contains
uracil unlike DNA.
• The bases are of two types: purines (adenine and guanine) and pyrimidines
(cytosine and thymine). They bind to each other using hydrogen bonding.
Adenine is linked to thymine by two hydrogen bonds, and cytosine is linked to
guanine using three hydrogen bonds. Hydrogen bonding follows Chargaff’s rule
[A + G] ¼ [C + T].
• DNA has sugar phosphate backbone made of adjacent nucleotides linked by
phosphodiester bond forming polynucleotide strand. Each polynucleotide strands
has free 30 -hydroxyl and 50 -phosphate group.
• DNA double helix crystal structure was solved by Rosalind Franklin. Based on
that crystal structure, Watson and Crick proposed DNA double helix model. Their
model showed that DNA forms a double helical structure with sugar phosphate
backbone lying on the outer edge of the helix and bases are stacked in the interior.
• There are 10 bp per 360
rotation of the helix. Each base pair is 0.34 nm apart, and
therefore there are ten base pairs in each turn encompassing 3.4 nm. The diameter
of B form of the helix is 2 nm. Diameter and pitch of the DNA vary in different
forms of DNA like A, B and Z DNA.
• DNA denatures to get single stranded as temperature increases; it is called DNA
melting. DNA absorbs UV light (Amax-260); the absorption increases when the
DNA becomes single stranded. It is called hyperchromic shift. DNA concentra-
tion (Co), the time allowed for reassociation (t) and the sequence organization of
the DNA are the factors deciding the extent of reassociation. Since the extent of
reassociation is directly proportional to both Co and t, their arithmetic product
(Cot) denotes the extent of reassociation. When log10Cot is plotted vs percent of
ssDNA, it can give the idea of repetitive sequence present in the DNA.
• Out of the five DNA polymerases found in E. coli, polymerases I and III are the
major enzymes which are involved in DNA replication. Rest are involved in
repair of damaged DNA.
• DNA polymerase III is the principal enzyme involved in replication. The α
subunit carries out the function of synthesizing phosphodiester linkage between
adjacent nucleotides. It also has 30 to 50 exonucleolytic activity which is called
proofreading activity.
• The number of bases added by the enzyme during replication is called the
processivity of the enzyme. In DNA pol II, the two β-subunits are responsible
for high processivity. α subunit is the catalytic subunit.
7 Replication of DNA 409

• DNA synthesis occurs with high amount of accuracy, and only one mismatch
occurs per 100 million bases. That is called as fidelity of the enzyme.
• Three types of DNA sequences are found in origin of replication in E. coli (OriC);
AT rich, DnaA box binding region and GATC methylation sites.
• DNA polymerase can extend polynucleotide only in 50 to 30 direction by adding
new nucleotides at the 30 end. In the leading strand, RNA primer is made by
primase, and DNA polymerase III extends it towards the opening of the replica-
tion fork in the 50 to 30 direction. The other strand is called lagging strand, which
is formed in 50 to 30 . Several RNA primers are made by primase to facilitate
synthesis of DNA fragments, also known as Okazaki fragments in the 50 to 30
direction.
• In eukaryotes there are multiple origins of replication. Depending on chromatin
packaging, histone modifications also play significant roles in deciding origins of
replication.
• Mammalian cells have over 12 different DNA polymerases. Four of these, alpha
(α), epsilon (ε), delta (δ) and gamma (γ), have primary function of replicating
the DNA.
• The 30 end of DNA is replicated by telomerase enzyme. Telomerase has RNA and
protein component. Telomerase RNA component contains sequence complemen-
tary to telomere repeat. Telomere lengthening is catalysed by the telomerase
reverse transcriptase (TERT). This enzyme was discovered by Carol Greider
and Elizabeth Blackburn in 1984. Cancer cells can undergo uncontrolled division
which may not stop even if the telomere length is short. In them, the telomerase
undergoes mutations which increase the activity of the enzyme.

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Chromosomal Organization of DNA
8
Payal Gupta

8.1 Chromosome: Overview

If we look upon DNA as the blueprint of life, then chromosomes are the entities that
hold the blueprint together. Waldeyer first coined the term “chromosome” in 1888
when he saw coloured bodies stained with an aniline dye under the microscope.
Chromosomes are responsible for physically carrying the DNA and all associated
proteins. Even the tiniest of beings, bacteria, can hold anywhere between 130 kbp
and 14 Mbp of DNA, while the human genome holds over 3 billion bps which would
measure ~2 m if placed in tandem. The Fig. 8.1 shows the sizes of the chromosomes
of various organisms. It is bewildering how this massive amount of DNA is assorted
and physically packaged in cells of microscopic sizes and yet made accessible for
various life processes like replication, transcription, etc. Even though our under-
standing of the structure and organization of DNA packaging in chromosomes is
constantly evolving, the last few decades have greatly enhanced our knowledge in
this field.
There are many variations in the form of DNA packaging observed in different
life forms. The bacterial genome comprises of a single DNA molecule which is
present in the form of a single circular covalently closed chromosome and
compacted with the help of packaging proteins. On the other hand, the haploid
human genome has its DNA segregated into 23 units that form morphologically
distinct linear chromosomes on compaction (Fig. 8.2). Then, there are specialized
chromosomes like the polytene chromosomes that perform specialized functions.
However, chromosomes universally perform two fundamental functions: the
precise transmission of genetic information and accurate control of gene expression.
Specialized regions containing repetitive sequences help form structures such as
telomeres, replication origin, etc. which help the chromosomes in the execution of its

P. Gupta (*)
University of Calcutta, Kolkata, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 411
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_8
412 P. Gupta

Fig. 8.1 The average size of the genome of organismal groups. The scheme depicts the average
size of the genome of various groups of organisms and shows what proportion of the genome
actually codes for peptides

Fig. 8.2 Varying forms of chromosomes. (a) The diagram is a schematic representation of a
bacterial nucleoid. It depicts the bottle brush model of the nucleoid with supercoiled loops that are
interwound and radiate out of a dense core. (b) A schematic SKY image of a normal female
karyotype
8 Chromosomal Organization of DNA 413

functions. The level of chromosome compaction also varies depending on which

areas are needed for active transcription and which areas will remain inactive. Our
current ideas of chromosomal structures revolve around understanding how chro-
matin is arranged in structural domain in the three-dimensional nuclear spaces and
how this structural landscaping controls different facets of gene expression.

8.2 DNA Supercoiling

Now that we appreciate the gigantic size of the genetic material that must be
packaged into a comparatively miniscule cellular space, it stands to reason that the
DNA strings must be highly coiled and condensed. Yet, the coiling should still leave
functionally important domains of the DNA accessible to proteins. This feat is
achieved through the process of supercoiling which literally implies the coiling of
a coil.
To understand the process, let us visualize the double helical DNA. An axis
passes through the middle of the helix. When this axis is folded upon itself, it results
in a supercoiled DNA (Fig. 8.3). This can be explained by using a small linear
double helix with two to three turns. If both ends of the helix are twisted in direction

Fig. 8.3 The supercoiling of

DNA. A linear double helix is
shown on the left-hand side
with an imaginary axis
passing through it. The right-
hand figure depicts a
supercoiled DNA where the
axis has folded on itself
414 P. Gupta

of the helix winding, the number of turns in the helix will seem to increase. This
supercoiled helix is over-twisted and under strain. If, however, the helix is twisted in
the direction opposite to its coiling, it will appear to unwind. This state of the coil
with lesser number of turns is a relaxed state. If the molecule is consistently over-
twisted, it will relieve the molecular strain by twisting upon its own helical axis, thus
creating a positive supercoil. Likewise an under-twisted moiety will result in a
negative supercoil. Most basic processes like replication and transcription require
the unwinding of the double helix. This is turn results in over-twisting of the
domains lying ahead. Thus, supercoiling is an integral aspect of the tertiary structure
of the DNA that is omnipotent in cellular DNA and tightly regulated by the cell.
To understand the physiological relevance of supercoiling, let us focus on closed
circular B-form of DNA which has 10.5 bps per turn (Fig. 8.4). The underwinding of
this DNA at any point will result in a strain. For example, if the DNA has 84 bps, in a

Fig. 8.4 DNA

underwinding. (a) A relaxed
84 bps DNA segment with
eight helical turns. (b) The
underwinding of the DNA by
removing one turn results in a
strained DNA which is
compensated by (c) separation
of the strand over 10.5 bps or
(d) by forming a supercoil
8 Chromosomal Organization of DNA 415

Fig. 8.5 Depiction of linking number (Lk). (a) The molecules shown physically interact at one
junction and have an Lk of 1. (b) If one strand of DNA is kept unwound, then the number of times
the second strand passes it defines the linking number for that molecule

relaxed state, it will consist of eight helical turns of 10.5 bps each. The DNA is now
underwound, and one of these eight turns is removed, and then the 84 bps will be
divided in seven helical turns containing 12 bps per turn instead of the 10.5 bps. This
alteration will result in a thermodynamically strained structure. This strain can be
compensated in two ways. Either the strands can simply separate over a stretch to
resort to 10.5 bps per turn or the axis of the double helix can coil on itself to realign
the base stacking to approximate of 10.5 bps per turn pattern.
DNA supercoiling could be better understood with the help of a branch of
mathematics called topology. Of special interest is the concept of linking number
(Lk) which denotes the number of helical turns in a closed circular DNA when no
supercoiling is present (Fig. 8.5). This property does not vary even if the DNA is
twisted or deformed as long as the two strands of DNA remain intact. By standards,
if the DNA strands are twisted in a right-handed helix, the linking number is a
positive integer; however, if the strands are twisted in a left-handed helix (as in
Z-DNA), the linking number is a negative integer.
Two structural components make up the linking number, twist (Tw) and writhe
(Wr). Writhe denotes the coiling of the helix axis, while twist defines the local
twisting or the spatial arrangement of the neighbouring base pairs. When
supercoiling happens, the linking number of the DNA changes causing a strain
which is compensated by variation in supercoiling (writhe) or by change in the
twist patterns. This gives rise to the equation; Lk ¼ Tw + Wr.
In all systems, the supercoiling is regulated by a class of enzymes called
topoisomerases which play an important role in the processes of replication, DNA
packaging, etc. The genre of topoisomerases is divided into two classes, Type I and
Type II. Type I topoisomerases act by creating a nick on one strand, twisting it
around the uncut strand and rejoining it, thus changing the Lk by 1. Type II
topoisomerase acts by cutting both strands of the DNA, twisting them around one
another and rejoining them, thus changing the Lk by 2 (Fig. 8.6).
416 P. Gupta

Fig. 8.6 The mechanism of strand passage through topoisomerase core. The type II topoisomerase
(blue and yellow core) captures G-segment (gate) DNA (green). It then sequesters the transfer or T
segment (indicated in shades of pink to denote movement of the DNA). Then they bind two ATP
molecules thereby shutting the N-terminal gate (yellow) which is followed by the double-strand
cleavage of the gate strand. The T segment passes across the cleaved DNA, and, together with the
ATP hydrolysis products, it is then released

8.3 Organization of the Prokaryotic Chromosome

Having understood the basic dynamics of DNA supercoiling, we move onto the
question of how the DNA is packaged in prokaryotes lacking a defined nucleus and
how the stability of the coiled structures are maintained. Most of our knowledge of
the organization of prokaryotic chromosomes comes from the studies conducted on
E. coli. The single circular chromosome is packaged in the form of a nucleoid which
is defined as the area of the cell where the chromosome DNA is located (Fig. 8.7).
The packaging of the bacterial genome into the small nucleoid space occurs via
two processes; the first is the process of supercoiling discussed in Sect. 8.2, and the

Fig. 8.7 Packaging of the bacterial chromosome. (a) A circular chromosome without any com-
paction. (b) The DNA-associated proteins form a central scaffold, and the bacterial DNA loops
around them. (c) The loops are further supercoiled to form a condensed structure
8 Chromosomal Organization of DNA 417

Fig. 8.8 Proteins associated

with bacterial chromosomes.
Several proteins like HU and
H-NS are associated with the
bacterial DNA to maintain its
topological structure

other is the interaction of the DNA with packaging proteins. The main histone-like
packaging proteins present in the E. coli are HU, IHF (integration host factor), Fis
(factor for inversion stimulation) and H-NS. These positively charged proteins
interact with the negatively charged DNA and, together with the topoisomerase
and gyrase enzymes, maintain the supercoiling homeostasis of the bacterial genome.
For example, the HU protein works with topoisomerase I and introduces acute bends
in the chromosome which generates the tension required for the negative
supercoiling. During normal growth most of the bacterial genome is negatively
supercoiled. The negative supercoiling of DNA gives rise to plectonemic loops
forming a network of supercoiled domains that are topologically insulated from
each other. The current view is that these interwound loops are not rigid. They keep
changing depending on the genetic transaction that occurs within and between them
(Fig. 8.8).

8.4 Hierarchical Packaging of the Eukaryotic Chromosome

When DNA from eukaryotic cells is isolated using isotonic buffers like 0.15 M KCl,
it is found to be associated with nearly equivalent proportions of protein in an
extremely compacted complex called chromatin. The DNA in eukaryotes exists in
the chromatin state throughout the interphase of the cell cycle. However, in order for
the all the tangled DNA to accurately partition into two cells during mitosis, the
DNA needs to condense into ordered structures called chromosomes during the
prophase of the cell cycle. As discussed, the total length of total DNA of the diploid
human cell is about 2 m. This is divided into 23 pairs of chromosomes each
containing anywhere between 15 and 85 mm of DNA. The largest human chromo-
some, of ~85 mm long DNA, is packaged into a distinct mitotic chromosome 10 μm
long and about 0.5 μm diameter. Even so, the chromosome has specialized
418 P. Gupta

1
At the simplest level,chromatin 2 nm
is a double-stranded helical DNA double helix
structure of DNA.

3
Each nucleosome consists of
eight histone proteins around
which the DNA wraps 1.65 times.
2
DNA is complexed with histones
to form nucleosomes. Nucleosome core of
4
eight histonemolecules A chromatosome consists
H1 histon e of a nucleosome plus the
6 H1 histon e
...that forms loops averaging
300 nm in length.
11 nm Chromatosome
300 nm
5
The nucleosomes
fold up to produce
a 30-nm fiber....

30 nm
250-nm-wide fiber
700 nm

7 8 1400 nm
The 300-nm fibers are Tight coiling of the 250-nm
compressed and folded to fiber produces the chromatid
produce a 250-nm-wide fiber. of a chromosome.

Fig. 8.9 The hierarchical packaging of eukaryotic chromosome. A simple dsDNA wounds around
a nucleosome complex giving rise to a beads on string structure. This nucleosome forms a solenoid
complex of 30 nm diameter. The 30 nm fibre further forms 300 nm long loops around the
non-histone proteins undergoing several levels of compaction to form the chromosome

structures, and its dynamic topology allows access to proteins required for life
functions. This is achieved by a hierarchical and orchestrated process of packaging
with the help of different types of proteins (Fig. 8.9).

8.4.1 The Nucleosome Assembly

The most predominant proteins found attached to the eukaryotic DNA are the
histone proteins. They represent a family of basic proteins, rich in positively charged
amino acids such as lysine and arginine. This positive charge helps the protein
interact with the negatively charged backbone of DNA. There are five principal types
of histones: H1, H2A, H2B, H3 and H4. The histones form a highly structured
8 Chromosomal Organization of DNA 419

Fig. 8.10 An electron micrograph of nucleosome “beads-on-string” structure. The black brackets
indicate nucleosome assembly, black arrowheads indicate the nucleosomal core, and white
arrowheads indicate linker DNA. The scale bar indicates 50 nm. Image credit: Chris Woodcock

assembly, and the DNA loops around it to give rise to the beads on strings moieties
known as the nucleosome assembly.
The nucleosome consists of a string of DNA wrapped around a protein core like a
thread on a spool. The disc-shaped core is an octamer comprising of two copies of
H2A, H2B, H3 and H4 each. Histone protein sub-assemblies come together to form
the histone core with the H3 and H4 forming a tetrameric sub-assembly and the
H2A-H2B dimeric sub-assembly joining it to complete the histone core. The DNA
wraps around this core ~1.65 times using around 146 bps length. The H3 and H4
tetramers interact in the middle and the rear ends, while the rest of the DNA is bound
to the H2A–H2B dimer via hydrogen bonds. Consecutive nucleosome cores are
connected by short segments of linker DNA which harbours the linker histone, H1
(Figs. 8.10 and 8.11).
Each histone protein has an N-terminal tail that provides a guide for the DNA
strand to wrap around the core, by creating grooves similar to those on a screw such
that the DNA wraps in a specific pattern. Thus the beads form ellipsoidal bodies
measuring 110 Å in diameter and 60 Å in height (Fig. 8.12).

8.4.2 The Solenoid Structure

The electron microscopy data show that the chromatin fibres have a diameter of
~30 nm. The 10 nm nucleosome fibre is wrapped into a highly structured solenoid or
a super-supercoil. The solenoid model depicts the nucleosome as packaged into a
spiral arrangement having six nucleosomes every turn. The nucleosomes fold on the
420 P. Gupta

Fig. 8.11 The nucleosome assembly. The nucleosome consists of an octameric core comprised of
the H2A, H2B, H3 and H4 units with about 146 bps of DNA wound around it

Fig. 8.12 Crystal structure of the nucleosome assemble. The image shows a schematic represen-
tation of the X-ray crystal structure of the nucleosome core. All reference frames are in alignment
and computed by the PCA of the globular core of the histone octamer

inside with the help of the linker, H1, histone giving rise to a 30 nm chromatin
(Fig. 8.13). A unit nucleosome and a unit of the H1 together are called the
chromatosomes.
8 Chromosomal Organization of DNA 421

Fig. 8.13 The solenoid model. The nucleosomal core arrange in the form of a solenoid by bending
over the H1 linker histone, thus giving rise to the 30 nm fibre

8.4.3 The Chromosomal Structure

The 30 nm solenoid chromatin again undergoes several orders of supercoiling to

finally form the metaphase chromosome. The chromosome at the metaphase stage
has the highest order of condensation. This is composed of loops of the 30 nm
chromatin solenoid radiating from a central scaffold. The central scaffold contains
non-histone proteins which anchor the chromatin at specific AT-rich regions called
the matrix-associated regions (MAR) or scaffold-associated region (SAR). Each
loop emanating from the scaffold structure is about 100–300 nm (~60 kbps) long. It
has been recently shown that this model of chromosome is neither a rigid model nor
a random one. The chromosomes organize themselves in the three-dimensional
nuclear space in the form of distinct territories with defined interactions with the
nuclear subdomains. This gives rise to what is known as topologically associated
domains (TADs). TADs are essentially self-interacting regions of DNA which are
far apart from each other in terms of their two-dimensional length but are brought
together by the process of looping and three-dimensional organization of the
chromosome. This helps to form two compartments: an open or transcriptionally
active compartment and a closed or transcriptionally inactive compartment. So,
TADs can be simply visualized as long stretches of DNA that come together in a
common 3D space so that the genes that they house can be triggered or silenced
in sync.
422 P. Gupta

8.5 The Heterochromatin and Euchromatin

As discussed earlier, one of the most important tasks of the chromatin and chromo-
somal structures is to allow the accurate control of gene expression. Therefore, the
chromatin fibre must make allowances for the proteins to reach and transcribe a
segment of DNA required to be active while leaving other non-relevant segments
inactive. In most regions the chromatin appears to be less compactly packaged and
relatively more dispersed in the nucleus. These regions are transcriptionally more
active and are known as the euchromatin. On the other hand, the more densely
packaged regions of chromatin resembling the level of condensation seen in the
chromosomes are transcriptionally inactive and known as the heterochromatin.
These alternate states of packaging are achieved by various mechanisms including
histone modification. Heterochromatin can be formed with the help of increased
methylation of one histone, decreased acetylation of histones and hypermethylation
of the cytidine bases of the DNA.

8.6 Chromosomal Banding

The study of the structure of chromosomes requires rigorous techniques to stain and
visualize them under a microscope. This is done by enzymatic digestion or denatur-
ation of the chromosome followed by staining with DNA binding dyes. Because of
the variations in the extent of packaging, this procedure produces light and dark
banding patterns on the chromosomes. Each chromosome has a unique banding
pattern which helps in its identification (Fig. 8.14). Following are some of the
banding techniques used routinely for the study of chromosomes:
G-banding—A controlled trypsin digestion of the chromosomes are performed
followed by staining with Giemsa. The bands that take a dark stain are called
G-bands, while the lightly stained bands are called G-negative. G-bands are mostly
AT-rich segments of DNA.
Q-banding—This technique requires the staining of DNA with fluorescent dyes
such as quinacrine, DAPI (40 ,6-diamidino-2-phenylindole) or Hoechst 33258 and
visualization under a fluorescence microscope. Since these dyes bind preferentially
to the AT-rich regions of DNA, they produce a banding pattern similar to G-banding.
R-banding—This technique is performed by heat denaturing the DNA in high salt
concentration before staining with Giemsa. This results in a banding pattern which is
the reverse of G-banding. R-bands are also Q-negative. The same results can be
achieved by using GC-specific binding dyes.
T-banding—This banding technique is a more severe form of R-banding used to
identify the telomeric regions of DNA. This is achieved by either a more severe heat
treatment of DNA prior to Giemsa staining or using a combination of standard and
fluorescent dyes.
C-banding—This technique is used to stain constitutive heterochromatin of the
centromere. The DNA is denatured with a barium hydroxide solution before being
stained with Giemsa (Figs. 8.15 and 8.16).
8 Chromosomal Organization of DNA 423

Fig. 8.14 G-banding pattern of the human chromosome as observed under a microscope. Giemsa
is a protein stain that darkly stains the heterochromatic or transcriptionally inactive regions and
lightly stains the euchromatic or transcriptionally active regions

Fig. 8.15 C-banding patterns in human female chromosome. This technique specifically stains the
centromeric region of the chromosome which is constitutively heterochromatinized
424 P. Gupta

Fig. 8.16 Banding pattern of chromosomes. (a) Treatment and staining of chromosomes create
alternating light and dark banding patterns which are unique for each chromosome. (b) The image
depicts the G-banding pattern obtained after staining a chromosome with Giemsa which binds
AT-rich regions

8.7 Morphology of the Eukaryotic Chromosome

At the metaphase stage of mitotic cell division, the chromosome consists of two
molecules of DNA: one is parent DNA and the other is the DNA obtained from the
replication of the parent DNA at the S-phase. These two molecules form two
symmetrical structures called sister chromatids with each chromatid containing
one DNA molecule. The chromatids are held together at the centromere which
forms the point of attachment for the spindle fibres. When the two chromatids
share a common centromere, they are called sister chromatids, but once they separate
during the metaphase, each chromatid has its own centromere and is now known as a
chromosome (Fig. 8.17).

8.7.1 The Centromere

The centromere, also known as the primary constriction, appears as a narrowed zone
or a gap in the chromosome. The centromere harbours the kinetochore complex
which has microtubules radiating towards the spindle pole of the cell. The kineto-
chore plays a crucial role in the movement of the chromosome towards the spindle
pole during the cell cycle. The centromere is a heterochromatic region consisting of
long stretches (~171 bps) of tandem repeats of short DNA sequences. Depending on
the position of the centromere on the chromosome, the morphology is classified into
the following groups:

• Metacentric chromosome: The centromere is present in the middle of the chro-

mosome and divides it into two equal halves.
8 Chromosomal Organization of DNA 425

Fig. 8.17 The morphology of a metaphase chromosome. (a) The morphology of a eukaryotic
metaphase chromosome shows the centromere, telomere, secondary constriction and satellite. (b)
The centromere has the kinetochore assembly which connects to the microtubules and regulate
separation of the sister chromatids during anaphase

Fig. 8.18 Types of chromosomes based on the position of the centromere. The figure shows the
variations of chromosomes based on the location of the centromere

• Sub-metacentric chromosome: A chromosome is said to be sub-metacentric when

the centromere lies near the middle but not exactly in the centre of the chromo-
some. This results in two unequal arms. The shorter arm is called the “p-arm”,
while the longer arm is known as the “q-arm”.
• Acrocentric chromosome: The centromere is located near the end of a chromo-
some resulting in unequal chromosome arms which has the shape of the letter “j”.
• Telocentric chromosome: The centromere is located at the end of the chromo-
some. The chromosome thus appears to have only one arm and has the shape of
the letter “i” (Fig. 8.18).
426 P. Gupta

8.7.2 The Secondary Constriction

Additional non-staining gaps may be seen in certain chromosomes. These are known
as secondary constriction (Sc). These are generally located towards the end of the
chromosome arm and often contain genes encoding rRNAs or those that induce
nucleoli formation. Since these serve as the epicentre for organization of the
nucleolus, they are also known as the nucleolus organising region (NOR).

8.7.3 Satellite DNA

The Sc constriction separates a segment of the chromosome from the rest of the arm.
This rounded body is known as the satellite or the trabant. The satellite DNA
consists of simple or moderately complex DNA sequence repeated multiple times
over a long stretch of the DNA in tandem (end to end).

8.7.4 The Telomere

A distinctive feature of the eukaryotic chromosome is the telomeric region present at

the end of each chromatid arm. They form sticky ends when the chromosomes are
broken and differ markedly in structure and function from the remainder of the
chromosome. As in case of the centromere, the telomeric region consists of consti-
tutive heterochromatin. A span of 10–15 kbps region of highly conserved hexameric
repeat (TTAGG)n makes up the telomere (Fig. 8.19).

Fig. 8.19 The telomeric sequence of various organisms. The sequence of telomerase RNA and the
telomere repeat for different organisms have been depicted
8 Chromosomal Organization of DNA 427

8.8 Specialized Chromosomes

Certain eukaryotic organisms have tissues that harbour chromosomes with charac-
teristic structures which differ greatly from the standard chromosome. These
chromosomes, also known as the giant chromosomes, attain their largest size in
the nuclei of the cells housing them. They are commonly found in the suspensors of
the embryo of a few plants, in cells of the Malpighian tubules, in cells of the salivary
glands of Drosophila and Chironomus, in oocytes of certain vertebrates, etc. These
specialized chromosomes can be classified into two categories: polytene chromo-
some and the lampbrush chromosome.

8.8.1 The Polytene Chromosome

In these giant chromosomes, DNA replication is not followed by separation of the

daughter chromatids giving rise to a giant chromosome. Essentially, the DNA
replicates multiple times, but the nuclear division never happens. This phenomenon
is known as endomitosis. The daughter chromatids never separate from each other
and appear as a multistranded giant chromosome with the chromatids aligned side by
side (Fig. 8.20).
They were first observed by E.G. Balbiani (1881) in the cells of the salivary
glands of Chironomus larvae. Hence, these are also known as salivary gland
chromosome. They are also found in the living cells of the gut, the cells of the
Malpighian tubule and the salivary gland of Drosophila larvae. In Drosophila, they
appear to have a length ~100 times greater than the length of the somatic metaphase

Fig. 8.20 A polytene chromosome. The figure depicts the polytene chromosome of an insect. The
dark and light zones of bands and interbands are shown along with a puff or the Balbiani ring
428 P. Gupta

chromosome. However, since these chromosomes pair during the interphase, their
number appears to be half of the normal somatic cells.
The polytene chromosomes have alternating dark and light bands along its length
when stained with Feulgen stain. The dark bands are heterochromatin regions, while
the lighter bands, also known as the interbands, are the euchromatic regions.

8.8.2 Lampbrush Chromosome

Another type of giant diplotene chromosome found in the nuclei of oocyte of urodele
amphibians is the lampbrush chromosome. They consist of a well-demarcated
chromosomal axis, chromomeres and many looped extensions. Since the appearance
of these chromosomes resembles the bristles of the brushes used to clean the
chimneys of oil lamps, therefore they are known as the lampbrush chromosomes.
A multitude of fine lateral loops gives them a “hairy” appearance. Visible in the
meiotic prophase, the lampbrush chromosomes are found in the form of bivalents
each having four chromatids. The homologous chromosomes are held together by
chiasmata, and the axis of each homologue contains a row of chromomeres from
which one to nine lateral loops emanate (Fig. 8.21).

Fig. 8.21 The lampbrush chromosome. (a) Enlarged part of a lampbrush chromosome. (b) One
loop of the chromosome enhanced
8 Chromosomal Organization of DNA 429

Box 8.1 Scientific Concept: Investigating DNA Supercoiling in Eukaryotic

Genomes: Samuel Corless and Nick Gilbert
DNA supercoiling is an inherent and important property of the DNA.
Supercoiling is often generated when polymerases or DNA-binding proteins
unwind or bend the DNA double helix. Even though supercoiling has a pivotal
role in genomic organization and function, its study is extremely challenging
specially in the highly complex eukaryotic chromosome. Corless and Gilbert
have described the following techniques for the study of the supercoiling
dynamics.

1. Centrifugation or migration-based techniques

The process of supercoiling results in a more compact DNA with a 3D
structure remarkably different from that of a non-supercoiled DNA of the
same length. Such compaction alters the property of migration of DNA
during sucrose gradient centrifugation or agarose gel electrophoresis.
When both supercoiled and non-supercoiled DNA molecules are subjected
to sucrose gradient centrifugation, the supercoiled DNA molecule has a
higher sedimentation rate compared to that of a non-supercoiled DNA
molecule of the same molecular weight. Similarly, supercoiled, nicked and
linear DNA molecules show differential rate of migration in agarose gel
electrophoresis with the supercoiled DNA travelling the fastest. Further,
using an intercalating agent like ethidium bromide, it is possible to differ-
entiate between topoisomers with varying levels of supercoiling. The
intercalating agents unwind the DNA in order to bind to them thereby
inducing negative supercoils. Therefore, with the incorporation of the
intercalating agent, the rate of migration for the DNA molecule will
change as positive and negative supercoiling result in different rates of
migration (Fig. 8.22).
2. The preferential intercalation of psoralen molecules in underwound DNA
Psoralen is an intercalating molecule used to measure DNA supercoiling in
normal eukaryotic chromosomes. A cell permeable psoralen derivative,
4,50 ,8-trimethylpsoralen (TMP), intercalates in the DNA molecule and
forms stable cross-links with pyrimidine nucleotides when exposed to UV
light (365 nm). It binds preferentially to underwound segments in a probe-
specific way. This is an invaluable technique as the TMP molecule can be
conjugated with biotin and used for either a pull-down assay to enrich
underwound DNA, or it can be used for immunofluorescence in vivo by
treating the TMP bound DNA with a fluorescein-conjugated streptavidin
moiety.
430 P. Gupta

Fig. 8.22 Experimental evaluation of DNA supercoiling. (a) Differential partitioning of the
supercoiled and linear DNA in sucrose gradient centrifugation. (b) Differential migration of nicked,
linear and supercoiled DNA when subjected to agarose gel electrophoresis

8.9 Chapter Summary

• Given the size of the chromosome in proportion to the cells that harbour them,
they need to be condensed manifold to be able to be physically and functionally
accessible. For this purpose, DNA is often supercoiled. Supercoiled DNA appears
as a coiled coil, and its topology is defined in mathematical of linking number and
writher number. The topoisomerase enzymes function to introduce or relax these
supercoils.
• The bacterial genome consists of a single nucleoid which is a single covalently
closed circular DNA. The DNA is highly supercoiled forming coiled loops and is
packaged with the help of non-histone basic proteins such as HU, HNS, etc.
• The packaging of the eukaryotic genome follows a distinct hierarchy. The first
level of packaging is the nucleosome assembly which represents the “beads-on-
string” structure. An octameric histone assembly forms the core with a segment of
DNA bound around it. A linker histone binds the DNA which lies between two
core histones.
• The solenoid structure or the “coiled coil” is the second level of packaging and
forms the 30 nm fibre. The solenoid finally binds a scaffold matrix with the help
of matrix-associated regions to form the final chromosomal structure.
• The highly condensed regions of the DNA are known as heterochromatin and are
genetically inactive, while the relatively open regions known as euchromatin are
genetically active.
• Each individual chromosome can be karyotyped and assigned a unique morpho-
logical signature based on its banding patterns. Giemsa is an important stain, and
various treatments of DNA prior to staining can result in various types of banding
patterns, viz. the G-band, C-band, R-band and C-band.
8 Chromosomal Organization of DNA 431

• The eukaryotic chromosome has key structural features. The centromere is a

highly repetitive DNA segment that forms the attachment site for binding of
spindle fibres. There is another constriction known as the secondary constriction
evident on certain metaphase chromosomes.
• The telomere is a specialized region present at the end of a chromosome. An
active telomerase enzyme can bind to extend this segment to prevent shortening
of the chromosomal ends which do not replicate in somatic cells of higher
eukaryotes.
• There are also specialized chromosomes such as the lampbrush and polytene
chromosome which result from DNA that undergo multiple cycles of replication
without segregating during mitosis. These have distinctive structures and are
termed as the “giant chromosome”.

Further Reading
Bates AD, Maxwell A (2005) DNA topology. Oxford University Press, New York
Bednar J, Horowitz RA, Grigoryev SA, Carruthers LM, Hansen JC, Koster AJ, Woodcock CL
(1998) Nucleosomes, linker DNA, and linker histones form a unique structural motif that directs
the higher-order folding and compaction of chromatin. Proc Natl Acad Sci 95:14173–14178
Bendich AJ, Drlica K (2000) Prokaryotic and eukaryotic chromosomes: what’s the difference?
Bioessays 22:481–486
Cairns J (1963) The chromosome of Escherichia coli. Cold Spring Harb Symp Quant Biol 28:43–46
Castle WE (1919) Is the arrangement of the genes in the chromosome linear? Proc Natl Acad Sci 5:
500–506
Cook PR (1995) A chromatic model for nuclear and chromosome structure. J Cell Sci 108:2927–
2935
Corless S, Gilbert N (2017) Investigating DNA supercoiling in eukaryotic genomes. Brief Funct
Genomics 16:379–389
Judd BH (1999) Genes and chromosomes: a puzzle in three dimensions. Genetics 150:1–9
Kornberg RD (1974) Chromatin structure: a repeating unit of histones and DNA. Science 184:868–
871
Lewin B (2007) Genes IX. Jones and Bartlett, Sudbury
Noll M (1974) Subunit structure of chromatin. Nature 251:249–251
Pombo A, Dillon N (2015) Three-dimensional genome architecture: players and mechanisms. Nat
Rev Mol Cell Biol 16:245–257
Snyder L, Champness W (2003) Molecular genetics of bacteria, 2nd edn. ASM Press,
Washington, DC
Strachan T, Read AP (1999) Human molecular genetics. Wiley, New York
DNA Mutation, Repair, and Recombination
9
Atish Ray

A mutation is a process of alteration in genome sequence by mistakes during DNA

replication or as the result of environmental stress including radiation, toxic
chemicals, and smoke. Over a lifetime, the DNA of an organism can experience
many alterations in the bases, (A, C, G, and T) sequence. The transient errors are
corrected via a defined DNA repair mechanism. If the errors are not corrected in
course of time, mutation takes place leading to a permanent impact on function,
physiology, or phenotype mediated by awful translated proteins.
Commonly the term “mutation” has been associated with obvious detrimental
outcomes. The harmful effect of mutation is undoubtedly a major issue but not
always. In the natural selection process, positive mutations are inherited through the
generations contributing to genetic variation within the species. This variation often
facilitates the organism to survive and reproduce. On the other hand, “sickle cell
anemia” is quite well discussed as an unfavorable outcome of mutations. This is the
result of a mutation in the hemoglobin gene. Similarly, several forms of cancers, very
common in the present day, are also known to be contributed by certain mutations.
However, as mentioned above, unlike the harmful or disease-causing mutations,
positive and neutral mutations are least discussed. For example, variation in blood
types and eye color has neither good nor bad effects. Thus this kind of mutation can
be generally regarded as neutral mutation. In such variation, it is often meaningless
to distinguish between mutant and original alleles. Similarly, there can also be a
positive effect of the mutation. Bacterial populations have certain mutations which
can confer antibiotic resistance. Although this type of mutation is not at all positive
for humans, however, logically they are beneficial (positive) mutations for bacteria.
There are some other examples of beneficial mutations. HIV resistance and
malaria resistance are well documented. A 32 base pair deletion in a specific position
of human CCR5 (CCR5-Δ32) confers HIV resistance to homozygotes and delays

A. Ray (*)
Savil Technology and Business Incubator, Vadodara, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 433
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_9
434 A. Ray

AIDS outbreak in the European population. Surprisingly, there are certain instances
where a single detrimental mutation has resulted in other beneficial effects. In
sub-Saharan Africa, where malaria is common, significant numbers in the population
carry the mutant allele of sickle cell trait. Such heterozygous individuals (carriers of
sickle cell trait) are found to be resistant to malaria.
In this context, other terminology, “recombination,” must be explained in contrast
to “mutation.” Genetic recombination (which is also known as genetic reshuffling) is
the process of exchanging genetic material between different organisms leading to
the production of offspring with combinations of parent’s traits. In eukaryotes, a
novel set of genetic information during genetic recombination can be naturally
transmitted from the parents to the offspring in due course of time.
Both mutation and recombination can produce a novel trait. However recombi-
nant traits can be assorted independently through the generation, but technically
mutant traits are imprinted within the genome which can only be revered if it is
corrected. In several cases, correction is possible through the precise mechanism
known as the DNA repair mechanism, and this will be explored further in this
chapter. In the following section, different facets of mutation, DNA recombination,
and repair are elaborated.

9.1 Mutation: Overview of the Process

First of all, it’s important to emphasize that mutation is one of the diverse alternatives
to natural selection theory which was the subject of heated discussions both before
and after the publication of On the Origin of Species by Charles Darwin in 1859.
According to these conceptions, the mutation was taken as the preliminary consid-
erable component which was believed to be acted as a source of novelty in producing
new species. During the evolution of species, changes occur instantaneously through
a sudden jump. Mutation plays two key roles in evolution: (1) it creates an evolu-
tionary force that changes gene frequencies, and (2) it reserves the ultimate capacity
of all genetic variation. With the progress of in-depth research on cell physiology,
scientists concluded the molecular mechanism of mutagenesis. Not entering the
history, lets us come directly to the overview of the mutation process. In this section,
the entire process of mutation will be discussed thoroughly.

9.1.1 A Different Class of Mutation

The entire event of “mutation” can be classified in different categories depending on

the different parameters including the size of the affected DNA, nature genetic
alteration, impact on the translated peptide, and target cell types. It is important to
remember that these parameters are not necessarily mutually exclusive.
Terminologies related to the types of mutation are summarized in Table 9.1.
9 DNA Mutation, Repair, and Recombination 435

Table 9.1 Important terminologies and their definitions related to mutations

Term Definition
Base substitution Alteration of the base of a single DNA nucleotide
Transition Base substitution of purine with purine or a pyrimidine with pyrimidine
Transversion Base substitution where purine replaces a pyrimidine or a pyrimidine
replaces a purine
Insertion Addition of nucleotides
Deletion Deletion of one or more nucleotides
Indel Insertion or deletion
Frameshift Insertion or deletion that changes the reading frame of a gene
mutation
In-frame mutation Deletion or insertion of a multiple of three nucleotides resulting in no
shifting of reading frame
Reverse mutation Changes of a mutant phenotype back to the wild-type phenotype
Missense mutation Changes a sense codon into a different sense codon
Nonsense mutation Changes a sense codon into a nonsense codon
Silent mutation Changes a sense codon into a synonymous codon, leaving unchanged
amino acid
Neutral mutation Changes the amino acid sequence of a protein without altering its
functional property
Loss-of-function A complete or partial loss of function
mutation
Gain-of-function The appearance of a new trait or function
mutation
Lethal mutation The mutation that causes early infantile/premature death
Suppressor The mutation that suppresses the effect of the earlier mutations
mutation

9.1.2 Parameter 1: Size of the Affected DNA

Mutation can be broadly grouped into two types: (1) point mutation and (2) large-
scale mutation. Point mutation refers to the change in a single (or few) bases in the
DNA leading to alteration in the translated peptide. On the other hand, large-scale
mutation can be demonstrated as the alteration/deletion/duplication of a compara-
tively larger part of the DNA. (Fig. 9.1a–c). However, in present days, the concep-
tion of large-scale mutation has been expanded and became more closely associated
with chromosomal aberrations where abnormal ploidy (total number of complete
sets of chromosomes in a cell) values are also found in certain cases (Fig. 9.1).

9.1.3 Parameter 2: Mode of Occurrence

The nature of mutation can be spontaneous or induced. Spontaneous mutations take

place with a non-zero probability within a cell where several types of molecular
mechanisms are involved such as depurination, deamination, mispairing, and tau-
tomerism. These are discussed in the respective section in this chapter. On the other
436 A. Ray

Fig. 9.1 Overview of mutation. Mutation can be accomplished by changes in (a) small portion of
DNA (single or few) or by (b) deletion of the larger part. (c) Polyploidy, a type of chromosomal
aberration well known in the plant Rhoeo discolor where chromosomes are associated to form a
chain-like structure

hand, induced mutations are alterations in the DNA exposed to mutagenic chemical
and/or other environmental stressors including ultraviolet light and ionizing radia-
tion, base analogs, DNA intercalating agents, and DNA cross-linking agents.

9.1.4 Parameter 3: Target Cell Type

Mutation can occur both in somatic cells and in germ cells. Somatic mutations (also
known as acquired mutations) occur in cells that are not part of a designated
reproductive group and are not passed down to descendants. On the other hand,
germ line mutations occur soon after fertilization and get transmitted through the
offspring.

9.1.5 Parameter 4: Impact of Translated Peptide

Based on the impact on the translated peptide sequence, the mutation process can be
classified as (1) frame shift mutation, (2) in-frame mutation, and (3) base substitution
mutation. Base substitution can be either synonymous or non-synonymous. Further-
more, synonymous base substitution can also be silent, while non-synonymous base
substitution can be divided into nonsense and missense type. Two important impacts
of mutation—(a) frameshift and in-frame mutation and (b) synonymous and
9 DNA Mutation, Repair, and Recombination 437

non-synonymous base substitution—are described especially in the following

paragraphs.

9.1.6 Frameshift and In-Frame Mutation

Let us recall the triplet nature of the genetic code. The reading frame (from start to
stop codon read during translation of peptide) can be shifted if the alteration
(insertion or deletion) of bases takes place in such a position by which the combina-
tion of three bases is impaired. In contrast, if the alteration of bases takes place
without impairing the three-base devisable combination, it creates no changes in the
existing reading frame which is known as in-frame mutation. Let us consider a
typical example. Proline, aspartic acid, tyrosine, and leucine are all encoded by
CCU-GAC-UAC-CUA. Now if U is deleted from CCU, the entire peptide sequence
will be altered. Instead of proline, aspartic acid, tyrosine, and leucine, the resulting
peptide will be proline, threonine, threonine, and any other (any amino acid, stop
codon, etc.). Usually, the frameshift mutation is the result of either insertion or
deletion which is known together as “indel” mutation. It has been found that loss of
proofreading activity is significantly associated with the frequency of UV-induced
frameshift in bacteria. Investigation on novel frameshift mutation in search of the
certain frameshift mutation is going on. Recently, a frameshift mutation in the gon4l
gene is found to be associated with dwarfism in Fleckvieh cattle. Another study
found a link between an autosomal recessive non-syndromic hearing loss and a new
frameshift mutation (c.804delG) in the immunoglobulin-like domain containing
receptor 1 (ildr1) (Fig. 9.2).

9.1.7 Synonymous and Non-synonymous Base Substitution

Sometimes a single base can be substituted spontaneously resulting in changes in the

triplet codon without changing the reading frame due to the degeneracy of the
genetic code. In some cases, this type of mutation results in an altered codon that
encodes the same amino acid. This type of base substitution is known as synony-
mous substitution (Fig. 9.3). When synonymous substitution does not impact the
phenotype, it is called a silent mutation. Similarly in non-synonymous base substi-
tution, a codon is altered in such a way by a different codon that necessarily leads to
modification of amino acid.
Two definite types of non-synonymous base substitution mutations are
(a) nonsense and (b) missense mutation. We are familiar with the phrase nonsense
concerning nucleotide biology. Nonsense codon (UAG, UAA, UGA) refers to the
codon, the presence of which determines the termination of translation of a peptide.
In nonsense mutation the single base substitution results in the addition of a stop/
nonsense codon in the reading frame leading to premature termination of translation.
This results in a truncated and non-functional protein. A nonsense mutation in the
gene Cyp11B1 is one of the mutations that result in affected steroid
438 A. Ray

ORIGINAL READING FRAME

FRAMESHIFT
MUTAION CC U GA C U A C CUA

MUTATED READING FRAME

ORIGINAL READING FRAME

CC U GAC UAC CUA

Proline Asparc acid Tyrosine Leucine

ORIGINAL POLYPEPTIDE

GAC UAC CUA

U deleted from first codon CCU

Indel NEW READING FRAME

mutaon
CCG A C U A C C U A…

Proline Threonine Threonine !!!

New ready frame produce enrely different polypepde

Fig. 9.2 Mechanism of frameshift mutation. A single nucleotide insertion or deletion can lead to
change in the entire reading frame of DNA known as frameshift mutation. Frameshift mutation
results in different combinations of amino acid as compared to the original sequence
9 DNA Mutation, Repair, and Recombination 439

Fig. 9.3 Substitution mutation, a generalized conception. Substitution is commonly a point

mutation. Substitution can be a synonymous or non-synonymous mutation. Figure (a) demonstrates
non-mutated DNA. There are two types of non-synonymous mutation: (b) missense and (c)
nonsense mutation. While (d) synonymous base substitution leads to changes in RNA without
any impact on the translated peptide, nonsense mutation introduces a nonsense codon in the RNA
leading to premature termination of the peptide

11beta-hydroxylase leading to a disease condition called congenital adrenal hyper-

plasia. This is an autosomal recessive disorder where deficiency of the sex steroid
hormone impairs the primary and secondary characteristics of infants, children, or
adults.
On the other hand, a missense mutation is a point base substitution mutation
where the resulting altered amino acid leads to the non-functional protein. Such
mutations are responsible for several diseases condition including epidermolysis
440 A. Ray

bullosa (resulting in fragile skin, blister, and skin erosion), sickle cell disease, and
SOD1-mediated ALS (an adult-onset, lethal, paralytic disorder). Sometimes the
missense mutation leads to an altered amino acid, but the chemical nature of the
amino acid in the mutant sequence is similar to the normal. For example, substitution
of the middle A with G of the code AAA results in AGA which encodes arginine
instead of lysine. In such a case, the mutation has eventually very little or no effect
on the phenotype. Therefore these types of mutations are known as neutral
mutations.

9.1.8 Parameter 3: Character of Base Alteration

Depending on the character of base alteration, mutation can be classified into

transition and transversion (Fig. 9.4). From other perspectives, these mutations are
undoubtedly a type of substitution mutation. As mentioned earlier, all mutations
have been only logically classified based on different parameters which are not
necessarily to be exclusive.

(a) Transitions
In a transition mutation, a purine replaces a purine, and a pyrimidine replaces a
pyrimidine. Approximately two out of three single nucleotide polymorphisms
(SNPs, we will discuss later) are expected to be transition types of mutation.
Deamination and tautomerization are two chemical processes that contribute to
the transition type of mutation. In bacteria, DNA polymerase III has the capacity
of editing that specifically excises such mismatched bases. This reduces the
probability of mutations (Fig. 9.4a).
(b) Transversion
In transversion mutations (Fig. 9.4b), a pyrimidine substitutes for a purine or
vice versa. These mutations can be routed through DNA replication error
(discussed further in this chapter). To traverse the base, at some point of DNA
replication, purine-purine or a pyrimidine-pyrimidine mispair is critical.
Although according to the dimensions of the DNA double helix this type of
mispairs is energetically unfavorable, X-ray diffraction studies demonstrated
that purine-purine pairs are possible. For instance, 8-oxo-20 -deoxyguanosine
(8-oxodG), an oxidized deoxyguanosine derivative, can cause a spontaneous
heritable G to T transversion mutations in germ line cells of mice. The example
is one of the classical examples of the spontaneous molecular lesion discussed
later (in the “route introduction of mutation section”).

9.1.9 An Outline of Transition-Transversion Bias

Although not conserved, in general, the ratio of transitions to transversions is high in

the naturally observed mutation. In many classical works, transition-transversion
bias has been demonstrated as the ratio of differences. This has been expressed as a
9 DNA Mutation, Repair, and Recombination 441

Fig. 9.4 Transition and transversion. AT to GC transition (a) is achieved through T-G alteration in
the wobble base during DNA replication. Transversion (b) may take place during DNA replication
via a similar mechanism as of transition causing a complete change of base in both the strands. In
this figure, the fourth base pair (CG) is changed into GC

complex function of the degree of sequence divergence. In other words, transition

bias was defined as bias in instantaneous rates. There is a possibility of two
transversions while only one transition (Fig. 9.5). Each purine site may experience
one type of transition (G-A) at rate α and two types of transversion (G-C, G-T) at rate
β. Based on this consideration, probability of a transversion mutation should have a
higher frequency than transition. However in reality that scenario is different. The
conservation pattern of transition/transversion bias is not completely understood to
date and is diverse among different species. For example, metazoan DNA sequences
exhibit excess transition over transversion. A relatively high rate of mutation of
methylated cytosine to thymine is thought to be one of the major causes of this bias.
There are also several other reasons for this bias.
442 A. Ray

Fig. 9.5 Transversion versus translation. The probability of transversion is theoretically double
than transition. However, in many cases, transition mutation is found in higher frequency than
transition due to differential transition bias

Simply there are two major considerable facts behind this: (1) a single ring to
single ring substitution (in transition) is energetically favorable as compared to
double ring substitution for a single ring, and (2) transition mutation is more
abundant in the population due to its possibility to reside as a silent mutation in
the gene. Transition mutation is less likely to give rise to actual amino acid
substitution due to its wobble base pair (refer to the silent mutation as described
above). Therefore in the majority of the cases, transition mutation can reside as
single nucleotide polymorphisms (SNPs) in a population without impacting the
phenotypes. On the other hand, transversion mutations are more pronounced and
have the potential to result in a catastrophic endpoint.
9 DNA Mutation, Repair, and Recombination 443

9.1.10 Similarity and Contrast: Synonymous Substitution Mutations,

Silent Mutation, and Neutral Mutation

The terms “silent mutation,” “neutral mutation,” and “synonymous substitution

mutations” are often used interchangeably in an oversimplified manner. Synony-
mous substitution mutation is technically defined as the substitution of base leading
to different code but representative of the same amino acid as described above.
However, synonymous mutations are not always silent, nor vice versa. Sometimes
synonymous substitution mutation affects any of the processes including transcrip-
tion, splicing, mRNA transport, and translation. From the evolutionary viewpoint,
this implicates the fitness of the individual carrying the new gene to survive and
reproduce. Several synonymous mutations in the fruit fly alcohol dehydrogenase
gene, for example, were identified to cause suboptimal synonyms, resulting in a low
enzyme yield. In this situation, the phenotypic effect is not silent. Similarly, there are
other cases where synonymous substitution affects the protein function. (a) Intronic
regions are often regulatory regions, and substitution mutation in this region may
affect the protein function. (b) Timing of translation may be affected due to the
substrate specificity of the tRNA to the infrequent codon which is reflected in codon
bias. On the other hand, the mutations are neutral mutations where mutation replaces
the amino acid of the same functionality. It is the type of non-synonymous substitu-
tion often missappropriately described as silent mutation.

9.1.11 Mutations Affect Gene Expression: “Loss of Function” Versus

“Gain of Function”

Mutation can affect gene expression, and the consequence is either loss of function
or gain of function. Loss-of-function mutations are achieved through the complete or
partial absence of normal protein. Generally, in the loss-of-function mutation, the
impairment occurs within the coding region of a gene so that the ultimate proteins
are no longer able to work correctly. The mutation, on the other hand, can arise in
regulatory areas that alter transcription, translation, or splicing events. In the gain-of-
function mutation, alteration in the gene sequence produces the protein of a new
molecular function. “Loss-of-function” mutations are often recessive. Therefore,
homozygous individuals are only expressive mutant phenotype. There are several
examples of loss-of-function mutation. The most widely discussed example of
mutation is sickle cell anemia due to a mutation in β-globin gene which is a loss-
of-function mutation resulting in the impaired oxygen-carrying capacity of hemo-
globin. Similarly, a mutation in the insulin gene results in suboptimal production of
insulin leading to diabetes type I. A more complex mutation is seen in PKU or
phenylketonuria where metabolism of phenylalanine is reduced. Children seem
normal at birth but begin to express with age leading to an underdeveloped brain.
In contrast, a “gain-of-function” mutation produces an entirely new trait. This
mutation can cause a trait to arise at an inopportune moment and in an inopportune
location. Gain-of-function mutations are frequently dominant or semi-dominant.
444 A. Ray

Gain-of-function mutations in scn11a, for example, have been reported in a small

percentage of patients with painful peripheral small fiber neuropathy who have a
congenital loss of pain sensibility. Certain mutations are conditional mutations in
which the trait expresses only under certain conditions. On the other hand, certain
mutations are lethal which can cause premature death or early infantile death in
higher animals.

9.1.12 Mutations Are Instrumental in Evolution: “Gradual Change”

Versus “Quick Jump”

Mutations are thought to be the “raw materials of evolution” and are essential to
evolution. Every organism’s genetic traits were initially the result of a mutation. The
new genetic variant (allele) gets distributed via reproduction. Mutation improvises
fundamental mechanisms of life including feeding, growing, or reproducing effec-
tively. The mutant allele becomes increasingly abundant over time. As a result, the
population diverges ecologically and physiologically from the original population
that was unable to adapt. By removing a person bearing adaptive alleles at other
genes, deleterious mutations also promote evolutionary change in small populations.
In nature, both the evidence of gradual and quick evolutionary change are available
as a result of mutation.
Most mutations causing evolutionary changes are single-point mutations affect-
ing a single protein which remain less important, for example, genes that control the
structure and effectiveness of the salivary glands. At a glance, mutations to salivary
enzymes appeared to have less potential in impacting survival. However precise
accumulation of slight mutations to saliva has an impact on evolving snake venom
and snake evolution. Snake venom is a cocktail of proteins with varying effects.
Other poisonous snake families have a distinct mix of genetically related species.
Elapidae venom, which includes progenitors of sea snakes, coral snakes, and cobras,
has evolved to be neurotoxic, but Viperidae venom, which includes rattlesnakes and
bushmasters, acts on the circulatory system. Both families contain various species
that, through mutations, inherited a minor edge in venom power from their ancestors,
increasing the diversity of venoms and species over time.
Large-scale mutation usually affects quick evolutionary change within the popu-
lation. For instance, there are certain organisms where chromosomal duplication
(a type of large-scale mutation, broadly known as chromosomal aberration) takes
place because their ancestor failed to undergo successful meiosis before sexual
reproduction which resulted in the doubling of a chromosome. In North American
grey tree frogs, this approach finally leads to “instant speciation.”
As an animal, the process is also known in the plant kingdom which produces
abnormally large seeds or fruits. These are having distinct traits with specific
advantages. Most human edible cereals have enormous seeds as compared to other
grasses. In most cases, this is due to genomic duplications in the ancestors, and the
outcome of this error was successfully passed down to future generations. The
phenomenon is also found in certain modern rice and wheat. In modern day’s
9 DNA Mutation, Repair, and Recombination 445

agricultural biotechnology practice, this process of Darwin’s natural selection is

often mimicked artificially to produce better offspring through plant interbreeding
which is known as artificial selection. In no way, evolution is possible without
random genetic change. However, the differential evolutionary fitness of mutant
offspring and the probability of evolving by genetically modified individuals are the
major issues in the successful production of the mutant population.

9.1.13 Mutation Rate

The mutation rate in genetics is defined as the number of new mutations in a single
gene or organism over time. To put it in another way, it’s the rate at which a gene
switches from wild type to a certain mutation. Mutation rates are variable and diverse
(Table 9.2). Therefore, mutation rates are determined for specific classes of
mutations, and the spectrum of mutation rate is subdivided with subclasses. The
mutation rates are usually expressed with different units including mutation base
pair1 per cell division1, mutation gene1 generation1, and mutaiongenome1
generation1. Several natural units of time for each of these rates are considered in
practice. It is important to remember that only spontaneous mutations are considered
to calculate the mutation rate of an organism. The genetic makeup of each organism,
as well as environmental circumstances, has a significant impact on its mutation rate.
The upper and lower bounds of mutation rates are still up for debate. However, it has
been observed that certain health risks including cancer and other hereditary diseases
in humans increase with an increase of the mutation rate. According to the recent
estimate, the human mutation rate is approximately 0.5  109 per base pair per year
(Fig. 9.6). In practice, mutation frequency and population history are used to
compute the frequency (ƒ) of the mutant organism in the overall organism. The
mutation rate is usually denoted by μ often with subscripts to denote the type of

Table 9.2 Different mutation rates in different organisms

Organism Mutation target/effect Mutation rate Unit
Bacteriophage T2 Lysis inhibition 1  108 Per replication
Host range 3  109
E. coli Lactose fermentation 2  107 Per cell division
Histidine requirement 2  108
Neurospora crassa Inositol requirement 8  108 Per asexual spore
Adenine requirement 4  108
Corn Kernel color 2.2  106 Per gamete
Drosophila Eye color 4  105 Per gamete
Allozymes 5.14  106
Mouse Albino coat color 4.5  105 Per gamete
Dilution coat color 3  105
Human Huntington disease 1  106 Per gamete
Hemophilia A 3.2  105
446 A. Ray

Fig. 9.6 Mutation rate. The mutation rate in humans is based on the recent data (Scally 2016).
Mutation rate was calculated using family sequencing comparing genome samples across
generations in one or more families. Within each, there are de novo mutations present in offspring
and neither parent. The human mutation rate is estimated to be 0.5  109 per base pair per year,
according to a recent report

mutation. However precise calculation of mutation rate is somehow challenging both

theoretically and practically. There are certain mutations which have low and
delayed phenotypic effect; hence, the frequency of mutant population remains
intangible.

9.1.14 Mutation Hotspot

Based on the aspects discussed so far, we consider mutations in terms of the

inactivation of the gene. For understanding mutation hotspots, some important
feature of the mutation needs to be considered. (a) Most of the genes within a
species show more or less similar rates of mutation relative to their size. As a result,
vulnerability to mutation is basically proportional to the size of the genome. (b) Not
necessarily all the base pairs in a single site are equally susceptible to mutation.
(c) Majority of mutations reside at different sites, while few are found on the same
site. (d) Due to (three) distinct point mutations, two independently isolated mutations
at the same place may result in the same or different change in DNA. (e) While some
sites may get multiple point mutations, others do not. This characteristic is
demonstrated in the lad gene of E. coli.
Random hit kinetics can estimate the statistical probability of more than one
mutation occurring at a given site, which is expressed as a Poisson distribution. From
a random distribution expressed as 10–100, some sites are anticipated to gain
more than the expected number of mutations. Hotspots are the term for these sites.
Hotspots are known to cause spontaneous mutations; however, various mutagens
9 DNA Mutation, Repair, and Recombination 447

may have variable hotspot selectivity. A modified base has evolved into mutational
hotspots. Sites containing 5-methylcytosine, for example, are hotspots for spontane-
ous point mutations in E. coli, where the mutation occurs as a GC to AT transition.
One of the most prominent reasons for the existence of such hotspots is the high
frequency of spontaneous deamination of cytosine bases. While discussing the
mechanism of spontaneous mutation, the process of deamination is covered in its
own section of the chapter.

9.1.15 Somatic and Germinal Mutation

It is already discussed that mutations can occur in both somatic and germ cells. In
this section, both somatic and the germinal mutation will be discussed with the
typical examples.
Somatic mutation: As the name implies, a mutation is a somatic mutation when
it takes place in any in the somatic cells. Somatic mutations cannot flow through the
progeny and therefore do not impair the infant’s genotype. The impact of somatic
mutation fairly depends on the developmental stage when the mutation occurred. For
instance, if the somatic mutation takes place in a single diploid cell at a very early
stage of development, the mutant progenitor cell clonally multiplies producing
mutant body parts. If mutation takes place in the later phase of life in the
non-pluripotent cells, the impact mutations are closely restricted to a small portion
(Fig. 9.7) known as the “mutation sector.” Thus the formation of the “mutation
sector” depends on the stage where the first mutation occurred. The phenotypic
effect is often not observable if the mutation takes place in a single or a few cells.
However, the mutation causing cancer is an exception where a mutation in few cells
may lead to a significant phenotypic endpoint, because in these cases the mutation
itself promotes cell division by impairing cell cycle regulation and reduced
apoptosis.
One of the best examples of somatic mutation in the plant system is apple skin
color. A rare form of somatic mutation is found in red delicious apple; half of the
apple appears red, and half is green or golden. In these cases, a mutation occurred in
the early developmental phase of the ovary wall cells which eventually differentiated
into the fruit skin. The mutation changes the skin color from red to green or yellow.
The exact mechanism and key mutations producing green or golden color are not
completely characterized. However, according to recent reports, anthocyanin is
found to be responsible for producing red color in the apple skin. Differential
accumulation in different zone produces different colors, and MdMYB1-mediated
MdGSTF6 expression acts as one of the important regulators in the anthocyanin
production network.
Germinal mutation: Like somatic cell mutation, when the mutation occurs in
germ cells, it is known as germinal mutation. In the germ line, if a specific tissue is
programmed to generate sex cells, a germinal mutation occurs (Fig. 9.8). If mutant
sex cells (either oocyte, spermatozoa, or both) fertilize, then the mutation will be
passed down to the following generation. If the fertilization takes place between both
448 A. Ray

Mutation
sector So mut on in ovary
Changes the apple color

Mutation
sector grows
with
development
through
clonal
propagation

B
Developmental age of mut

Size of muta sector

Earlier muta produce larger the mut sector

Fig. 9.7 Somatic mutation—a simplified representation: A specific example of somatic mutation
in red delicious apple. Mutation in ovary cells produces a partially green or golden color. (b)
Conceptual representation of the growth of mutation. The size of the mutation sector depends on the
stage of development when the mutation occurred

the mutant counterpart and the progeny becomes homozygous mutant else it
produces heterozygous mutant. An individual acquired mutation in the sex cells
may otherwise remain perfectly normal until detected in the germ cell. For instance,
X-linked mutation expressing hemophilia in European royal families is believed to
9 DNA Mutation, Repair, and Recombination 449

Normal Normal
A

Normal Development

B Normal Mutant

Mutant Developed

C Mutant Normal

Mutant Developed

Ferlizaon

Normal cells Mutant cells

D
Normal Normal

Fig. 9.8 Mutation in a different stage of development and their predicted result. Mutation in a
different stage of life and different cells may result in different expected consequences. (a) No
germinal or somatic mutation development of normal cells. (b) Mutation in oocytes may lead to the
development of mutant fertilizing with the normal sperm or (c) vice versa. (d) Mutation in the very
early stage somatic cells with high differentiation potency has a significant probability to affect the
organism to a large extent
450 A. Ray

have aroused from the germinal mutation of Queen Victoria or one of her parents.
However, as hemophilia (a bleeding disorder with impaired blood clotting capacity)
is the X-linked recessive mutation, these are expressed only in the male descendants.

9.1.16 Spontaneous or Induced Mutation

9.1.16.1 Spontaneous Mutation

In general perception, the appearance of a new mutation is a rare event. In the
majority of cases, geneticists were able to spot these mutations in nature. Mutations
that were originally studied occurred spontaneously and are known as spontaneous
mutations. The rate of spontaneous mutation is variable. A larger genome provides
a large target; thus the probability of mutation frequency is high. A study of the five
coat color loci in mice exhibited the range of mutation rate from 2  106 to 40 
106 mutations per gamete per gene. A eukaryotic organism’s spontaneous mutation
rate is 2–12  10–6 mutations per gamete per gene, according to data from multiple
studies. Similarly, with 100,000 genes in the human genome, 1–5 human gametes
are projected to be mutated in some genes. Spontaneous mutations can be any sort of
point mutation, including frame shift, substitution, and mismatch. There are three
major routes by which spontaneous mutation has been introduced in a DNA such as
(a) error in DNA replication, (b) deletion or duplication, and (c) molecular decay or
spontaneous lesion. In this section, we will be discussing important routes of
introducing spontaneous mutation.

(a) Error in DNA Replication

When an inappropriate nucleotide pair (e.g., A–C instead of A–T) develops in
DNA synthesis, an error might occur, resulting in a base substitution. Let us now
look at how this occurs. Every base in DNA exists in one of multiple isomeric
forms known as tautomers, which differ in the positions of their atoms and the
bonds that link them but remain in equilibrium. These forms are keto, imino, and
enol. Among which keto forms are abundant (Fig. 9.9). At a certain point of
DNA replication, one tautomer often shifts to another (inappropriate) resulting
in mispairing of bases. This phenomenon is known as “tautomeric shift” first
identified by Watson and Crick while they were formulating the model of DNA
structure. The ability of the wrong tautomer of a standard base to mispair
eventually potentiates the probability of mutation.
(b) Deletions and Duplications
Large deletions (deletions of more than a few base pairs) account for a signifi-
cant portion of spontaneous mutations. The deletions occur in the majority of
cases at repeated sequences. Several studies have shown that the longest
repeated sequences are hotspots for deletions. Duplications of DNA segments
have also been observed in several organisms, and they frequently occur at
sequence repeats.
(c) Molecular Decay or Spontaneous Lesion
9 DNA Mutation, Repair, and Recombination 451

Fig. 9.9 Tautomerism in bases. Common and rare forms of bases participate in tautomeric shift
leading to erroneous DNA replication and thereby resulting in point mutation in the nucleotide
chain

Molecular decay or spontaneous lesions as the name implies constitute naturally

occurring damage to the DNA. Major types of spontaneous lesions frequently
result from depurination, deamination, and oxidative damage.

9.1.16.2 Depurination
Sometimes the glycosidic bond between the base and deoxyribose is interrupted, and
subsequently, the purine residues (A and G) are lost from DNA. It has been shown
and observed that a mammalian cell spontaneously loses ~10,000 purines from DNA
in a single cell cycle period. Theoretically, this can lead to significant genetic
damage. Base specificity is hindered at the apurinic sites. But that never happens
because the efficient repair mechanism ((Fig. 9.10a) discussed later in this chapter)
removes apurinic sites. However, under some circumstances, a base is frequently
inserted across from an apurinic site, resulting in DNA mutation.

9.1.16.3 Deamination
Let us recall the chemical structure of cytosine and uracil. The sole structural
difference is an amino group at C4. Uracil can be produced via the spontaneous
deamination of cytosine. During replication, unrepaired uracil residues can pair with
adenine, converting a G-C pair into an A-T pair (a GC AT) (Fig. 9.10b). This is one
452 A. Ray

Fig. 9.10 The spontaneous lesion in bases. (a) Depurination in GTP produces depurination sugar
leading to impaired specificity in base pairing. (b) Delaminated bases compromise the base-pairing
specificity leading to mutation. (c) Formation of thymine dimer induced by ultraviolet radiation

of the classic examples of transition mutation. Among depurination and deamina-

tion, depurination is found more frequently.
(d) Oxidatively Damaged Bases
Several metabolic pathways produce reactive oxygen species (ROS), which
include superoxide radicals (O2•), hydrogen peroxide (H2O2), and hydroxyl
radicals (OH•). They have the potential to cause oxidative damage to DNA and
DNA precursors (such as GTP). These can result in mutations, which have been
linked to many human disorders. The 8-oxo-7-hydrodeoxyguanosine (8-oxodG,
or GO) product, for example, frequently mispairs with A, leading to a high rate
9 DNA Mutation, Repair, and Recombination 453

of G-T transversions. If left unrepaired, thymidine glycol prevents DNA repli-

cation, but it has not yet been linked to mutagenesis.
(e) Pyrimidine Dimers
Pyramiding dimers are a special case of spontaneous lesions formed from
thymine, cytosine, or uracil bases in the nucleotide chain through a photochemi-
cal reaction. Ultraviolet (UV) light induces covalent linkages formation between
two consecutive pyrimidine bases along a nucleotide chain (Fig. 9.10c). These
pre-mutagenic lesions impair the structure and the base pairing. UV is the natural
causative agent of these lesions; therefore, our skin cells exposed to sunlight can
undergo up to 50–100 such reactions per second. However, these lesions are
normally corrected immediately by a repair mechanism known as nucleotide
excision repair (NER) (NER mechanism is later discussed in this chapter). In
bacterial this correction takes place via a much simpler mechanism known as
photolyase reactivation. Uncorrected lesions, beyond the threshold, can cause
mutation due to misreading during transcription or replication. These lesions can
also arrest the normal replication. This phenomenon is most commonly
described in the bacterial system. However, pyrimidine dimers are thought to
be the primary cause of melanomas in humans. The two most common UV
products are CPDs (cyclobutane-pyrimidine dimers) and 6–4 photoproducts.

9.1.17 Mutation: A Reverse Phenomenon

9.1.17.1 Reverse Mutations and Suppressor Mutations

The impact of one mutation can be reversed by another mutation leading to restora-
tion of the original phenotypic characteristic of an organism. These mutations are
known as reverse mutations or back mutations, in contrast to the forward mutation
where alteration of the original phenotypes takes place. For example, a nonsense
mutation can be reversed by further changing the nonsense codon in the mRNA to a
codon of amino acid. If these changes bring back the original amino acid sequence,
then it is considered a true reversal. If the change of nonsense codon results in the
addition of some amino acid other than the original, it is known as the partial
reversion.
The phenotypic effect of an organism can also be restored by suppressor muta-
tion. However, it is important to note that in this case restoration of the phenotypic
effect is not true reversal; thus restoration is not robust. There must not be any
confusion between the reverse mutations and the suppression mutation. A suppres-
sor mutation balances out the effects of the first mutation, having left the organism
with no evident phenotypic differences. Only the effects of the initial mutation are
diminished by a suppressor mutation, but the first mutation is never reversed by the
second (suppressor) mutation. Suppressor mutations may occur in the different sites
of the same gene where the original mutations have taken place (intragenic suppres-
sor), or they may occur in a different gene (intergenic suppressors). The mechanism
of both types of the suppressor is different. Intragenic suppressors can change
various nucleotides in the same codon as the original mutation, or they might change
454 A. Ray

a nucleotide in a different codon entirely, whereas intergenic suppression is the

consequence of the second mutation in another gene. For example, the effect of a
nonsense mutation can be suppressed if the second mutation alters the anticodon in
the respective tRNA which induced codon recognition capacity. For instance, if the
anticodon 30 -AUG-50 of a tyrosine tRNA gene is altered to 30 -AUC-50 , the suppres-
sor tRNA still carries tyrosine but reads the nonsense codon 50 -UAG-30 . As a result,
rather than terminating the chain, tyrosine is introduced into the polypeptide.
Although the original mutation is not reversed in this case, it fairly suppresses the
effect of the nonsense mutation.

9.1.18 Mutagenesis in Bacteria

Genomes of bacteria exist in circular DNA where the genes are arranged in an
operon. The average size of a bacterial genome is around 4000 kb. Other than the
circular DNA molecule, other elements including plasmids, transposons, integrons,
or gene cassettes are also evident.
Bacterial genes with similar functions frequently share a promoter and are
transcribed at the same time. An operon is a name for this type of system. The
binding of transcription factors to the operator sequence of the DNA allows these
operons to be regulated. Mutation in bacterial genome is introduced and inherited
quickly through error-prone DNA replication due to having short generation turn-
over of the majority of the genes. Mutations in bacteria can produce alteration in
structural or colony characteristics or loss in sensitivity to antibiotics. Bacterial
spontaneous mutations occur at a rate of 1 in 105 to 108, resulting in random
population variation. Few potential contributions in bacterial mutation are as
follows:

(a) Auxotrophs: Auxotrophs have essential nutrient process dysfunctional.

(b) Resistant mutants: The population has developed a high level of tolerance to the
stress of inhibitory chemicals or antibiotics.
(c) Regulatory mutants: Population having defective regulatory sequences such as
promoter regions.
(d) Constitutive mutants: In constitutive mutants, the genes that normally turn on
and off as in operons are expressed continuously.

Spontaneous mutations are most common in bacteria when DNA pol III
synthesizes a new strand of DNA. In some cases, incorrect nucleotides are added
or omitted during the procedure. In studies with E. coli, the lagging strand was found
to be mutated 20 times more likely than the leading strand.
9 DNA Mutation, Repair, and Recombination 455

9.2 Mutation: Phenotypic Effects

9.2.1 Phenotypic Effects

Mutations have diverse phenotypic effects. These effects are inheritable when in the
case of the germinal mutation. Only by comparing the mutant to the most prevalent
phenotype in a natural population can the phenotypic effect of a mutation be
realized. In contrast to the mutant, the original phenotype is known as the wild-
type phenotype. As discussed above, a broader consequence phenotypic change in
an organism may eventually lead to evolutionary change although in several cases of
a slow propagating spontaneous point mutation in a population, it is difficult to
identify the original phenotype. Drosophila melanogaster, for example, has red eyes
by nature; hence flies with red eyes are considered wild type. Any other genetically
determined eye color in fruit flies is regarded as a mutant phenotype, in contrast to
the red eye color. Forwarding mutation refers to a mutation that affects the wild-type
phenotype, whereas reverse mutation refers to a mutation that returns a mutant to its
wild-type phenotype.
In nature, most mutations do not exhibit pronounced phenotypic effects and are
believed to be silent mutations. There are two important theories behind these.
(a) Only a small portion of the genome of a higher organism possesses coding
functionality. For example, in humans with 3234.83 Mb of total haploid genome
size, only ~2% possesses coding functionality. Therefore, a spontaneously occurring
mutation has a higher probability to affect the non-coding region resulting now
phenotypic effects. (b) All higher organisms have double sets of chromosomes that
constitute the diploid genome. In case of several mutations, the phenotypic change is
only expressed where genes in both the homologous chromosome are mutated;
otherwise the mutations remain recessive. In this way, several diploid species
increase the burden of genetic disease accumulating the large pool of mutation
without expressing the same. Therefore, on the one hand, large portion of
non-coding DNA may be advantageous in respect to the phenotypic effect of the
mutation. However, the recessive character of a mutation is significantly disadvan-
tageous increasing the mutation burden in the population without exhibiting pheno-
typic change.

9.2.2 Inheritance of Mutation

Most animals and several plants exhibit sexual dimorphism. There are two categories
of chromosomes in this organism such as autosomes and sex chromosomes. Sex is
determined by special sex chromosomes at the specific stage of development. The
rules of inheritance which were primarily demonstrated with Mendel’s example are
the rules of autosomes. The sex chromosomes in diploid organisms are generally one
pair only. We know that human has 46 chromosomes among which 44 autosomes
(A) and 2 sex chromosomes (X and Y) in a diploid cell. XX combination determines
the female, and XY combination determines the male progeny. A mutant trait can be
456 A. Ray

Table 9.3 Type of inheritance of mutational disorders

Types of
mutant
inheritance Pedigree characteristics Example
Autosomal 1. Usually children of one affected Huntington disease, achondroplasia,
dominant parents are affected myotonic dystrophy, Marfan
2. Disease appears in every syndrome
generation
3. Caused by one mutant allele
4. Vertical flow
Autosomal 1. Generally children are Cystic fibrosis, Beta thalassemia,
recessive unaffected (heterozygotes); parents homocystinuria
are affected
2. This is not typically found in
every generation
3. Caused by two mutated alleles
4. Horizontal flow
X-linked 1. Females are more frequently Hypophosphatemia, Rett syndrome
dominant affected than the males
2. No male-to-male transmission
X-linked 1. Males are more frequently Duchenne muscular dystrophy,
recessive affected than females hemophilia
2. For a female to be affected, both
her parents must be a carrier
3. Fathers cannot pass X-linked
traits to their sons
Mitochondrial 1. Both males and female children LHON: Leber hereditary optic
can be affected neuropathy
2. Can appear in every generation
of a family

inherited and either linked to autosomes or linked to a sex chromosome. Both types
of inheritance can be either recessive or dominant. Different types of inheritance are
summarized in Table 9.3. Recessive mutation produced haploinsufficiency that
means the individual gets only 50% of the mutant product from one counterpart of
the homologous chromosome. As a result of these, only homozygous genotype
(having both the mutant counterpart) is only expressive of the trait. On the other
hand, dominant mutation can be expressed where at least one counterpart of the
homologous chromosome is mutated. Several diseases are inherited and either linked
to autosomes or sex chromosomes, most X chromosomes. Some of the examples of
different types of inheritance are as follows: autosomal dominant (Huntington
disease, neurofibromatosis, and polycystic kidney disease); autosomal recessive
(autosomal recessive cystic fibrosis, Tay-Sachs disease); X-linked dominant
(X-linked hypophosphatemia, Rett syndrome); and X-linked recessive (color blind-
ness, hemophilia). Like X-linked inheritance, there are certain characteristics not
9 DNA Mutation, Repair, and Recombination 457

Fig. 9.11 Pedigree demonstrating a different type of inheritance. Basic pedigree pattern
demonstrating inheritance of (a) autosomal dominant, (b) autosomal recessive, (c) X-linked
dominant, and (d) X-linked recessive trait. (e) Pedigree of Queen Victoria and her descendants,
showing inheritance of hemophilia trait

always linked to the Y chromosome. This is known as holandric inheritance. One of

the classical examples of holandric inheritance in humans is a hairy ear. Figure 9.11
represents the typical pedigree demonstrating the flow of mutation through
generations.
458 A. Ray

9.2.3 Morgan’s Experiment

Thomas Hunt Morgan (1909) started working on the inheritance pattern of certain
characteristics in fruit fly (Drosophila melanogaster). During his experiment, Mor-
gan found one fly in his laboratory colony with a white eye as compared to the red
eyes of the normal population. This observation played a big role in the discovery
and demonstration of inheritance mutation. Through a series of systematic genetic
crosses, Morgan demonstrated the pattern of inheritance. As a result of the first cross
between purebred red-eyed fly and a white-eyed male, he found almost all the
individual in F1 had red eyes (only 4 was white among 1237 flies). From these
results, he predicted that white eyes are a simple recessive trait. When F1 progenies
were crossed with another, all of the females had red eyes, while half of the males
had white eyes (Fig. 9.12). The result was a clear deviation of the simple recessive
trait (expected value 25% of the total progeny). Thus it was hypothesized the locus
of the mutation producing white eye in Drosophila melanogaster was X-linked
recessive. Therefore males with the mutant X chromosome are haplosufficient
expressing the trait. On the other hand, 50% of the females having only one mutant
chromosome are compensated by the normal X counterpart, whereas the female
having both the mutant X expresses the trait. Morgan’s hypothesis was confirmed by
a subsequent cross between a white-eyed female and a red-eyed male, which
produced all red-eyed females and all white-eyed males, just as Morgan predicted.
Morgan’s hypothesis was statistically validated. Not all the individuals exhibited the
expected result. A very small fraction (about 2–2.5%) deviated from the anticipated
result. Later this deviation was explained by Bridges. In certain females, two X
chromosomes fail to split during anaphase I of meiosis, resulting in some eggs
receiving two copies of the X chromosome and others receiving none. This disorder
is known as chromosomal nondisjunction. If these eggs are fertilized by sperm from
a red-eyed male, three extra genotypes with different combinations of sex
chromosomes are produced including XwXwY, X+O, and XwXwX+. Among these
genotypes, XwXwX+ is lethal. However, X+O produce red-eyed male (because sex
determination in Drosophila depends on X: A ratio and X+O genotype only receive
the male X chromosome counterpart), and XwXwY genotype produces a white-eyed
female.

9.3 Mutation: Mechanism

The mechanism of mutation largely depends on the causative agents. The specific
mechanism is studied in induced mutation, not in spontaneous mutation. In this part,
different types of induced mutation and the associated mechanism are discussed.
9 DNA Mutation, Repair, and Recombination 459

First Cross

Morgan’s fly
Morgan’s fly

Almost all were red eyed

Only 4 among 1237 found
white eyed

Second Cross

Morgan’s fly Morgan’s fly

Morgan’s fly Morgan’s fly
Morgan’s fly

ALL FEMALE 50% REST 50%

OUTCOME RED EYED MALE WHITE EYED MALE RED EYED

Fig. 9.12 Overview of Morgan’s experiment on X-linked inheritance with Drosophila

melanogaster of red and white eye color where red-eyed flies are wild type (purebred). In the
first cross, almost all flies were red-eyed. In the second cross between red-eyed males and red-eyed
females, 50% of males were red-eyed while the rest were white-eyed. On the other hand, 100% of
females were found red-eyed

9.3.1 Induced Mutation

Mutations can be induced using different methods. The three most common
approaches used to induce mutations are radiation, chemical, and transposon
460 A. Ray

insertion. The early mutations in Drosophila were caused by radiation (X-rays),

which Mueller used to cause fatal mutations. Other forms of radiation, including
gamma radiation and fast neutron bombardment, have also been discovered to be
proven mutagens. Similarly, chemical mutagens are frequently used to generate
work point mutations. Alkylating agents and base analogs are the most routinely
used mutagen in chemical mutagenesis. Other than radiation and chemicals,
transposons or transposable elements are also used to generate mutants.
Transposable elements are the mobile DNA fragments that can migrate from one
site of the genome to another. As a result, new mutants are formed. In this section,
we will be discussing the characteristics and mechanisms of different types of
induced mutation.

9.3.2 Induced by Radiation

Ultraviolet light (UV) is the most common form of non-ionizing radiation. Cytosine
and thymine, the two bases of DNA, are most susceptible to this radiation. Exposure
to UV especially the longer wave UV light (UVA) can induce pyrimidine dimer
between two adjacent pyrimidine bases in a DNA strand (as discussed earlier). UV
exposure can also result in oxidative damage in DNA. Gamma radiation, on the other
hand, is an ionizing radiation that can cause cancer.
As mentioned above, X-rays have enough potential to induce mutation depending
on the magnitude of exposure. The differential mutagenic effects of X-ray are the
matter of investigation in the last 50 years. In a recent genome-wide study, up to
6000 SNV (single nucleotide variation), CNV (copy number variation), and indel
(insertion and deletion combined) mutation was found to be evident in the diploid
mammalian germ line cells. These cells however have the ability to repair 30,000
similar endogenous DNA defects per day. As a result, an additional 6000 damage is
less likely to have a long-term effect. Radiation-induced double-strand breaks and
clustered damaged sites, on the other hand, are the most severe types of DNA
damage because their repair is significantly delayed or hindered, and if not repaired
properly, these lesions can produce DNA rearrangements.

9.3.3 Induced by Chemical

9.3.3.1 Incorporation of Base Analogs

Some chemical compounds are suitably similar to regular nitrogen bases, and such
compounds are the base analogs. They are inserted occasionally into DNA to replace
regular nucleotides. These bases lack the same stringent paring qualities as conven-
tional bases, thus causing mutations during DNA replication. 5-bromouracil (5-BU),
for example, is a thymine analog with bromine at the C5 position instead of the CH3
group. During transition mutation, 5-BU can frequently undergo a keto-enol tauto-
meric shift.
9 DNA Mutation, Repair, and Recombination 461

9.3.3.2 Specific Mispairing by Alkylating Agents

Certain alkylating agents, such as ethyl methanesulfonate (EMS) which used
nitrosoguanidine (NG), can cause specific mispairing instead of getting incorporated
into the DNA. These agents add alkyl groups to several positions on bases. The
mutagenic effect is most pronounced when they add the oxygen at the six positions
of guanine to produce an O6-alkylguanine. This addition leads to direct mispairing
with thymine, resulting in GC ! AT transitions during replication. Mutagenic
specificity for EMS and NG exhibits a strong preference for GC ! AT transitions.
Alkylating agents can modify the bases in any dNTPs.

9.3.3.3 DNA Intercalating Agents

DNA intercalating agents interact non-covalently with the DNA molecule either
through threading intercalation or through classical intercalation. Threading interac-
tion is a groove-bound connection that is stabilized by hydrophobic, electrostatic,
and hydrogen-bonding interactions. Classic interaction, on the other hand, is an
intercalative association in which a planar, heteroaromatic moiety slides between the
DNA base pairs.
Intercalation causes the DNA double helix to unwind. Depending on the
intercalating agents, the degree of unwinding varies. For example, ethidium unwinds
DNA by around 26 and proflavine by about 17 . These structural abnormalities
eventually result in functional changes, such as suppression of transcription and
replication, as well as mutations in DNA repair pathways (Fig. 9.13a).

9.3.3.4 DNA Cross-Linkers

DNA cross-linking agents have two independent reactive groups within the same
molecule which can bind with a nucleotide residue of DNA. Both natural and
synthetic chemicals can act as exogenous cross-linking agents, whereas endogenous
cross-linking agents are the compounds that are the intermediary metabolites of
biochemical pathways within a cell or organism. For example, cisplatin
(cis-diaminedichloroplatinum (II)) and its derivatives act as exogenous cross-linking
on the adjacent guanines at their N7 positions. On the other hand, nitrous acid acts as
the endogenous cross-linking agent via conversion of the amino group to the
carbonyl group in the DNA (Fig. 9.13b).

9.3.3.5 Aflatoxin B1
Aflatoxin B1 is a mycotoxin produced by Aspergillus flavus and Aspergillus
parasiticus. Aflatoxin B1 is highly hepatotoxic and hepatocarcinogenic. It can
generate apurinic sites resulting in the formation of an adduct at the N7 position of
guanine (Fig. 9.13c).

9.3.4 Induced by Transposable Elements

Transposons are DNA elements that can move from one region to another region in
the genome. When transposon shifts from a heterochromatin region to a
462 A. Ray

Fig. 9.13 Chemical-induced changes in DNA leading to mutation. (a) Ethidium bromide interca-
lation in DNA. (b) Cisplatin-mediated intra-strand adduct and intra-strand cross-links. (c)
Aflatoxin-DNA interaction producing apurinic sites via formation of base aflatoxin adduct

transcriptionally active part of DNA, mutagenesis takes place. It is successfully used

as a tool for mutagenesis and gene function. Transposons are most well
demonstrated in Drosophila melanogaster (P elements), Arabidopsis thaliana, and
9 DNA Mutation, Repair, and Recombination 463

a Transposon Tn10 b

9,300 bp Transposon, Tn3

1,400 1,400
bp 6,500 bp bp 4,957 bp
tnpA tnpB bla

IS10L IS10R
Tetracycline Transposase β-lactamase
resistance Left inverted Resolvase Right inverted
Inverted gene (Tc R) Inverted repeat (38 bp) repeat (38 bp)
repeats repeats
mRNAs
of IS of IS
element element

Inverted IS elements

Fig. 9.14 The general features (structural) of transposons. (a) Composite and (b) non-composite
transposons flanked by inverted IS sequence at both ends

Escherichia coli. Transposon mutagenesis was first discovered as “jumping genes”

by Barbara McClintock in corn for which she was awarded Nobel Prize in 1983.
In bacteria, transposition mutagenesis is generally achieved by the plasmid from
which transposons are extracted and placed into the host chromosome. Chemical
mutagenesis is less efficient than transposon-mediated mutagenesis. It has a higher
frequency of mutations. Furthermore, the organism’s lethality is reduced. It can
cause single hit mutations while also incorporating selectable markers in the con-
struction. However, it is having lower transposition frequency inaccuracy in trans-
position. Initially, the transposon mutagenesis experiments are used to be achieved
by bacteriophages and conjugative bacterial plasmids. Non-specificity was the key
issue in this method. Shuttle mutagenesis is a relatively new approach that uses
cloned genes from the host to introduce genetic elements. The transposon and
transposition system is wide and complicated. Tn3, Tn5, Tn10, and many transposon
systems have been developed. There are two types of bacterial transposons: com-
posite transposons and non-composite transposons. Tn10 is an example of compos-
ite transposons (Fig. 9.14a), which have a core region with genes (e.g., genes that
confer antibiotic resistance) flanked on both sides by IS elements known as IS
modules. Tn3 (Fig. 9.14b) is an example of a non-composite transposon that
contains antibiotic resistance genes but does not end with IS elements. In this
context, the overview of the two most important transposition systems, the Tn5
transposon system, and the Sleeping Beauty transposon system are provided as
follows.

9.3.4.1 Tn5 Transposon

Transposon mutagenesis is studied using the Tn5 transposon as a model system. Tn5
is a bacterial composite transposon with IS50R and IS50L insertion sequences
flanking genes. IS50R codes for two proteins, Tnp and Inh. A transposase is
represented by the letter Tnp. Transposase inhibitor (Inh) is a gene that is encoded
464 A. Ray

by the Inh gene. Nineteen base pair components separate the IS50R and IS50L
sequences. Mutation in these regions leads to impair the ability of transposase genes
to bind to the sequences. Genes of interest are introduced into the transposon
between IS50 sequences controlled by the host promoter. Incorporated genes usually
include the target gene and the selectable marker to identify transformants. Almost
all transposon systems share the most likely pathway for Tn5 transposition.

9.3.4.2 “Sleeping Beauty” Transposon

The SBTS (“Sleeping Beauty transposon system”) is the first nonviral vector to
successfully introduce a gene cassette into a vertebrate genome. Transposing the
cassettes directly into the genome of the organism from the plasmid allows for long-
term expression of transgenes. This transposition system has evolved over time as
the transposon sequences have been improved and the utilization of transposase
enzymes has increased, resulting in a transposition frequency of around 50%.
Transposase enzymes can be expressed in both a cis and a trans manner in relation
to the gene cassette. SBTS transposition has a similar underlying process to Tn5
transposition. Inverted repeat repeats, known as IR/DR sequences, flank the trans-
poson. The gene sequences are the only eukaryotic components. Transposition
allows for long-term transgene expression. This can be enhanced further by using
transposase enzymes to improve transposition capacity. SB100X is a hyperactive
mammalian transposase with a hundred times the efficiency of the first-generation
transposase. Incorporating these enzymes into the cassette can lead to longer-lasting
transgene expression (over 1 year). When pronuclear injection into mouse zygotes is
used, transgenesis frequencies can reach 45%.

9.3.5 Trinucleotide Repeat: Mutation

9.3.5.1 CAG Repeats

Trinucleotide repeat expansion, also known as triplet repeat expansion (Fig. 9.15), is
a form of DNA mutation that can result in a variety of disorders. In this type of
mutation, the number of the trinucleotide repeat in gene or intron exceeds the normal
stable threshold leading to microsatellite repeats. These mutations are dynamic and
gradually increase the number of repeats in the gene. By generating spherical
clusters known as RNA foci, trinucleotide repeats in introns can induce toxic effects
in nucleases.
Slippage during DNA replication causes triplet expansion. During replication or
DNA repair synthesis, instable tandem repetitions in the DNA sequence form “loop-
out” structures in DNA. Endonuclease generates a nick on one side of the DNA
strand, and DNA polymerase extends and seals a repeating triplet throughout this
process. The number of repeats increases when the loop-out structure is produced
from the sequence on the daughter strand, whereas the number of repeats reduces
when the loop-out structure is formed on the parent strand. A fragile X syndrome is a
classic example of an expanded trinucleotide repeat. Characteristically in the patient
with this disease, the tip of the X chromosome is found attached only by a lean
9 DNA Mutation, Repair, and Recombination 465

DNA WITH 8 CAG

1 REPEAT STRAT
REPLICATING

HAIRPIN LOOP
FORMS ON THE
2 NASCENT STRAND

PART OF TEMPLATE
STRANDS
REPICATED TWO
3 TIMES INCREASING
THE NUMBER OF
REPEAT (FOLLOW
MARKING)

STRAND WITH
EXTRA REPEATS
4 SERVES AS
TEMPLATE

RESULTING DNA MOLECULE CONTAIN ADDITIONAL COPIES OF CAG REPEAT

5

Fig. 9.15 Trinucleotide repeat expansion of CAG repeat via formation of hairpin loop. DNA with
eight CAG repeat replicates and forms a hairpin loop in the nascent strand. As a result, the shortened
complementary segment replicates again introducing the extra repeats. The strands with extra
repeats serve as a template for further replication

thread. The disease is caused by several (around 60 or more as compared to the

normal) CGG repeats in the fmr-1 gene. Huntington’s disease is another critical
disease found to be caused by trinucleotide repeats.

9.3.5.2 Why ‘Three’ No Other Numbers in Nucleotide Repeats

An important question is why three, not the other numbers nucleotides are
expanded? Dinucleotide repeats are a common feature of the genome as are larger
repeats (VNTRs—variable number tandem repeats). One possible answer is any
other numbers of nucleotide repeats may cause frame shift mutations causing
obligation in developmental pathways and therefore which are not due to three
466 A. Ray

Table 9.4 Non-polyQ (non-polyglutamine) diseases

Normal Pathogenic
Type Gene repeats repeats Codon
Fragile X syndrome fmr1 6–53 230+ CGG (50
UTR)
Fragile X-associated tremor/ataxia fmr1 6–53 55–200 CGG (50
syndrome UTR)
Fragile XE mental retardation aff2 6–35 200+ CCG (50
UTR)
Friedreich’s ataxia fxn 7–34 100+ GAA
(Intron)
Myotonic dystrophy type 1 dmpk 5–34 50+ CTG (30
UTR)
Spinocerebellar ataxia sca8 16–37 110–250 CTG
(RNA)
Spinocerebellar ataxia ppp2r2b 7–28 66–78 nnn (50
UTR)

nucleotide repeats may increase the probability of getting masked by early infantile
or developmental lethality. On the other hand, mutation in three tandem nucleotides
cannot produce catastrophic frameshift, and unless a stop codon is added, gene
expression is not attenuated. Single nucleotide expansions to a coding region less
likely to cause detrimental because a maximum of two amino acids are affected.
However, the overwhelming number of repeats is deleterious.
Currently, an increased number of CAG repeats in the coding areas of unrelated
proteins have been discovered to cause nine neurologic diseases. A polyglutamine
tract is formed when enlarged CAG repeats are translated into a series of consistent
glutamine residues (“polyQ”). This polyQ is more prone to aggregation. The activa-
tion of downstream regulatory processes can be disrupted by a prolong tract. The
autosomal-dominant inheritance patterns define these disorders. Trinucleotide repeat
diseases are characterized by a genetic anticipation phenomenon, in which the
influence of a mutation grows with the individual’s age but is not expressed at an
earlier stage. The common symptoms of polyQ disease are progressive degeneration
of nerve cells. Like polyQ repeats, there are certain non-polyQ diseases; however,
they do not express specific symptoms. Some important polyQ and non-polyQ
diseases are documented in Tables 9.4 and 9.5.

9.3.5.3 The Practical Implication of Mutagenesis: Site-Directed

Mutagenesis
Mutagenesis is being rapidly used as an essential tool in several modern biological
investigations. With the advancement of technology, mutagenesis can be achieved in
specific sites via site-directed mutagenesis (SDM). To mutate plasmids, reverse PCR
is employed. This approach amplifies the entire plasmid using two back-to-back
primers, and the linear result is then ligated back to the circular form. Altering the
primer sequences to include the desired mutation can affect the primer binding areas.
9 DNA Mutation, Repair, and Recombination 467

Table 9.5 PolyQ (polyglutamine) diseases

Pathogenic polyQ Normal polyQ
Type Gene repeats repeats
Dentatorubropallidoluysian atrophy atn1/ 49–88 6–35
drpla
Huntington’s disease htt 36–250 6–35
Spinal and bulbar muscular atrophy ar 38–62 9–36
Spinocerebellar ataxia type 1 atxn1 49–88 6–35
Spinocerebellar ataxia type 2 atxn2 33–77 14–32
Spinocerebellar ataxia type 3 or Machado- atxn3 55–86 12–40
Joseph disease
Spinocerebellar ataxia type 6 cacna1a 21–30 4–18
Spinocerebellar ataxia type 7 atxn7 38–120 7–17
Spinocerebellar ataxia type 17 tbp 47–63 25–42

By adding flanking sequences to the primers, insertions can be made around the
primer binding areas, and deletions can be made by simply leaving a space between
the two primers. To change a target region, primer extension employs nested
primers. Primers B and C contain the mismatched sequence to insert bases, as
shown in the diagram. The mutant sequence is used in the first round of PCR,
which uses primers A–B and C–D to create two products.
The smart thing happens in the second PCR round, and a new sequence is
generated. Because primers B and C have complementary sequences, the first
round’s products will hybridize after being denatured after the first PCR cycle.
The full-length product with the required mutation can then be amplified using
primers A–D. Changes to this procedure could result in deletions or lengthy
additions. The mechanism of SDM is depicted in Fig. 9.16a. Figure 9.17, on the
other hand, depicts one of the potential applications of SDM.

9.4 Test for Mutagenicity: The Ames Test

• The Ames test is a widely employed method for testing mutagenic chemicals
using bacteria (Fig. 9.18). A positive test predicts the carcinogenic potential of the
chemical because cancer is one of the deadly outcomes of mutation. The test is a
quick and easy way to screen a carcinogenic substance before performing animal
tests.
• A histidine synthesis mutant (his) strain of Salmonella typhimurium is used in
this test. These mutants are auxotrophic mutants requiring histidine for growth.
The test asseses the capability of the bacteria to be converted in a “prototrophic”
state which can grow in the histidine-free medium upon chemical exposure.
• The tester strains are developed to detect frameshift (e.g., strains TA-1537 and
TA-1538) or point mutations in the genes that synthesize histidine (e.g., strain
TA-1531). The tester strains have mutations in genes associated in
lipopolysaccharide production, which makes the bacteria’s cell wall more
468 A. Ray

ERROR INTRODUCED
SUBSTITUTION

A
1
DELETION

2
SMALL INSERTION

3
LARGE INSERTION

B a c
5’ 3’
Modified fo
fforward
rward primer
3’ 5’
Modified reverse primer
b d
Normal fo
fforward
rward primer
a First PCR Normal reverse primer
5’ 3’
3’ 5’
5’ 3’
3’ 5’ SDM in linear DNA
d achieved via double
Second PCR PCR . In this once
additional set of
3’ primers having
5’
desired mutation is
3’ 5’
used
Final Product

Fig. 9.16 Site-directed mutagenesis. Site-directed mutagenesis (SDM) can be achieved either in
(a) plasmid and (b) liner DNA

permeable, in addition to his mutations. The tester strain also possesses a

mutation that results in a flawed excision repair pathway, making the cells more
sensitive.
9 DNA Mutation, Repair, and Recombination 469

Hypothesis
D Regulatory seq gene
APPLICATION T

OF SITE P1 ….. Pn X

DIRECTED PROBLEM : To understand the

MUTAGENESIS mode of regulaon of gene X via
transcripon factor t in presence
IN EXPERIMENT of compound D
[ where P1 - Pn are the regulatory
sequences ]

Experiment 2 : Checking Expression

Mulple cloning Reporter gene

sites (MCS) (GFP, Lusiferase etc)

P1- P2-P3 regions are mutated with SDM.

Both mutated and normal P1, P2, P3 are
Construct Construct independently cloned in the
with with expression vector
Normal mutated
sequence sequence

D D vector construct carrying both

mutated and the normal The is
expressed in the host cell
exposed to drug D

Cell with normal construct

Hypothecal result
will fluoresce at maximum
intensity (In case of X
florescence reporter ) . In case
P1 mutated. Effect: least severe
of luminaoon, it can be
measured with lumino-meter X
P2 mutated .Effect most severe
X
P3 mutated. Effect most severe
General hypothecal inference

Gene X is majorly regulate by P2. P3 has the supporng role

Fig. 9.17 A hypothetical experiment demonstrating the application of site-directed mutagenesis

(SDM). Promoter activity can be assessed using SDM. The anticipated promoter sequence is
mutated in a different position and cloned in an expression vector with a reporter gene. A positive
correlation between SDM and affected expression of the reporter genes predict the involvement of
the promoter a specific promoter sequence in driving its downstream genes
470 A. Ray

Fig. 9.18 Ames test. Chemical-induced mutation can be checked cost-effectively using the Ames
test. Chemical-induced mutation in bacteria can convert it to a phototrophic state which can survive
in histidine-free media. In colonies grown in the histidine-free media, it is assumed that the chemical
added to the bacteria is a potential mutagen

• Initially, bacteria are grown on an agar plate with a limiting amount of histidine.
On chemical exposure, when the medium histidine is depleted, bacteria will grow
only if the exposed chemical has induced mutation and conferred survivability.
Result is obtained by counting colonies after 48 h of incubation.
9 DNA Mutation, Repair, and Recombination 471

9.5 DNA Repair Mechanism

DNA repair is the process by which a cell detects the damage in its DNA and corrects
it. The cell can tackle multiple assaults to DNA by a repair system. DNA damage in a
human cell can occur both due to normal metabolic activities and in response to
environmental stress. A probable estimate of about one million lesions day1 cell1
has can impair the cell physiology, affect cell growth and cell viability in humans.
Among these, many of these lesions are mutation inducer. For example, malignancy
can be induced by irreparable DNA damage (e.g., inter-strand cross-links or ICLs)
due to failure of the DNA repair system. The rate of DNA repair is dependent
majorly on three factors: (a) cell type, (b) environmental factor, and (c) age of the
cell. A cell harboring a large amount of DNA damage is unable to repair completely
resulting in either of the three major consequences: (1) senescence (an irreversible
state of dormancy), (2) apoptosis (a type programmed cell death; a process through
which cells commit suicide), and (3) tumorigenicity and/or malignancy (due to
uncontrolled cell growth). The mechanism of DNA repair is a vital area of ongoing
research in the present day. The 2015 Nobel Prize in Chemistry was awarded to
Tomas Lindahl, Paul Modrich, and Aziz Sancar for their work on this area. In this
section, we will be discussing the various mechanisms of DNA repair mechanism
with diagrammatic representations.
There are mainly two types of DNA damage: (a) endogenous damage including
replication errors and (b) exogenous damage. It is important to remember that the
replication of damaged DNA before cell division can lead to the incorporation of
wrong bases in the sister DNA. These alterations are irreversible by DNA repair
mechanisms once they are passed down to daughter cells. Only back mutation can
revert the changes. Many complex pathways are involved in repairing DNA. How-
ever, two general facts are important. (a) Most of the DNA repair mechanisms
require both strands of DNA to replace whole nucleotides, and a template strand is
essential to specify the base sequence in the DNA. (b) Redundancy of DNA
repair events—several types of DNA damage can be corrected by several DNA
repair pathways. This satisfies the significance of the DNA repair mechanism. DNA
repair pathways are versatile and complex. Here, we will consider the general
mechanisms of DNA repair. Commonly we can categorize DNA repair mechanisms
into four types: (a) light-dependent repair, (b) base excision repair, (c) mismatch
repair, and (d) nucleotide excision repair. The DNA repair mechanism is complex
and depends on the cellular response due to the specific types of damage. These
responses operate through complex molecular pathways. Let us first discuss the
global response of DNA damage before discussing the specific types of DNA repair
mechanisms.

9.5.1 An Overview of a Global Response to DNA Damage

Ionizing radiation, UV, or chemicals can cause multiple lesions in the cell at multiple
sites. These include massive DNA lesions and double-strand breaks. Furthermore,
472 A. Ray

DNA-damaging agents can also damage other biomolecules including proteins,

lipids, carbohydrates, and RNA. The common initiation signals for a “global
response to DNA damage” are found among these double-strand breaks that halt
replication forks. The cell’s self-protective mechanism is an event-directed global
response to any damage. This triggers multiple pathways determining the cell fate
including lesion bypass, tolerance, or apoptosis cycle arrest and inhibition of cell
division.
Chromatin relaxation and remodeling are the initial steps followed in response to
DNA damage; these are achieved through several complex processes. The major
three processes are mentioned briefly as follows:
1. Chromatin relaxation takes place rapidly at the damaged site of DNA. It can
initially be accomplished by the JNK-SIRT6 axis where stress-activated protein
kinase, c-Jun N-terminal kinase, phosphorylates SIRT6 on tenth serine residue in
response to double-strand breaks. This facilitates recruitment of SIRT6 to dam-
aged sites of DNA which in turn recruit PARP-1 (poly [ADP-ribose] polymerase
1) to DNA break sites immediately (time of half max accumulation at damage site
is 1.6 s after the damage takes place). Eventually PARP-1 recruits DNA repair
enzymes, MRE11, initiating DNA repair in around 13 s.
2. γH2AX (phosphorylated H2AX on serine 139 residue) is detected within 20 s of
the formation of DNA double-strand break. (Half maximum accumulation of
γH2AX occurs in 1 min.) Then RNF8 protein is associated with γH2AX
mediating extensive chromatin decondensation.
3. 2 DDB1-DDB is the heterodimeric complex associated with CUL4A (the
ubiquitin ligase protein) and PARP-1. This larger complex rapidly gets associated
with UV-induced damage. The half maximum association time is 40 s. DDB1 and
DDB2 both get attached with PARP-1 attracting the DNA remodeling protein
ALC1. ALC1 relaxes the chromatin at the site of UV damage to DNA.

Cell cycle checkpoints are activated after rapid chromatin remodeling, allowing
DNA repair to take place before the cell cycle proceeds. ATM and ATR kinases are
the key contenders in this process. These are activated in 5–6 min after DNA
damage. The cell cycle checkpoints—a protein detected in 10 min of DNA dam-
age—are then phosphorylated as a result of this event.
[Further reading suggestion: DNA damage checkpoints, cell division cycle, cell
division checkpoints, p53 pathways].
Two types of response to DNA damage are important (a) prokaryotic SOS
response in bacteria and (b) eukaryotic transcriptional responses.

(a) The prokaryotic SOS response

Changes in gene expression in E.coli and other bacteria as a result of significant
DNA damage are known as the SOS response. The bacterial SOS system is
regulated by two proteins named LexA and RecA. LexA homodimer binds to
SOS boxes, which are transcriptional repressors. LexA has been found to
regulate roughly 48 genes in E. coli, including LexA and RecA. Although the
SOS response mechanism is common in bacteria, it is absent in some taxa, such
9 DNA Mutation, Repair, and Recombination 473

as spirochetes. Specific single-stranded DNA (ssDNA) regions resulting from

stalled replication forks or double-strand breaks can activate the SOS response.
First, RecA binds to ssDNA mediated by an ATP-dependent reaction leading
to the formation of RecA-ssDNA filaments which are then processed by DNA
helicase producing separate DNA strands. After that, RecA-ssDNA filaments
turn on the autoprotease activity of LexA leading to the cleavage and subsequent
degradation of LexA dimer. Degradation of LexA repressor switches on the
transcription of the genes involved in SOS response and allows for further signal
induction. It is important to note that in E coli, SOS boxes are constituted of
20 nucleotide sequences close to the promoters having palindromic architecture.
A high degree of sequence conservation has been found in these regions,
whereas in other phyla, there is a considerable variation in structure and length
of SOS box. The timing of the SOS response is fine regulated by the differential
binding pattern of LexA to different promoters. At a very early stage, SOS
response induces the lesion repair genes and induces the error-prone translesion
polymerases including UmuCD’2 (DNA polymerase V) thereafter. Once the
DNA is repaired, LexA cleavage activity is reduced restoring binding to the
SOS box resulting in the normal homeostasis of gene expression.
(b) Eukaryotic transcriptional responses to DNA damage
Eukaryotic cells can activate critical defensive pathways in response to DNA
damaging agents by activating various proteins involved in DNA repair, cell
cycle checkpoint control, protein trafficking, and degradation. These transcrip-
tional responses are exceedingly diverse, sophisticated, and well-coordinated
across the genome. Saccharomyces cerevisiae demonstrates overlapping tran-
scriptional profiles. Similarities to environmental assaults exhibit general stress-
responsive pathways which are operated at the transcriptional level. The global
pathway is lacking in a human probably because of heterogeneity of cell types in
the higher organism.
Multiple genes involved in post-replication repair, homologous recombina-
tion, nucleotide excision repair, DNA damage checkpoint, transcriptional acti-
vation, regulating mRNA decay, and many more are expressed in response to
DNA damage in general. A wide number of complicated signaling pathways
triggered by stress and/or DNA damage share a common mechanism that leads
to death or survival. Depending on the extent of stress in case the error is
repaired adequately, the cell survives or else cell death takes place in the
concourse of death-survival interplay.

9.5.2 Light-Dependent Repair

Light-dependent repair demonstrates the process through which the correction of

erroneous DNA takes place in response to light (Fig. 9.19). Three types of damage
can be eliminated by chemically reversing without using a DNA template. Therefore
these types of DNA repair mechanisms are also known as “direct reversal.” Bacteria
and some eukaryotic cells can repair DNA through this mechanism. The most
474 A. Ray

Fig. 9.19 Light-dependent DNA repair: Chemical reaction of photoreactivation of T-T dimer
involving FAD as a cofactor

common form of this type of repair mechanism is known as the photoreactivation of

pyrimidine dimers (y-y). The covalent bond of y-y is broken down in the photoreac-
tivation process by the enzyme photolyase, whose activation is primarily dependent
on energy absorbed from blue/UV light (300–500 nm wavelength) to enhance
catalysis (Sancar 2003). The enzyme is a primitive type of enzyme which is lacking
in humans. In humans, damage by UV radiation is usually repaired by nucleotide
excision repair (will be discussed in the respective section). Another example of
direct repair is a correction of O6-methylguanine, an alkylation product of guanine
that pairs with adenine, producing G-C ~ T-A transversions. The enzyme involved in
this process is O6-methylguanine-DNA methyltransferase. Because MGMT-
mediated reactions are stoichiometric rather than catalytic, this method is expensive.
The adaptive response in bacteria is a worldwide response to methylating
compounds that confer resistance to alkylating agents when exposed to them
consistently.

9.5.3 Base Excision Repair

In base excision repair (BER) (Fig. 9.20), a modified base is excised followed by the
entire removal of the nucleotide. There are several enzymes involved in this cascade.
The enzyme known as DNA glycosylases catalyzes the base removed from the DNA
9 DNA Mutation, Repair, and Recombination 475

Damaged base is recognized and

removed by DNA glycosylase enzyme

Phosphodiester bond cleaved by

AP endonuclease on the 5’ end of the AP

deoxyribose sugar removed

dNTPs
PO 2 +dNMPs
New nucleo es added by DNA
polymerase to the exposed 3’-OH group.

The gap in the sugar – phosphate

backbone is sealed by DNA ligase
and original sequence is restored

Fig. 9.20 Base excision repair (BER). BER is initiated by the enzyme DNA glycosylase. The step-
by-step event is demonstrated in the figure. Once the damaged base is recognized by the DNA
glycosylase enzyme, successive actions of AP endonuclease, DNA polymerase, and DNA ligase
result in the repair of the gap

strand. The enzyme recognizes a specific type of base modification before removing
those. For instance, uracil glycosylase recognizes and removes uracil produced by
the deamination of cytosine. Other glycosylases can recognize hypoxanthine,
3-methyladenine, 7-methylguanine, and other modified bases. Endogenous oxida-
tion and hydrolysis are used by BER enzymes to repair the damage. In the first step,
the glycosylase enzyme, for example, 8-oxoguanine DNA glycosylase 1 (OGG1),
cleaves the bonds between nucleotide base and ribose leaving intact ribose-
phosphate chain resulting in apurinic or apyrimidinic (AP) site. In successive
steps, these AP cites are repaired by AP endonuclease 1 (APE1). APE1 cleaves
the phosphodiester chain 50 to the AP site. Finally, through its associated AP lyase
476 A. Ray

activity, DNA polymerase β (Polβ) inserts the right nucleotide based on the appro-
priate W-C pairing and eliminates the deoxyribose phosphate. A mutation (polymor-
phism) in the human OGG1 gene has been linked to an increased risk of
malignancies such as lung and prostate cancer.

9.5.3.1 Short-Patch BER and Long-Patch BER

In the short-patch BER process, XRCC1 (X-ray repair cross-complementing group
1) acts as a scaffold protein to bring the Polβ and LIG3 (ligase III) enzymes (which
connect the deoxyribose of the replacement nucleotide to the deoxyribosylphosphate
backbone) together at the site of repair. The interaction among PARP-1 (poly
[ADP-ribose] polymerase), XRCC1, and Polβ is a necessary event in the execution
phase of the process.
Long-patch BER, on the other hand, substitutes a strand of nucleotides with a
minimum length of two nucleotides. So far, 10–12 nucleotide length repairs have
been reported. Proliferation cell nuclear antigen (PCNA) is used as a scaffold protein
in the long-patch BER process. In this process, DNA Pol and Pol generate an
oligonucleotide flap, and flap endonuclease 1 eliminates the existing nucleotide
sequence (FEN1). Thereafter, DNA ligase I binds the oligonucleotide to the DNA
(LIG1). It’s important to remember that whereas BER can replace numerous
nucleotides via the long-patch pathway, damage to a single nucleotide with minimal
DNA impact can trigger both short-patch and long-patch pathways. Figure 9.21
represents the schematic diagram of short- and long-patch BER.

9.5.4 Mismatch Repair

The technique of replication is amazingly accurate. Each nascent copy of DNA, on

the other hand, has a chance of containing a mistake (1 billion nucleotides 1).
Mismatched nucleotides are integrated into a new DNA with a frequency of
104–105 without permanently affecting the freshly produced DNA during replica-
tion. Mismatched nucleotides are detected and corrected by DNA polymerases.
Before executing the 50 to 30 polymerization, the DNA polymerase begins its
proofreading activity and 30 –50 exonuclease activity to delete the erroneous bases
that have been introduced. Incorrectly paired bases distort the three-dimensional
structure of DNA, and mismatch repair enzymes can identify these distortions. A
mismatch repair system can detect and mend tiny unpaired loops in DNA generated
by strand slippage during replication, in addition to improper base pairing (men-
tioned earlier in mutation section). Some trinucleotide repeats may make it easier for
this repair system to miss them. Mismatch repair enzymes slice out the deformed
region of the nascent strand and patch the gaps with new nucleotides using the
original DNA strand as a template after the integrated error has been identified.
The presence of methyl groups on the unique sequences of the old strand helps
mismatch repair proteins in E. coli discriminate between old and new strands. Dam
methylase methylates adenine nucleotides in the sequence GATC after replication.
Because the methylation process is delayed after replication, only the old strand is
9 DNA Mutation, Repair, and Recombination 477

Both long and short

patch BER may be
DNA glycosylase iniated by
minimum DNA
damage and under
opt the respecve
APE1 pathway depending
on the magnitude of
damage

POL β
POL γ / ε , PCNA

POL β FEN1,PCNA

LIG3/XRCC1
LIG1,PCNA
COMPLEX

Short patch Long Patch

Fig. 9.21 Short-patch and long-patch base excision repair (BER). Short-patch and long-patch BER
are both initiated by minimum DNA damage and opt the respective pathway depending on the
magnitude of damage

found to be methylated. In E. coli, the proteins MutL, MutS, and MutH are the key
elements of mismatch repair. MutS binds to the mismatched bases forming a
complex with MutH and MutL. This complex takes the unmethylated GATC
sequence to the proximity of mismatched bases. Unmethylated strands are nicked
by MutH at the GATC site and degraded by the unmethylated strand. Finally, DNA
polymerase and DNA ligase replace the missing nucleotides on the unmethylated
strand.
In eukaryotic systems, nicks on the nascent lagging strand of DNA (before being
sealed by DNA ligase) give a signal that directs mismatch proofreading of the proper
strand. According to recent research, the nick allows for RFC-dependent orientation-
specific loading of the replication sliding clamp, PCNA. At the nick, one face donut-
shaped protein was located juxtaposed to the 30 -OH end. In the presence of a
MutSbeta or MutSalpha, the loaded PCNA guides the MutL alpha endonuclease to
function on the daughter strand. MutL and MutS have several eukaryotic homologs.
Two major heterodimers—Msh2/Msh6 (MutSα) and Msh2/Msh3 (MutSβ)—are
478 A. Ray

formed by MutS homologs, whereas five homologs of MutL have been found in
eukaryotes including MLH1, MLH2, MLH3, PMS1, and PMS2. A simplified
schematic representation of mismatch repair has been provided in Fig. 9.22.

9.5.5 Nucleotide Excision Repair

DNA damage in at least two bases which resulted in structural distortion is repaired
by nucleotide excision repair (NER) (Fig. 9.23). NER is versatile and can repair
many different types of DNA damage. It repairs single-strand breaks and serial
damage from exogenous sources including bulky DNA adducts and UV radiation.
Oxidative stress-induced DNA damage can also be repaired via the NER mecha-
nism. NER system is one of the most important repair systems and ubiquitously
found in all cells of all organisms from bacteria to humans and comprise of versatile
enzymes. In bacteria, the system is comparatively simpler. For example, the E coli
NER system is represented by four major proteins UVrA, UVB, UVrC, and UVrD.
XPA, XPC-hHR23B, replication protein A (RPA), transcription factor TFIIH,
XPB, and XPD DNA helicases, ERCC1-XPF and XPG, Pol, Pol, PCNA, and
replication factor C are among the more than 20 proteins involved in the NER
pathway in mammalian cells. Overexpression of the excision repair cross-
complementing 1 (ERCC1) gene has been linked to cisplatin resistance in
non-small cell lung cancer cells and is associated with increased DNA repair ability.
NER mechanisms repair DNA damage in two ways: (a) global genomic NER
(GGR) repairs damage across the genome, and (b) transcription-coupled repair
(TCR) repairs genes while active RNA polymerase transcription.

9.5.6 Other Types of DNA Repair System

Apart from the four repair mechanisms discussed so far, there are more special two
types of DNA repair mechanisms: (a) translesion synthesis (TLS) and (b) double-
strand break repair system.
Translesion synthesis (TLS) is a DNA damage tolerance mechanism that prevents
the DNA replication machinery from skipping replication of earlier DNA lesions
like thymine dimers or apurinic sites. Instead of conventional DNA polymerases, it
employs specialist translesion polymerases from the polymerase family, such as
DNA polymerase IV or V. The active sites of this enzyme are well designed, making
it easier to insert bases opposite damaged nucleotides. Different types of specialized
DNA polymerases are engaged in bypassing or fixing various mistakes. For instance,
Pol η arbitrates error-free bypass induced by UV irradiation, whereas Pol ι
introduces mutations at these sites. Pol η, on the other hand, uses Watson-Crick base
pairing to add the first adenine to the T-T dimer and Hoogsteen base pairing to add
the second adenine syn conformation. Complex DNA lesions, like as G-T intra-
strand cross-links, can be bypassed by human DNA pol η (G [8, 5-Me] T). A
9 DNA Mutation, Repair, and Recombination 479

Fig. 9.22 Mismatch repair. Mismatch correction enzyme recognizes by reading the methylation
state of a nearby GATC sequence. If the sequence is unmethylated, the mismatches are removed
from the DNA strand, and new DNA is introduced. The figure shows the involvement of MutS,
MutH, DNA pol III, and ligase in a step-by-step manner
480 A. Ray

Fig. 9.23 Nucleotide excision repair mechanism. Thymine dimer can be repaired via NER with
Uvr proteins. Briefly, the T-T dimer is recognized by the Uvr AB complex. Association of UvrC
dissociates UvrA dimer from the site. Nick is formed at the 30 and 50 end of the Uvr complex. The
nicked part is replicated with DNA pol I, and the end gap is filled up by DNA ligase
9 DNA Mutation, Repair, and Recombination 481

replicative polymerase performs extension after translesion synthesis. Finally, a

specialized polymerase, Pol ζ, extends terminal mismatches.
On the other hand, sometimes DNA damage is more severe when breakage
occurs at both the strand. This kind of DNA damage leads to genomic rearrangement
often resulting in a gross chromosomal aberration. These types of DNA breaks are
repaired by three mechanisms: (a) homologous end joining (NHEJ),
(b) microhomology-mediated end joining (MMEJ), and (c) homologous recombina-
tion (HR). Important features of these repair mechanisms are discussed below.
(a) In NHEJ, DNA ligase IV, a specialized DNA ligase which forms a complex with
the cofactor XRCC4, directly joins the two ends. The accuracy in repair is
achieved by guidance of short homologous sequences known as
microhomologies present on the single-stranded tails of the DNA ends to be
joined.
(b) MMEJ begins with MRE11 nuclease excision of short range ends on either side
of a double-strand break. MMEJ requires PARP-1 as an early component. In
MMEJ, microhomology areas are paired, and then flap structure-specific endo-
nuclease 1 (FEN1) is recruited to remove overhanging flaps. Following the
recruitment of XRCC1-LIG3 to the location for ligating the DNA ends, undam-
aged DNA is produced.
(c) Homologous recombination requires an identical or substantially identical
sequence to serve as a template for repairing a DNA break. The enzyme
machinery that performs this repair is nearly identical to that which performs
chromosomal crossover during meiosis.

9.5.7 Pathological Impact of Impaired DNA Repair Mechanism

According to experiments, the accumulation of mistakes can engulf a cell if the rate
of DNA damage surpasses a threshold level of DNA repair potential, resulting in
early apoptosis, various disorders including cancer, and increased susceptibility to
carcinogens. As a result, several genetic illnesses linked to defective DNA repair
pathways cause accelerated aging.
For instance, in NHEJ pathway and telomere maintenance mechanisms, deficient
mice exhibited shorter life spans than wild-type mice. Similarly mice deficient in a
key DNA unwinding protein, helicase has been shown to affect DNA repair mecha-
nism and results in premature onset of aging.
Several individual genes have been identified which influence the life span of
individuals. The effects of these genes are essentially environment-dependent,
especially on the organism’s diet. Caloric restriction causes nutrient-sensing
mechanisms to extend life span and metabolic rate to decrease in a range of species.
Although detail mechanism is still a matter of conjuncture, many DNA repair
mechanisms are responded associated with caloric restriction. For example, several
anti-aging agents have been shown to attenuate the constitutive level of mTOR
signaling which is an evidence of reduction of metabolic activity. This results in a
reduction of DNA damage by endogenous ROS. In an experiment, it has been shown
482 A. Ray

Fig. 9.24 Skin condition in xeroderma pigmentosum

that C. elegans extends its life span after the increase in gene dosage of SIR-2 that
encodes the downstream DNA repair factor in NHEJ. The effect is promoted in
caloric restriction conditions; there are several disorders associated with faulty DNA
repair mechanism.
One of the best studied human DNA repair-related diseases is xeroderma
pigmentosum. It is a rare autosomal disease responsible for anomalous skin pigmen-
tation and acute sensitivity to sunlight (Fig. 9.24). Affected people are more likely to
get skin cancer, with an incidence of 1000–2000 times that of unaffected people.
Photolyase activity is absent in human cells (the enzyme which repairs pyrimidine
dimers in bacteria). The NER system in humans corrects the majority of pyrimidine
dimers. However, most persons with xeroderma pigmentosum have a deficiency in
cellular NER, which leads to pyrimidine dimer buildup and, eventually, malignancy.
Defects in numerous genes can cause xeroderma pigmentosum.
At least seven different xeroderma pigmentosum complementation groups have
been experimentally identified. Two other genetic diseases associated with impaired
NER system are trichothiodystrophy (brittle hair syndrome) and Cockayne syn-
drome. Persons having either of these diseases exhibit multiple developmental and
neurological problems. Some genes commonly affect all three diseases. Two special
cases associated with faulty DNA repair are HNPCC (hereditary nonpolyposis colon
cancer) and Li-Fraumeni syndrome due to mutation in the p53 gene which exhibits a
different form of cancer in different tissue.

9.6 Mechanism of DNA Recombination

Recombination is the process by which DNA molecules exchange its part with their
counterpart. When this exchange occurs between homologous DNA molecules, it is
known as homologous recombination. Homologous recombination takes place dur-
ing crossing over where homologous regions of chromosomes are swapped
9 DNA Mutation, Repair, and Recombination 483

Fig. 9.25 Formation of

chiasma. Recombination
occurs during the crossing
over of chromosomes via the
formation of “X”-shaped
chiasma

(Fig. 9.25). Through this process, genes are shuffled producing new combinations.
Apart from mutation recombination is another vital genetic process implicating
genetic variation. Rates of genetic recombination information are essential to creat-
ing genetic maps to deduce the linkage relations among genes. As mentioned earlier
certain recombination process is essential for DNA repair.
Homologous recombination is a precise routine process during meiosis through
multiple steps as follows: (1) one chromosome’s DNA strand coincides with a
homologous chromosome’s nucleotide strand; (2) breaks appear in corresponding
sections of DNA molecules; (3) parts of the molecules change their position accu-
rately; and (4) all of the parts are securely attached. No genetic information is lost or
acquired during this sequence. In this process, the exchange of DNA takes place via
the formation of a heteroduplex. During meiosis, homologous recombination (cross-
ing over) takes place in prophase forming an “X” structure known as chiasma that
often remains microscopically visible till early anaphase. In this section, we will be
discussing the different types and models of DNA recombination.

9.7 Types of DNA Recombination

Apart from general or homologous recombination, there are at least three more types
of DNA recombination that have been identified in the living organism which are as
follows:
Illegitimate or non-homologous recombination: It occurs in regions where
there is no significant sequence similarity. However, when the DNA sequence at
the breakpoints is examined in detail, small sequence similarity regions have been
484 A. Ray

discovered in some situations. Recombination of two distantly placed comparable

genes, for example, can result in the deletion of the intervening genes in somatic
cells.
Site-specific recombination: Recombination between specific short sequences
(approximately 12–24 bp) on otherwise distinct parental molecules is called site-
specific recombination. Recombination at a given site necessitates specialized
enzymes. The integration of certain bacteriophage DNA into a bacterial chromo-
some, for example, and the rearrangement of immunoglobulin genes in vertebrate
animals are examples of site-specific recombination phenomena.
Replicative recombination: This type of recombination generates a new copy of
a segment of DNA. Many transposable elements generate a new copy of the
transposable element at a new location via replicative recombination.

9.8 Models of DNA Recombination

Two general models of homologous recombination have been proposed: (1) single-
strand break initiated DNA recombination and (2) double-strand break initiated
DNA recombination.
Double-stranded DNA molecules from two homologous chromosomes align
precisely in the pathway began by a single-strand break in DNA molecule. A
break in a single strand creates a free end that invades and connects with the other
DNA molecule’s end. Strand invasion and joining occur on both DNA molecules,
resulting in the formation of two heteroduplex DNAs. A unique type of structure
known as a Holliday junction is produced during this process, which is also known
as the Holliday model of DNA recombination (discussed later in detail in this
chapter).
In the recombination process initiated by double-strand breaks, the breaks occur
in one of the two aligned DNA molecules. In this model, strand invasion is followed
by the removal of certain nucleotides from the ends of the broken strands. Two
heteroduplex DNA molecules are formed by successive displacement and replica-
tion, which are connected by two Holliday junctions and separated by additional
cleavage. This model has been observed in yeast where double-strand breaks occur
during meiosis prophase I.

(a) Holliday Model

The Holliday model of DNA recombination (Fig. 9.26) requires the
(a) unwinding of DNA helices, (b) cleavage of nucleotide strands, (c) strand
invasion, and (d) branch migration, followed by further (e) strand cleavage and
(f) union to remove Holliday junctions. The important features are described as
follows:
1. At the site of recombination, a heteroduplex is formed.
2. The recombinant joint has the ability to move with the duplex. Branch
migration is the term for this type of movement. As one strand is displaced
by the other, the branching point can migrate in either direction.
9 DNA Mutation, Repair, and Recombination 485

1 2 3

4 5 6
Inter connected
Branch migraon duplex pulled away

Boom half of
the structure
rotates
7

Horizontal
plane

Cleavage and
rejoining Vercal
plane
Cleavage and
rejoining

Noncrossover
recombinant

Crossover
recombinant

Fig. 9.26 Holliday model of DNA recombination. In the Holliday model of recombination, a nick
is formed in one strand of pairing DNA, and subsequently, a heteroduplex is formed. The crossover
junction is known as the Holliday junction which migrates to the desired location (branch point) of
recombination along with the heteroduplex. The heteroduplex is separated by torsion, and recom-
bination of DNA takes place at the branch point as demonstrated in the figure. Recombination of
DNA can be crossed over or non-crossed over depending on the plane of rotation

3. Because the branch may have migrated since the molecule was isolated,
branch migration confers a dynamic property on recombining structures that
cannot be examined in vitro. The recombination enzymes catalyze branch
migration.
4. The strand exchanged and joint must be resolved into two separate duplex
molecules. A second set of nicks is required for the resolution.
5. The nick releases splice recombinant DNA molecules.
486 A. Ray

Experiments with short homologous sequences containing plasmid

introduced to bacteria revealed that the length of the homologous region
>75 bp and the distance >10 bp increase the efficiency of recombination.
Important enzyme systems are associated with this procedure. Experiments
with E. coli revealed that three genes play a vital role in recombination. These
are recB, recC, and recD. These genes code for three distinct polypeptides that
come together to generate the RecBCD protein. This protein cleaves the nucle-
otide strands as well as unwinds the double-stranded DNA. The RecA protein,
which is produced by the recA gene, permits a single strand invasion to occur,
causing one of the original strands to be displaced.
(b) Double-Strand Break Initiated Model
In this process, recombination (Fig. 9.27) is initiated by an endonuclease which
cleaves one of the partner DNA duplexes. The cut in one DNA known as
“recipient” DNA by the endonucleases becomes distended forming a gap by
the action of the exonuclease. The exonuclease then dissolves one strand on
each side of the break, resulting in a 30 single-stranded terminus.
A homologous region of the other “donor” duplex is invaded by one of the
free 30 ends. This is called as a single-strand invasion. A D loop is formed when
heteroduplex DNA is formed, in which one strand of the donor duplex is
displaced.
Repair DNA synthesis generates double-stranded DNA by extending the D
loop and using the free 30 end as a primer.
The D loop eventually grows large enough to match to the full gap on the
recipient chromatid. After reaching the far side of the gap on the extruded single
strand, the complementary single stranded is annealed, resulting in heteroduplex
DNA on both sides of the joints, which is resolved by cutting. The initial
non-crossover is released if both joints are resolved in the same way. A genetic
crossover is created when the two joints are resolved in opposite directions.

Box 9.1 Scientific Concept: Testing the Mutagenicity Potential

of Chemicals: Geert R Verheyen et al.
Testing of potentially hazardous chemicals is driven by the assessment of
endpoints. Ames test using bacterial auxotrophic strain is a preliminary exper-
iment that studies only the mutagenic potential of the chemical. However, it
can’t provide information regarding other harmful effects. The most important
endpoints considered in testing the hazardous and/or genotoxic chemicals are
cell death, apoptosis, mutagenicity, and DNA damage. The organization,
OCED use to adopt the standard test guidelines set by the global scientific
community. OCED has adopted the following tests as standard procedures for
testing mutagenicity: (a) bacterial reverse mutation test, (b) mammalian cell
gene mutation test, (c) transgenic rodent somatic and germ cell gene mutation

(continued)
9 DNA Mutation, Repair, and Recombination 487

Two DNA strands from

homologous chromosome
arranged ; Nick in one double
strand

Few nucleodes enzymac

ally removed from the nick
end end s of both strands

Free 3’ end invades and

displace the strand

Elongaon of 3’ end

Displaced strand forms a loop

DNA synthesis at 3’ end of

the boom strand

Two holiday juncons formed

Fig. 9.27 Double-strand break model of DNA recombination. In the double-strand break model,
nick is formed both in the strands of each pairing DNA molecule, and recombination takes place
based on two Holliday junctions as demonstrated in the figure
488 A. Ray

Box 9.1 (continued)

assay, and (d) mammalian chromosome aberration test and mammalian
micronucleus test.
However, the mode of toxic action is diverse. Therefore the genotoxic
effect of a completely unknown chemical is a matter of in-depth toxicological
research. Let us discuss the basic pattern of testing the genotoxicity of
chemicals. Further reading is suggested in this regard.
Investigation system: (a) in silico, (b) in vitro, and (c) in vivo

1. In silico: Computer-generated program to compare the structural properties

of the chemicals with the known chemical to interpret the probable effects
in a predictive way.
2. In vitro: Cell types or cell lines are utilized to check the endpoints and
mechanism of toxic action. Cell types are selected depending on the
objective. For example, for investigating hepatotoxicity, hepatocytes cul-
ture or cell line is taken.
Key Tools
(a) Ames test: Mutagenic potential with bacterial strain is performed.
(b) Checking DNA ladder: The DNA damage can be checked in agarose
gel by the appearance of multiple bands or smears.
(c) Micronucleus assay: The fragmented nucleus is assessed with the
microscopic investigation.
(d) Chromosomal aberration: Chromosomal break and/or damage is
checked under a microscope.
(e) PARP-1 cleavage: PARP-1 cleavage can be detected with
immunoblots.
(f) HPRT mutation: Mutation at hypoxanthine-guanine
phosphoribosyltransferase (HPRT) and a transgene of xanthine-
guanine phosphoribosyltransferase (XPRT) are studied.
3. In vivo: in silico prediction and subsequent in vitro is validated in the live
animals. For different proposed mutations, different models are selected
including, rats, mice, fish, etc. Zebrafish (Danio rerio) which resembles
structural features of 70% of humans is a valuable model for genotoxicity
study in the present days. Furthermore, apart from rats and mice, catfishes
have been established models for testing immunotoxicity. Transgenic
animals are used depending on the purpose
• In present days, high-throughput gene expression profiling approach is
considered a fruitful way to evaluate alteration in expressed gene and
functional pathways due to chemical exposure.
9 DNA Mutation, Repair, and Recombination 489

9.9 Conclusion

From this chapter, we conclude changes in genetic material are essential for evolu-
tionary change. Alteration in genetic information is accomplished either through
mutation or via recombination. While recombination is the routine procedure for
genetic alteration, mutation is incidental. Therefore mutation is more likely to
impose the detrimental character in the genome although beneficial mutation is
evident. Mutation can be achieved by several modes through which alteration in
bases takes place. Several mutations are without significant phenotypic expression,
or it remains subdued due to the countereffect of a second mutation. However many
mutations are inherited either via generations, via autosomal linked fashion, or via
sex chromosome linked to fashion. These produce the diseased phenotype. Small
errors are corrected by the specialized repair mechanism. However, the errors
accumulated beyond the critical tolerance potential eventually significant impact of
mutation occurs. Therefore impairment in DNA repair mechanism machinery or
mutation in the components of DNA repair machinery itself may act as the most
severe consequence.

9.10 RuvC Enzyme and Holliday Junction

RuvC enzyme of E. coli is an important endonuclease that takes part in cleaving the
four-way junctions in DNA structures during homologous recombination, and the
enzyme system is also known as resolvase. It resolves the Holliday junction by
collaborative action with the branch migration protein RuvAB. Two recombining
DNA sections are eventually divided into discrete duplex molecules, which are
subsequently joined together by ligation to form a continuous molecule. Certain
bacteriophage three-strand or Y junctions, as well as four-strand Holliday junctions,
have similar endonuclease to E. coli.

9.11 Summary

• Mutations can cause great suffering and are the sustainers of life. A mutation is
the source of all genetic variation and the raw material of evolution.
• Without mutations and resulting variation, organisms are unable to adapt them-
selves to environmental change which may increase the risk of extinction of a
species. In many cases mutation leads to detrimental effects resulting generation
of a severe disorder.
• If a mutation occurs in gametes or somatic cells at an early point in development,
the consequences are severe. Mutation can be inherited through the generation if
takes place in the germ cells.
• Many disorder associated to the mutation either in autosome or in sex chromo-
some is inherited as a recessive or dominant fashion.
490 A. Ray

• There are several modes of mutation through which bases are altered and eventu-
ally alter the composition of the region of DNA. Mutation can be introduced in a
DNA spontaneously or by induction of radiation and chemicals including
alkylating agents and intercalating agents.
• Although mutation is the chief source of genetic variation, detrimental variation is
noteworthy. Therefore our system possesses a specialized mechanism to correct
the transient or temporary errors of DNA.
• The sustained impact of mutation occurs only when the DNA repair mechanism
fails to correct the error.
• Apart from mutation, genetic recombination is the regular mode of reshuffling
and alteration of genetic material.
• DNA of an organism is prone to different types of assaults (environmental and
chemical). However, due to certain characteristics of genetic codes including
wobble base pairing and transition-transversion bias, an organism acquires a
higher probability to defend the mutational change. Silent mutation and reverse
mutation potentially subdued the effect of the mutation.
• In present days, the phenomenon “mutation” is being widely used as an important
genetic tool to investigate a gene function known as site-directed mutagenesis
(SDM).
• Mutagenesis in bacteria has been tactfully utilized to screen the mutagenic
potential of chemicals cost-effectively through the Ames test.

References
Sancar A (2003) Structure and function of DNA photolyase and cryptochrome blue-light
photoreceptors. Chem Rev 103:2203–2237
Scally A (2016) The mutation rate in human evolution and demographic inference. Curr Opin Genet
Dev 41:36–43
RNA Transcription
10
Manasa G. Sharma

10.1 RNA Transcription: Overview

The process of producing proteins from nucleotides is termed as gene expression.

Genetic information present in the DNA, is first rewritten, generating RNA, the
process of which, is termed as transcription. During transcription, starting with a
DNA template, RNA is essentially synthesized (Fig. 10.1). The three major types of
RNA generated during transcription are tRNA, mRNA, and rRNA. Other
non-coding RNAs (ncRNAs) are also generated. The term “coding RNA” refers to
RNAs that are translated to proteins, whereas “non-coding RNA” refers to RNAs
that do not code for proteins. Messenger RNAs (mRNAs) serve as templates for
translation, and ncRNAs are involved in many regulatory functions in the cell.
Recent emerging trends have suggested that many proteins play dual roles, and
these are referred to as bifunctional (bifRNAs). Every single gene is transcribed into
several copies of mRNA, and each mRNA molecule is capable of generating
identical copies of a single protein. The amount of a protein synthesized from a
gene is regulated during transcription and translation.
The first step in gene expression where the enzyme RNA polymerase converts a
DNA segment into RNA is called transcription. DNA and RNA both make use of
nucleotide base pairing as a complementary language.
The direction of flow of genetic material was established as the central dogma by
Francis Crick in 1957. This states that the flow of genetic material is in the direction
of DNA to RNA to protein and is fundamental to all organisms like complex
multicellular organisms as well as unicellular prokaryotes.
This phenomenon came into the picture after the discovery of reverse transcrip-
tion in retroviruses, which does not follow central dogma. Exceptions also include
prions, where information flow is from protein to protein (Fig. 10.2).

M. G. Sharma (*)
Ramaiah University of Applied Sciences, Bangalore, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 491
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_10
492 M. G. Sharma

Fig. 10.1 RNA transcription overview. All living organisms carry genes, in the form of nucleotide
sequences that contain the information required to synthesize proteins. To carry out essential
functions such as survival, growth, and proliferation, these proteins are essential

10.2 Types of RNA: Overview

The product of transcription is RNA. Four kinds of nucleotides make up the RNA,
and phosphodiester bonds link them linearly. The difference between DNA and
RNA lies in their structures, mainly the sugar moiety and the type of nucleotides that
make up the respective nucleic acids. DNA primarily contains deoxyribose sugar,
whereas RNA contains ribose sugar—with an extra –OH group. These structural
differences have contributed to the nomenclature of the biomolecules. Also, thia-
mine (T) is one base pair that is part of DNA, whereas it is replaced by uracil in
RNA. The other three bases—adenine (A), cytosine (C), and guanine (G)—are in
both of these nucleic acids.
10 RNA Transcription 493

DNA 3’ 5’
A T G A G T C C A A G T

Transcription T A C T C A G G T T C A
5’ 3’

mRNA U A C U C A G G U U C A
5’ 3’
Codons
Translation

Protein Asn Pro Gly Thr Amino acids

Fig. 10.2 Central dogma. The central dogma is a concept in molecular biology that explains how
the residue-by-residue transfer of genetic information happens in living organisms. It states that
such information transfer follows a particular order and that it does not move from protein to nucleic
acid or protein to protein

The substitution of thiamine with uracil in RNA leaves us with very few chemical
differences between the two molecules, and the base complementary properties
remain the same as DNA except the U base pairing with A. However, the structure
differs a lot. This can be attributed to the fact that RNA molecules are single-stranded
and they have different confirmations. Upon completion of the human genome
project, a major discovery was made where studies suggested that only 2% of the
entire human genome coded for proteins. But, the amount of the genome that was
being transcribed was exceptionally high (62%) indicating that there were RNA
molecules in huge amounts that did not code for proteins. These non-coding RNAs
were found to be involved in cell cycle regulation, maintaining structural integrity,
and other cellular and molecular functions.
Many different types of RNAs are responsible for various functions. The impor-
tant ones include (Fig. 10.3):

• mRNA—They are protein-coding messenger RNAs.

• rRNA—These are ribosomal RNAs that are the main structural components of
ribosomes. They are essential for the catalysis of translation.
• tRNA—These clover leaf-shaped molecules are adapter molecules that facilitate
the transfer of amino acids to the site of translation.

Others include:

• snRNA—The processing of pre-mRNA is mediated by small nuclear RNAs.

• snoRNA—Processing and modification of pre-rRNAs are facilitated by small
nucleolar RNAs.
• miRNA—MicroRNAs are responsible for post-transcriptional gene regulation.
494 M. G. Sharma

Fig. 10.3 The three major types of RNA: (a) mRNA or messenger RNA, (b) rRNA or ribosomal
RNA, and (c) tRNA or transfer RNA

• piRNA—These are known as Piwi-interacting RNAs, and they are known to

regulate epigenetic and post-transcriptional silencing of transposons that protect
germlines from certain transposable elements.
• lncRNA—Chromatin remodeling is influenced by these long non-coding RNAs.
They also regulate gene expression at the transcriptional and post-transcriptional
level.
• circRNAs—These are circular RNAs that regulate gene expression and alterna-
tive splicing.

10.3 Process of RNA Transcription

10.3.1 Prokaryotes

The mechanism of transcription has remained the same in all living species. Pro-
karyotic transcription has been extensively studied in bacteria such as E. coli. The
transcription of RNA involves three steps: initiation, chain elongation, and
termination.

10.3.2 Promoter Recognition by RNA Polymerase

RNA polymerase locates the target DNA by recognizing the promoter region.
Promoter sequences are usually located in the region preceding the transcription
start site (TSS) on DNA. The TSS is usually referred to as +1, and the nucleotides
towards the 30 end of the template strand from this point are said to be downstream of
10 RNA Transcription 495

Fig. 10.4 Feklistov et al. study the 10 promoter element recognition “T12A11T10A9A8T7
consensus sequence,” i.e., the major process involved in the opening of the bacterial promoter by the
RNA Pol “σ subunit”

the transcript start site. The nucleotides preceding +1 are referred to as 1, and the
bases towards the 50 direction are upstream. Promoters are characterized by the
presence of two hexameric AT-rich signature sequences at 10 and 35 bases and
are referred to as 10 and 35 elements, respectively (Fig. 10.4) The 10 element
is commonly known as the Pribnow box. The 10 and 35 elements are separated
by a nonspecific stretch of 17–19 bases. This region is known as the spacer region.
Several studies analyzing promoter regions of a large number of bacteria suggest that
there are conserved sequences (TATAAT and TTGACA) at 10 and 35 elements,
respectively. The activity of a promoter is highly dependent on its ability to bind
strongly to the RNA polymerase, thereby increasing its efficiency of inducing
conformational changes in the DNA-polymerase close complex leading to opening
the DNA duplex and quick disassociation of the promoter. Heterogeneity and
variance in the promoter sequences lead to differential levels of gene expression.
In eukaryotes, transcription initiation by Pol II is triggered when the core promoters
are recognized through the interactions with DNA, transcriptional (co)activators, and
modified histones.
496 M. G. Sharma

Initiation: The core enzyme is aided by the σ factor to locate the transcription
binding site. This activity is mediated by specific nucleotide sequences on the DNA
known as the promoter regions. Promoter recognition is crucial for the initiation of
transcription. Initially, the holoenzyme is attached to the DNA weakly and rapidly
slides across it. Once the σ factor locates the promoter region, the holoenzyme
adheres tightly to the DNA and specifically interacts with the outer edges of the
bases of the DNA.
The tightly bound holoenzyme then initiates transcription and unwinds around
12–14 bases of the DNA duplex forming a transcription-competent open promoter
complex. The σ factor binds to the unpaired bases of any one strand of the
transcription complex, and the core enzyme starts assembling the complementary
ribonucleotides.
This process is tedious and generates a lot of stress. This is because the RNA
polymerase stays attached to the promoter region pulling the upstream DNA region
into its active site expanding the transcription bubble. This is known as scrunching
(Fig. 10.5). This stress coupled along with the steric clashes between the RNA
undergoing elongation and the σ element set up abortive transcription in which the
newly synthesized short stretches of the RNA are discarded from the transcription
machinery, while the RNA polymerase remains in the same place and starts again.
After the abortive initiation rounds, and synthesis of around 17 bases, the initiation
transitions into elongation. Due to scrunching pressure, the RNA polymerase breaks
free off the promoter region leaving behind the σ factor.
Elongation: After the transcription cycle is set up, the elongation process has to
be stabilized. The Pol II machinery equips additional factors to stop the premature
dissociation of Pol II. These factors are known as elongation factors, and they
associate with Pol II just after initiation. They help the polymerase move along the
DNA template on the chromatin network. Pol II goes through something known as
“promoter-proximal pausing” after the transcription of 30–50 nucleotides down-
stream to the TSS (transcription start site). This is depicted in Fig. 10.6. Pol II is
released by cyclin-dependent kinase 9 (CDK9) a subunit of positive transcription
elongation factor b (P-TEFb). This is facilitated by phosphorylation of several
components that make up the transcribing elongation complex (TEC). The P-TEFb
works on the core promoters by the COF bromodomain-containing protein
4 (BRD4). A negative elongation factor (NELF) complex is also responsible for
promoting Pol II pausing with the help of the DRB sensitivity-inducing factor
(DSIF). DSIF directly binds to Pol II, but NELF has a preferential binding activity,
and it binds to the assembled DSIF/RNAP II complex.
The NELF complex functionally competes with TFIIF. TFIIF subsequently binds
to Pol II and induces conformational changes in the polymerase which are ideal for
elongation. Phosphorylation of DSIF, Pol II, and NELF causes dissociation of NELF
from Pol II which creates optimum conditions for it to move on to the elongation
phase. Certain histone chaperones also help by rapidly disassembling and assem-
bling nucleosomes ahead of the moving Pol II. In eukaryotes, transcription elonga-
tion goes hand in hand with RNA processing, and the newly formed RNA molecules
10 RNA Transcription 497

Fig. 10.5 Studies establishing that transcription initially follows a “scrunching” mechanism. RNA
polymerase stays on the promoter region of the DNA and directs the downstream DNA past its
active center and into itself (Kapanidis et al. 2006)

are processed in the nucleus, before being exported to the cytoplasm as

mature RNAs.
Termination: The terminator signal triggers cascades that cause the core enzyme
to dissociate from the template, which releases the newly synthesized RNA tran-
script and re-associates with the σ factor so that it can start a new round of
transcription. The major two kinds of transcription termination mechanisms studied
498 M. G. Sharma

Fig. 10.6 Pol II-DSIF-NELF: Paused transcription complex (Vos et al. 2018). The negative
elongation factor (NELF) as well as the DRB sensitivity-inducing factor (DSIF) protein complexes
help stabilize the paused Pol II. NELF binds to the polymerase funnel and forms a bridge between
the polymerase units that are mobile, thereafter contacting the trigger loop. This restrains the
mobility of Pol II which is necessary for pause release
10 RNA Transcription 499

Fig. 10.7 Mechanisms of bacterial transcription termination. (a) Transcription is induced by RNA
hairpin formation; (b, c) DNA translocase Mfd and RNA translocase Rho move towards the RNAP,
engaging it and forcing dissociation of the elongating complex

in bacteria are factor-dependent termination (Rho-dependent) and intrinsic

(Rho-independent) termination (Fig. 10.7, Table 10.1).
500 M. G. Sharma

Table 10.1 Key differences between the Rho-independent and Rho-dependent transcription
termination mechanisms
Rho-dependent Rho-independent
The Rho factor is a protein with helicase The terminator region is characterized by an
activity and works by utilization of ATP inverted repeat sequence followed by an
energy adenine-rich region (AAAA)
Rho binds to the RNA template and moves in The RNA polymerase moves on the transcript
the 50 -30 direction along with the RNA producing mRNA. Due to hydrogen bonding,
polymerase. It breaks the hydrogen bonds the complementary regions in the inverted
between the DNA and RNA transcript repeat sequence region form a hairpin loop
structure
The DNA/RNA hybrid is pulled apart when Hairpin structure stops the RNA polymerase
the Rho factor reaches the transcription bubble, activity. Weak interactions between adenine-
and this releases the transcript from the uracil bonds in the U-rich region also
transcription bubble, terminating transcription destabilize the DNA template and the RNA
transcript separating them from each other

10.3.3 Eukaryotes

Transcription is carried out by one of three RNA polymerases. The RNAP is

recruited based on what kind of RNA is being transcribed. Eukaryotic transcription
also involves three stages: (a) initiation, (b) elongation, and (c) termination.

10.3.4 Initiation of Transcription in Eukaryotes

Prokaryotic RNA transcription is facilitated by RNA polymerases that is capable of

binding to a DNA template autonomously, but eukaryotic RNAPs make use of
transcription factors. This promoter region is the binding site for these factors, and
then polymerase selection takes place based on the requirement. RNAPs and tran-
scription factors form a completed assembly. This binds to the promoter in order to
form the pre-initiation complex (PIC). The TATA box is a promoter element which
is basically a short stretch of DNA sequence. This found 25–30 base pairs upstream
from the transcription start site is the best characterized and most studied core
promoter element in eukaryotes. However, the TATA box is only present in about
one in ten mammalian genes. Various other core promoter regions are also known to
exist.
TBP binds to its binding site “the TATA box.” The binding of TFIID containing
the subunit TBP to the TATA box results in the assembly of five more transcription
factors. Around the TATA box (Fig. 10.8), these complexes and the RNA polymer-
ase combine forming the pre-initiation complex. Transcription factor II H (TFIIH)
drives the unwinding of the DNA double helix in order to provide a DNA template
that is single stranded to the moving RNA polymerase. The pre-initiation complex
alone is not entirely responsible for transcription initiation. Other than activators,
coactivators, repressors, and corepressor proteins are also responsible for regulating
10 RNA Transcription 501

Fig. 10.8 The TATA box region is known to be the major core promoter element in eukaryotic
transcription. TATA-binding protein (TBP), a transcription factor, binds to this region. Transcrip-
tion factor II D (TFIID) contains the subunit TBP

transcription. Activators increase the rate of transcription, and repressors decrease

the transcription rate.

10.3.5 Transcription Through Nucleosomes

The release of the polymerase is triggered by the formation of pre-initiation complex,

and elongation begins. This phase is characterized as soon as the RNA is synthesized
502 M. G. Sharma

Mechanisms of transcription through nucleosomes

a b
Barrier magnitude
Recruitment
+1 +1
S-5/7P High S-2P Low > +3
+2

Cooperativity +1
High Low
Histone
variants

FACT

Torsion +1
High Low
Ubiquitin

Torsion Chromatin
Low High High
sensitivity:
remodelers
Ti BS

Fig. 10.9 Teves et al. discuss the mechanism of transcription through nucleosomes and suggest
that the stability and the dynamics of transcribed nucleosomes are affected by Pol II transit

by the polymerase in the direction of 50 to 30 . In eukaryotes, DNA is present in the

form of chromatin and is packed around charged histone molecules. DNA along with
histones makes up the nucleosomes. FACT (facilitates chromatin transcription)
protein helps in the removal of histones which is mediated by. The disassembly of
nucleosomes is also carried out by FACT ahead of a moving Pol II, by the removal of
eight histones (Fig. 10.9). This unwinds the chromatin and gives the DNA template
access to RNA polymerase II. The nucleosome reassembles the FACT behind the
moving and mobile RNA polymerase II. Pol II elongates the newly synthesized
RNA until transcription termination signals are encountered.

10.3.6 Elongation

Twelve protein subunits make up the dynamic RNA polymerase II. This enzyme is a
sliding clamp, and single-stranded DNA-binding protein possesses helicase activity.
New RNA strands synthesis does not require extra proteins due to this unique
multifunctionality of Pol II when compared to replication by the DNA polymerase.
But, RNA Pol II requires several accessory proteins for transcription initiation until it
is positioned at the +1 initiation nucleotide. After the elongation process has begun,
Pol II leaves behind the initiation proteins by a process called “escaping.” This is
shown in Fig. 10.10.
10 RNA Transcription 503

Fig. 10.10 Promoter escape is triggered by the generation of the nascent mRNA strands. This
stage can be recognized by abortive transcripts formation and also by the functionally and
physically unstable transcription complex

The template DNA guides the RNA polymerase to move in the 30 to 50 direction.
New nucleotides are added by the RNAPs to the 30 end of the RNA strand and
synthesize new RNA strands in the 50 to 30 direction. Ahead of the moving RNA Pol,
DNA double helix is unwound and simultaneously rewound behind it. When syn-
thesis is happening, 25 unwound DNA base pairs are known to be attached along
with new RNA strands which are about 8 nucleotides long.

10.3.7 Termination

Transcription termination differs based on which RNA polymerase is recruited. A

specific (11 bp long in humans; 18 bp in mice) sequence is transcribed by RNA
polymerase I that transcribes ribosomal rRNA which contains the sequence. This
sequence is recognized by a termination protein called TTF-1 (transcription termi-
nation factor for RNA polymerase I) and begins the termination of transcription.
504 M. G. Sharma

Fig. 10.11 Mammalian RNAPII termination at protein-coding genes

From the template DNA strand, RNA polymerase I dissociates causing the release of
the new RNA that has just been synthesized. Before the transcription is complete,
RNA Pol II cleaves the transcript and is cleaved at an internal site, releasing the
upstream portion of the preliminary transcript. This acts as the initial RNA
(or pre-mRNA) before further processing can take place. Upon encountering the
cleavage site, the end of the gene is reached. A 50 -exonuclease (Xrn2 in humans)
digests the remaining transcript as it is being transcribed by Pol II. After the
overhanging RNA is digested by Pol II, the 50 -exonuclease catches up to the
polymerase II and helps it dissociate from the DNA template strand and concluding
transcription.
Where pre-mRNA synthesis is involved, the end of the gene is determined again
by the cleavage site. This site is located between an upstream AAUAAA sequence
and a downstream GU-rich sequence separated by about 40–60 nucleotides. After
transcription of both of these sequences, the CPSF protein binds to the AAUAAA
sequence, and the CstF protein binds to the GU-rich sequence (Fig. 10.11).
10 RNA Transcription 505

Fig. 10.12 Illustration of eukaryotic and prokaryotic bacterial RNA polymerase holoenzyme

10.4 RNA Polymerase: Mechanism

Bacterial RNA polymerase is made up of a core complex consisting of multiple

subunits and an initiation factor called the sigma (σ) factor (Fig. 10.12). The core
complex has nonspecific polymerase enzyme activity and can bind to DNA and
nicks in a nonspecific manner and is known as the core enzyme (E). The σ factor is
essential for the sequence-specific transcription activity. The σ factor along with the
core enzymes make up the RNA polymerase holoenzyme (Eσ). Most bacterial RNA
polymerases are functionally similar, but there are significant structural differences.
The E. coli core complex is made of five subunits α2, β, β0 , and ω that possess
different functions. Bacteria express several forms of σ factor that recognize and
bind different promoter sequences in response to various signals and environmental
triggers. In E. coli, the main σ factor is σ70 or σD. It expresses housekeeping genes.
The active site and the DNA-binding cleft of the bacterial RNA polymerase
structurally look like a crab claw that possesses two pincers. The β and β0 subunits
occupy more than 80% mass of the core enzyme and form the pincers generating a
cleft for the entry of the template DNA into the active site of the enzyme. The pincer
formed by the β0 subunit is known as the clamp which changes its orientation by
swinging between the open and closed conformations. The β and β0 subunits form
two double-psi beta-barrel (DPBB) domains. The DPBB domain interacts with the
506 M. G. Sharma

Fig. 10.13 Direction of synthesis of RNA transcript in a transcription bubble

incoming nucleotides with their basic residues on the surface. The DPBB domains
are used by most of the cellular RNA polymerases for RNA synthesis. The
ribonucleosides enter into the active site of RNA polymerases through a secondary
channel, which is a funnel-shaped opening separate from the main channel. The
secondary channel contains a binding cavity for accessory factors that control RNA
polymerase activity. Two additional motifs from the β0 subunit play very important
roles in the RNA synthesis reaction. One motif is the trigger loop involved in
catalysis, and the other is the bridge helix used for the translocation of DNA and
RNA during the nucleotide addition cycle.
RNA polymerase carries out transcription which is also enzymatically catalyzed.
Transcription happens in three distinct phases: initiation, elongation, and termina-
tion, which constitute the transcription cycle. In the initiation phase, RNA polymer-
ase recognizes and binds to the DNA at the promoter region which lies upstream of
the DNA template. RNA polymerase unwinds the DNA double helix and creates a
transcription bubble and exposes 12–14 bases on each strand. One of the helices of
DNA acts as the template strand for transcription for the complementary ribonucle-
otide bases to align. The template strand is commonly referred to as the non-coding
strand. The other strand of the DNA double helix will have the same base sequence
as the RNA (except uracil instead of thiamine) and is known as the coding strand.
RNA polymerase covalently links the base pairs on the template strand. After this
step, nine to ten bases of the newly synthesized RNA and the template DNA remain
attached forming a temporary DNA-RNA duplex structure. After the synthesis of ten
bases, RNA polymerase proceeds to enter the elongation phase. It also involved in
the unwinding of the double helical DNA in front and rewinding it from behind. The
RNA polymerase moves in the 30 to 50 direction of the template, but the direction of
chain elongation is in the 50 to 30 direction (Fig. 10.13).
At the molecular level, it is understood that the RNA polymerase works by
creating phosphodiester bonds between the incoming ribonucleotide triphosphates
and the growing chain of RNA. This is a thermodynamically feasible and irreversible
reaction. The RNA polymerase adds around 10–100 bases every second. It does not
10 RNA Transcription 507

dissociate from the DNA until the transcript is completely formed, and this charac-
teristic is known as processivity. The movement of the RNA polymerase across the
template strand happens in such a way that the enzyme is capable of detecting
mismatches and other errors. The fidelity of transcription is extensively taken care of
because a misincorporated ribonucleotide base leads to disastrous consequences. In
case an error is detected, the enzyme moves back onto the template and excises the
misincorporated base at the 30 end exhibiting proofreading activity and replaces it
with the correct base. The binding of the RNA polymerase is lenient and allows it to
move on the DNA template at different rates. Transcription is terminated at the site
where the RNA polymerase recognizes a terminator sequence. The transcript is then
released from the transcription bubble. In eukaryotes, RNA polymerases are also
involved in the modification of transcripts, a process known as the post-
transcriptional modification in which primary transcripts, the firsthand product of
the transcription; undergo certain modifications to become functional.

10.4.1 The Three Eukaryotic RNA Polymerases (RNAPs)

Compared to prokaryotes, mRNA synthesis in eukaryotes is significantly compli-

cated. Eukaryotic transcription involves three polymerases that are comprised of
more than ten subunits.
RNA polymerase I: It is a characteristic nuclear substructure in which ribosomal
RNA (rRNA) is transcribed, processed, and subsequently assembled into ribosomes.
It is found in the nucleolus region. The rRNA molecules are structural RNAs
because they offer structural maintenance and support, but they are not translated
into proteins but are essential to carry out translation. Majority of the rRNAs, except
the 5S rRNA, are synthesized by RNA Pol I.
RNA polymerase II: Main polymerase involved in the synthesis of protein-
coding nuclear pre-mRNAs. Eukaryotic pre-mRNAs are subjected to extensive post-
transcriptional after. RNA polymerase II transcribes a majority of eukaryotic genes,
which includes protein-encoding genes as well as genes that encode for various
regulatory RNAs, like microRNAs (miRNAs) and long non-coding RNAs
(lncRNAs).
RNA polymerase III: Transcribes structural RNAs including the 5S pre-rRNA,
transfer pre-RNAs (pre-tRNAs), and small nuclear pre-RNAs. Small nuclear RNAs
are involved in “splicing” pre-mRNAs and regulating transcription factors.

10.5 Prokaryotic vs Eukaryotic RNA Transcription

The process of transcription is essentially the same in both prokaryotes and

eukaryotes. But more steps are involved in eukaryotic transcription (Fig. 10.14).
Bacteria and species belonging to archaea require only one type of RNA polymerase,
whereas eukaryotes require at least three main enzymes—RNA polymerases I, II,
and III (Pol I, II, III), along with polymerases IV and V which are present in plants
508 M. G. Sharma

Fig. 10.14 Transcription in prokaryotes vs transcription in eukaryotes. (a) Bacterium. (b)

Eukaryote

that transcribe different subsets of RNA. In all three RNA polymerases, the core
enzyme is structurally conserved and comprises ten subunits. Additional subunits are
located on the periphery. Out of the three, Pol II is known to transcribe the maximum
number of genes.
DNA is replicated, and RNA is translated in the same shared space in prokaryotes
because of the absence of a nuclear membrane. In eukaryotes, the nucleus is the site
of DNA replication and transcription, whereas protein synthesis occurs in the
cytoplasm. RNA is exported across the nuclear membrane before it can undergo
translation. Transcription and translation are separated by physical barriers. The
primary transcript in eukaryotes, which is also known as “heterogeneous nuclear
RNA (hnRNA),” is subjected to post-transcriptional processing in order to make a
messenger RNA (mRNA) molecule that can pass through the nuclear membrane.

10.6 Regulation of RNA Transcription

In regulation, transcription factors are key players. Transcription factors are

DNA-binding proteins that work by repressing or activating gene transcription.
Preferential activity is displayed by some transcription factors. These bind to each
other, cis-acting DNA sequences as well as to both DNA and other transcription
factors. In order to promote repression or activation, specific promoters act as
binding sites for these transcription factors. Certain transcription factors act exclu-
sively as activators or repressors, and some others function as either activators or
10 RNA Transcription 509

repressors. The genome of the bacteria E. coli is comprised of around 300 genes that
code for proteins that function as transcription factors that up- or downregulate
transcription. The functional properties of most of these proteins are still unknown.
For the most part, they are known to regulate a large number of genes. Half of all
regulated genes are controlled transcription factors such as CRP, FNR, IHF, Fis,
ArcA, NarL, and Lrp. A single promoter is known to be controlled by 60 transcrip-
tion factors. Data inferred from sequence analysis suggest that bacterial transcription
factors can be classified into various families and based on these studies. Among
these, 12 groups of families have been extensively analyzed and characterized
including the LacI, AraC, LysR, CRP, and OmpR families. Bacterial promoter
activity also depends on multiple environmental factors and seldom on one signal.
Multiple signals are necessary for promoter response. Various transcription factors
mediate these events. Many promoters are controlled by two or more transcription
factors, with each factor responding to a particular environmental signal.

10.6.1 Repression of Transcription Initiation

A repressor protein negatively regulates gene expression, by binding to the DNA in

order to inhibit the initiation of transcription. An effector molecule essentially binds
to the repressor which decides whether or not the repressor is capable of binding to
the DNA. Transcriptional control can be best explained by the lac operon expression
in E. coli. In this case, the initiation of transcription is brought about from the lac
operon promoter by a repressor protein known as LacI. A site known as operator on
the DNA acts as the binding site for LacI. This region shares a stretch of sequences
with the promoter. Because of the region of similarity, competitive binding of RNAP
and LacI repressor takes place, which makes it a requirement that the repressor
should be released from the operator for RNAP to bind to the promoter (Fig. 10.15).
The repressor is subsequently compounded by the binding galactoside, which
causes the binding of the RNAP to the promoter region and the initiation of
transcription due to the destabilization of the repressor-operator complex
(Fig. 10.16). LacI has two additional binding sites in the lac, an upstream site and
a downstream site, located in the first gene of the operon. These sites have a lower
affinity for the repressor protein, and, likely, they do not directly inhibit transcription
initiation.

10.6.2 Small RNAs

In bacteria several studies have indicated that a subset of small RNAs has been found
to regulate transcription in bacteria. An important example is the 6S RNA that
inhibits transcription at 70-dependent promoters by binding to the active site of
70-RNAP and competing for DNA binding. It has been proposed that, in the
conserved secondary structure of 6S RNA, a single-stranded central bulge within a
highly double-stranded molecule that is essential for 6S RNA function is present
510 M. G. Sharma

Fig. 10.15 Gene regulation by transcription factors and microRNAs (Hobert 2008)

from which it can be hypothesized that 6S RNA mimics the open conformation of
promoter DNA. 6S RNA blocks access to the promoter DNA, and in some cases, it is
also used as a template for RNA synthesis.

10.6.3 Regulation of Transcription Initiation via Changes in DNA

Topology

Negatively supercoiled DNA acts as the template for transcription. DNA melting is
necessary for the open transcription complex assembly. The degree of supercoiling
influences and affects the efficiency of some of the promoters (Fig. 10.17). They are
also stimulated by negative supercoiling. The effect of superhelicity on transcription
initiation has been demonstrated in several in vitro studies and in in vivo models by
gyrase inhibitors, which introduce negative supercoils. Some promoters are also
sensitive to the degree of supercoiling, and some are not; the reason for this lies in the
fact that the sequence of some promoters is easier to melt.
10 RNA Transcription 511

Fig. 10.16 The Lac operon concept and the regulation of gene expression in bacteria

Fig. 10.17 Epigenetic regulation of gene regulation. Gene expression is regulated by DNA
methylation, histone post-translational modifications (PTMs), and the actions of non-coding
RNAs, among other mechanisms. To fit within the nucleus, DNA is wrapped around histone
proteins creating a higher-order chromatin structure, which can facilitate or prevent access to
gene regulatory machinery through steric mechanisms (Torres-Berrío et al. 2019)
512 M. G. Sharma

10.7 RNA Processing: Mechanism

RNA processing can be defined as “any type of alteration performed on the RNA
after it has been transcribed from DNA to obtain its complete functionality in the
cell.”

What Happens During RNA Processing?

After transcription, the RNA is processed before it is exported to the cytoplasm for
translation (Fig. 10.18). The hnRNA which is the product of transcription of DNA
consists of introns (non-expressing codons) and exons (expressing codons). The
introns are excised off from the primary transcript. This process of excising the
introns and joining the exons in eukaryotic mRNA (also tRNA and rRNA) is referred
to as splicing, usually mediated by a set of protein complexes known as the
spliceosomes.

10.7.1 Processing of mRNA

10.7.1.1 50 Capping
Eukaryotic mRNA is not stable at the ends and is susceptible to damage thus
requiring modification to protect it from ribonucleases. The pre-mRNA hence
undergoes capping at the 50 end immediately after transcription and is then released
by Pol II. GTP condensation with triphosphates at the 50 end is an event that triggers
the capping reaction followed by guanine methylation at N-7. This methylation
produces the modified guanine or 7-methylguanosine which is attached to the
triphosphates of the first transcribed base. Capping of the nascent mRNA protects

Fig. 10.18 Overview of mRNA processing (Desterro et al. 2020)

10 RNA Transcription 513

it from enzymatic degradation by RNAse and also helps in the identification of

mRNA by the eukaryotic factors and initiates translation by ribosomes.
It is already known that a protein called m7G cap-binding protein eIF4E binds to
the 50 cap of mRNA and recruits the 40S ribosome subunit to the 50 end of the
mRNA. The Υ phosphate is removed from the first nucleotide by the enzyme
50 -triphosphatase. The enzyme guanylyl transferase facilitates the attachment of
guanosine nucleotide to the first nucleotide of the pre-mRNA. The beta phosphate
of the RNA transcript displaces the pyrophosphate group at the 50 position of the
GTP molecule. The cap formation involves a 50 -50 linkage between the two
substrates.
To the terminal end of the RNA, and in the opposite direction, a G residue is
added. 50 diphosphate RNA acts as a substrate for the specific enzyme
guanylyltransferase. However, GMP transfer is not catalyzed to give rise to a 50
monophosphate RNA. Only 50 end of the pre-mRNAs contain caps, whereas
processed 50 ends followed by endolytic mRNA cleavage do not contain caps.
In the terminal guanine, seven positions are methylated. This event is the first
methylation event. The methyl groups are obtained from S-adenosylmethionine in
the presence of the enzyme methyltransferase. A cap known as cap 0 carries a single
methyl group. The enzyme usually adds methyl groups to 20 hydroxyl groups on the
ribose sugar of the next two nucleotides in the mRNA adjacent to the cap. The
addition of methyl group to 20 OH of ribose of the first nucleotide is known as cap-1.
The addition of methyl group to 20 OH of the ribose of the second nucleotide and
third nucleotide is called cap-2, cap-3, and so on. The decapping enzyme removes
the cap and the cap-binding complex, and the mRNA is subjected to degradation
after translation (Fig. 10.19).

10.7.1.2 30 Polyadenylation
Polyadenylation is a post-transcriptional mechanism in which the addition of poly
(A) tail to the messenger RNA at the 30 end takes place. The poly(A) tail is around
100–250 residues long. The mechanism takes place by endonucleolytic RNA cleav-
age coupled with the synthesis of polyadenosine monophosphate on the newly
formed 30 end also known as the polyadenylation site. A poly(A) tail is added to
the 30 UTR of newly synthesized pre-mRNAs by the enzyme poly(A) polymerase,
which is in turn followed by the recognition of the poly(A) signal and endonucleo-
lytic cleavage of the pre-mRNA at the poly(A) site. Polyadenylation increases the
efficacy of mRNA by protecting the 30 downstream sequences against several
nucleases and also plays important roles in mRNA export to the cytosol, its locali-
zation, stability, as well as translation. A set of proteins cleave the 30 segment of the
newly synthesized pre-mRNA and then the poly(A) tail. Another important function
of the poly(A) tail is to recruit RNases that cleave the RNA. Almost all eukaryotic
mRNAs except animal replication-dependent histone mRNAs are polyadenylated.
Other important functions of the poly(A) tail include the export of mature mRNA
from the nucleus to the cytoplasm, increasing the stability of mRNA and offering
protection from cleavage, and signal recognition for the binding of translational
factors (Fig. 10.20).
514 M. G. Sharma

Fig. 10.19 The mRNA cap is a methylated modification of the 50 terminus of mRNA. RNA
processing and translation factors are recruited to the mRNA cap. The mRNA cap protects
transcripts from degradation and defines mRNA as “self.” The formation of the mRNA cap is
regulated by cellular signaling pathways. mRNA cap regulation results in changes in gene expres-
sion and cell function (Galloway and Cowling 2019)

A multiprotein complex present in the nucleus of eukaryotes primarily targets

precursor mRNA. This multi-protein complex excises the 30 end and adds
polyadenyl groups to the cleaved end. The enzyme CPSF (cleavage/polyadenylation
specificity factor) specifically binds to the recognition site with the following
sequence—50 AAUAAA30 also known as the polyadenylation signal (PAS) is
recognized by the RNA cleavage complex.
When RNA polymerase II recognizes the termination sequence 50 TTTATT 30 on
the DNA template, transcription is terminated. The polyadenylation machinery is
also linked to the spliceosomes.
Alternative polyadenylation (APA) is yet another mechanism of RNA processing
which produces distinct 30 ends on mRNAs. APA is also a gene regulation mecha-
nism in eukaryotes. It is tissue-specific and is extensively studied to understand
10 RNA Transcription 515

Fig. 10.20 The process of alternative polyadenylation (Gruber and Zavolan 2019)

proliferation and differentiation in cells. Alternative polyadenylation is sometimes

used to reduce the length of the coding region which can lead to the expression of
different proteins.

10.7.1.3 RNA Splicing

RNA splicing is an important post-transcriptional process where the non-coding
intron sequences are removed from the transcript and the exons are subjected to
processing and rejoining. The splicing complex is similar to restriction enzymes that
recognize specific sites within the RNA and cleave and ligate the RNA at the cleaved
sites. Splicing of pre-mRNA takes place in the nucleus before export.
There has been a significant amount of progress in computational analyses and
sequencing methods, and they have led to the discovery of novel splicing isoforms
and non-canonical splicing mechanisms. One such example is co-transcriptional
splicing. This allows for the epigenetic and epitranscriptomic fine-tuning of gene
expression. Studies have revealed that intrinsically disordered domains of RNA Pol
516 M. G. Sharma

Fig. 10.21 Emerging evidence highlights that the RNA splicing and export machinery can display
regulatory potential. Core spliceosome components can display regulatory potential if their levels
become limiting for the function of complexes. These findings have important implications for the
contribution of selective mRNA processing and export to the development of human cancers and
neurodegenerative disorders (Carey and Wickramasinghe 2018)
10 RNA Transcription 517

II form local condensates, and several splicing factors are known to optimize
splicing reactions (Fig. 10.21).
Exon junction complexes facilitate recursive splicing and also inhibit cryptic
splice sites. Circular RNA splicing efficiency is enhanced by the low-efficiency
splicing of the flanking introns. Pre-mRNA splicing is crucial in eukaryotic gene
expression. Identification of exact splice sites and the accurate removal of introns are
also essential for the generation of mRNA and its isoforms. Splicing regulation is
mostly well understood. Emerging studies have also revealed that certain
non-canonical splicing mechanisms exist. These are important in the regulation of
gene expression.

10.7.1.4 Alternative Splicing

Alternative splicing is a process that allows a messenger RNA (mRNA) to express
different forms of proteins (Fig. 10.22). Alternate splicing occurs by the reorganiza-
tion of the intron and exon sequences in various combinations. This alters the coding
sequence of the mRNA. Alternative splicing of the precursor mRNA increases the
complexity of gene expression and plays an important role in cell differentiation and
growth. Alternative processing is tightly regulated, and this regulation is supervised
by the regulatory elements linking both transcription and splicing.

10.7.1.5 Sequestration as RNP

After nuclear pre-mRNA undergoes complete processing, it is recognized due to the
absence of association with splicing factors, and the RNA associated with the
functional spliceosome complex is retained in the nucleus. As splicing begins
from the cap site and moves towards the polyadenylation site, the hnRNP A1 protein
factor binds to the single-stranded RNA molecules that are already exposed. The

Fig. 10.22 A gene that contains numerous exons and introns can be spliced together in various
ways. For example, in a gene containing eight exons, the mRNA transcribed from that gene can
contain exons 1–7
518 M. G. Sharma

Fig. 10.23 Crystal structure of hnRNP A1

final processed and fully mature pre-RNA molecules are devoid of any bound
splicing factors. Some of the sequences on the proteins are markers for nuclear
export signals (NES) and nuclear localization signals (NLS). hnRNP A1 protein also
acts as a carrier molecule for mature pre-mRNA (Fig. 10.23).

10.7.2 Processing of tRNA

Transfer RNA or the tRNA is the primary molecule that facilitates the process of
translation. It consists of a single RNA strand made up of 75–95 nucleotides. tRNA
is the smallest of the three types of RNA. The 20 amino acids that make up the
primary peptide chain all have a specific tRNA that binds to it and transfers it to the
growing polypeptide chain during translation. tRNAs are also called adapter
molecules. tRNAs have a cloverleaf structure which is stabilized by strong hydrogen
bonds between the nucleotides.
All the tRNA molecules have a 30 end with a conserved 50 -CCA-30 sequence.
Some tRNAs have unusual and modified bases in their primary structure. These
unusual bases are mostly a result of post-transcriptional enzymatic modifications of
the normal bases in the polynucleotide chain. Two common modifications include
pseudouridine (ψU), a derivative of uridine, in which uridine is modified such that
the uracil attaches to the ribose to the carbon at the fifth position instead of the
nitrogen in the first position, and dihydrouridine (D), also a derivative of uridine
where enzymatic reduction of the double bonds between the fifth and the sixth
carbon occurs. Other modified bases include hypoxanthine, thymine, and
methylguanine. Studies have suggested that cells that do not have these modified
bases have shown retarded growth leading to the conclusion that the modified bases
have a role in enhanced and better tRNA function.
10 RNA Transcription 519

Parts of tRNA Function

Acceptor The incoming amino acids are attached to the acceptor stem by the paring of the
stem 50 and 30 ends of the tRNA molecule. 50 -CCA-30 end is present which is a single-
stranded region protruding from the double-stranded stem
ψU loop It is named as T loop due to the presence of the modified base pseudouracil in
(T loop) this region or loop. The sequence 50 -TψUCG-30 contains the modified base
D loop It is named as D loop because of the presence of the modified base
dihydrouridine
Anticodon It is named as anticodon loop because it contains the anticodon which
loop recognizes the codon in the mRNA and the corresponding specific amino acid is
attached to the tRNA. The anticodon loop always has uracil in its 50 end
Variable loop It is present between the anticodon loop and the T loop, also called the
pseudouracil loop, and it is so called because of its variation in the base stretch
between 3 and 21 bases

10.7.2.1 Secondary and Three-Dimensional Structure of tRNA

There are regions of complementarity within an RNA molecule that enables RAN to
form small stretches of double helical patterns which are subsequently stabilized by
base pairing. tRNA molecules have a unique conserved pattern showing both single-
and double-stranded regions which are also commonly referred to as the cloverleaf
model. The cloverleaf model includes significant structures such as the acceptor
stem, the ψU loop, the D loop, and the anticodon loop and a fourth variable loop
(Fig. 10.24).

Fig. 10.24 (a, b) Secondary and tertiary structures of tRNA. (c) Crystal structure of tRNA (Liu
et al. 2015)

X-ray crystallography studies revealed the tertiary structure which takes the shape
of the letter L. This structure enabled us to better understand that the orientation of
the acceptor stem and anticodon loop and that they are at opposite ends of the
adaptor molecule. The acceptor stem and the pseudouracil loop form an extended
continuous helix. The anticodon stem associates with the D loop stem to form an
extended second helix. The two helices are perpendicular to each other bringing the
D loop and the ψU loop together. Interactions such as base stacking, hydrogen bond
formation between the bases, and the interaction between bases and the
520 M. G. Sharma

sugar-phosphate backbone stabilize the three-dimensional L-shaped structure and

the final confirmation of the tRNA molecule.

10.7.3 Processing of rRNA

rRNAs account for around 80% of the total RNA present in cells, and they are the
main components of ribosomes. Ribosomes are made up of two subunits, a large
subunit (the 50S) and a small subunit (30S). Each subunit is made up of specific
rRNA molecules. The rRNAs along with proteins and enzymes combine to form
ribosomes, which are sites of protein synthesis. The small and large rRNAs contain
around 1500 and 3000 nucleotides in prokaryotes such as bacteria and 1800 and
5000 nucleotides in eukaryotes such as humans. The 16S rRNA is the only rRNA in
the small subunit of the ribosome and is also called the small subunit rRNA or
ss-rRNA. The 5S and 23S are both components of the large subunit of the ribosome.
Ribosomes are denoted by the sedimentation unit “S.” In eukaryotes and archaea,
four rRNAs are present: 18S in the small subunit and 5S, 5.8S, and 28S in the large
subunit. Mitochondria contain 12S and 16S rRNAs. The processing of rRNA is
depicted in Fig. 10.25.

Fig. 10.25 The processing of rRNA

10 RNA Transcription 521

10.8 RNA Editing: Mechanism

RNA editing involves series of molecular processes where the RNA sequence is
altered to allow the mature RNA to show variance from the RNA that is encoded by
the genomic DNA. Editing includes processes like deletion, insertion, and substitu-
tion of the nucleotides. The variation observed in the messenger RNA (mRNA),
ribosomal RNA (rRNA), transfer RNA (tRNA), and microRNAs (miRNA) can be
attributed to RNA editing. The process of RNA editing occurs in the time interval
between the transcription of DNA into mRNA and the translation of this mRNA to
protein.
With the discovery of RNA editing, more light is being shed on novel post-
transcriptional modifications. RNA editing is facilitated by adenosine and cytidine
deaminases acting on DNA and RNA (Fig. 10.26). Adenosine to inosine (A-to-I)
editors are members of the ADAR and ADAT protein families. They are important
molecules that are crucial in the regulation of alternative splicing and transcription.
Other kinds of editors such as cytidine to uridine (C-to-U) editors are members of the

Fig. 10.26 RNA editors such as cytidine and adenosine deaminases are functionally important in
regulating cellular processes. (a) Apolipoprotein B is produced in the gut which is mediated by
APOBEC1 editing. Glutamate is transformed to a stop codon by C-to-U editing at residue 2153 of
hepatic Apo-B100, and a truncated protein Apo-B48 is produced in intestinal cells. (b) The
glutamate receptor 2 (GluR2) mRNA at position 607 is edited by ADAR2 in neurons, resulting
in change of adenosine to inosine (Christofi and Zaravinos 2019)
522 M. G. Sharma

Fig. 10.27 RNA editing: overview

AID/APOBEC family and are key players that mediate innate and adaptive immu-
nity and are also responsible for antibody diversification, antibody generation, and
antiviral response. These editors are enzymes, and they are present in the nucleus or
the cytoplasm. They play a role in the modification of several RNA molecules,
including miRNAs, tRNAs, and most importantly mRNAs. Some editors are also
capable of editing DNA. Latest technologies such as next-generation sequencing
(NGS) have provided us with a large amount of data regarding these post-
transcriptional modifications. RNA editing is often implicated in disorders such as
cancer and other neurological diseases concerning the brain and the CNS. RNA
editing is directly affected by cancer heterogeneity, carcinogenesis, response to
treatment, and drug efficacy. Research on RNA editing will lead to the discovery
of novel biomarkers identification and diagnostic techniques.

What Is Substitution Editing or Site-Specific Base Modification Editing?

A base substitution that causes a significant change in the coding properties or the
structure of RNAs is known as substitution editing. These alterations often arise due
to chemical changes in the individual nucleotides. Three common deamination
reactions give rise to this substitution editing (Fig. 10.27).
This figure depicts the process of RNA editing, where the original guide RNA
undergoes post-transcriptional editing which upon translation gives rise to proteins
with different functional properties.
10 RNA Transcription 523

10.8.1 A-to-I Editing

Conversion of A to I: Adenosine deaminases convert an A to inosine (I), which is

translated in the form of G by the ribosomes. The abundantly seen type of RNA
editing system is A-to-I editing by double-stranded RNA-specific adenosine deami-
nase (ADAR) enzymes. Data gathered from transcriptomic studies has revealed
several “recoding” sites supposedly at which A-to-I editing results in substitutions
of bases in protein-coding sequences (Fig. 10.28). The recoding sites are also

Fig. 10.28 A-to-I editing by double-stranded RNA-specific adenosine deaminase (ADAR)

enzymes (Eisenberg and Levanon 2018). The deamination of adenosine and formation of inosine,
leading to unstable double-stranded RNA base pairing. This alteration leads to reduced production
of siRNA, by the dsRNA, and this disrupts the RNAi pathways
524 M. G. Sharma

conserved within lineages and are subjected to positive selection, and they have
functional and evolutionary importance. Mapping studies of the editosome complex
in various species of the animal kingdom has suggested that most A-to-I editing sites
are present within mobile genetic elements in the non-coding parts of the genome
and evidence points to the fact that editing of these non-coding sites might have a
critical role to play in protection against innate immunity activation by the self-
transcripts. Recoding, as well as non-coding events, has been implicated in genome
evolution and their deregulation, which could lead to diseased conditions. ADARs
are being extensively studied and being adapted for RNA engineering.

10.8.2 C-to-U Editing

Conversion of C to U: Cytidine deaminases convert a C base in the RNA to uracil

(U) apolipoprotein B gene in humans with Apo B100 being expressed in the liver
and apo B48 in the intestines of humans (Fig. 10.29).

Fig. 10.29 C-to-U RNA editing of apolipoprotein B. In this figure a 35-nucleotide region of
apoB RNA flanking the edited base is shown also highlighting apobec-1 and ACF binding to RNA
both 50 and 30 of the edited base and depicts the presence of additional proteins that may modulate
assembly of the holoenzyme
10 RNA Transcription 525

RNA editing by cytidine deamination, the extent to which editing takes place, its
regulation, and enzymatic and molecular basis have not been properly established.
Hundreds of gene transcripts are known to undergo site-specific C-to-U RNA editing
in macrophages and monocytes during M1 polarization and in response to hypoxia
and interferons, respectively. This allows for the altering of the amino acid
sequences of proteins, especially those that are involved in the viral disease patho-
genesis. In single-stranded DNA, cytidines are deaminated by APOBEC3A and also
inhibit retrotransposons and viruses. Amino acid residues of APOBEC3A involved
in anti-retrotransposition and DNA deamination were also found to affect its RNA
deamination activity. In plants, C-to-U editing is seen in the mitochondrial RNA of
flowering plants.

10.8.3 Editing in Mitochondria

10.8.3.1 Types of RNA Editing in Mitochondria

Protein-coding transcripts (mRNA) are always edited, but introns, rRNA, and tRNA
species are also edited in a few cases. In a mitochondrial system, editing restructures
many different mRNA transcripts and generates a translatable reading frame. This
structured re-tailoring is usually seen in insertion/deletion editing. Editing also gives
rise to translation initiation or termination codons. RNA editing affects internal
codons; however, substitution editing specifically occurs at first or second positions
of codons. Editing often changes the protein properties and causes the protein to
significantly deviate from the predicted gene sequence.
In mitochondria, RNA editing systems are known to be mechanistically distinct,
and several RNA editing systems are not related mechanistically (Fig. 10.30). The
molecular events that occur during editing are phosphodiester bond cleavage and
re-ligation. In some cases, editing happens by direct base conversion methods such
as deamination. These unique characteristics are discussed along with the concept of
the evolution of RNA editing processes. Mitochondrial editing systems are also said
to be phylogenetically isolated.
The phylogenetic distribution of most mitochondrial RNA editing systems is
restricted, and evidence suggests that these intrinsic mechanisms in the editing
systems have evolved from discrete eukaryotic lineages during evolution. Consider-
ing U insertion/deletion editing, it has been observed only in kinetoplastid protozoan
(Kinetoplastida), in the early-diverging and free-living bodonids (Bodonida), and the
late-diverging and parasitic trypanosomatids (Trypanosomatina). This type of
editing has not been observed within diplonemids or euglenids. Mitochondrial
mRNA editing that occurs in dinoflagellate mitochondria has not been observed in
the apicomplexans or ciliates. A different type of editing has been observed in plant
organelles (C-to-U and U-to-C) which are not found in any of the green algal
mitochondrial systems.
In this way, the mitochondrial editing systems are narrowly restricted and deflect
suspicions away from the hypothesis that the editing systems were present in a
common eukaryotic ancestor.
526 M. G. Sharma

Fig. 10.30 Varying levels of RNA editing is depicted in this schematic illustration. Heterotrophs
are known to display higher levels of RNA editing

10.8.4 Editing in Plastid

Mitochondria and plastids in terrestrial plants show evidence for post-transcriptional

editing from C to U as well as U to C in several transcript sequences. In plastids such
as chloroplasts, around 6–20 C bases were found to be deaminated to U bases. RNA
analysis studies in mitochondria reveal that in some plants, around 500–1000 U to C
conversations is common. Two important factors—(a) PLS-type pentatricopeptide
repeat (PPR) protein and (b) multiple organellar RNA editing factors (MORFs, also
known as RNA-editing factor interacting protein (RIP))—are trans-acting factors
that are involved in this process (Fig. 10.31). MORF9 binding induces significant
compressed conformational changes of (PLS)3PPR, revealing the molecular
mechanisms by which MORF9-bound (PLS)3PPR has increased RNA-binding
activity. Similarly, increased RNA-binding activity is observed for the natural
PLS-type PPR protein, LPA66, in the presence of MORF9.
Most plant species do not support U-to-C conversions, and these edits are rare
events, specifically two to three edits in the entire mitochondrial RNA sequence.
Sites for RNA editing are usually found in the coding regions of mRNAs and rather
less in the introns and non-translated regions. It has been observed that in certain
situations, RNA editing in tRNA molecules corrects errors and restores normal base
pairings. Here, RNA editing becomes crucial because only proper editing can ensure
further maturation, accurate folding, and processing of the precursors of tRNA.
10 RNA Transcription 527

Fig. 10.31 Yan, J., Zhang, Q., Guan, Z. et al. characterize the interactions between a designer
PLS-type PPR protein (PLS)3PPR and MORF9 and strongly suggest that RNA-binding activity of
(PLS)3PPR is drastically increased on MORF9 binding. Crystal structures of (PLS)3PPR, MORF9,
and the (PLS)3PPR-MORF9 complex are shown in the figure

Figure 10.32 depicts the molecular interactions that influence both RNA editing and
chloroplast signaling, essentially suggesting that RNA editing is crucial for normal
functioning. In plant organelles, rRNAs are subjected to minimal RNA editing. The
mechanisms underlying the recognition of these editing sites, the enzymatic action,
and the molecular pathways involved are yet to be determined.
In plants, other nucleotide insertions or edits have not been observed. One
hypothesis suggests that one reason for RNA editing is that it exists to trigger the
activity of a particular RNA-specific C deaminase. These deaminases, however, do
not catalyze reverse U-to-C reactions.
Research points to the idea that RNA sequences are involved in guiding the
“editosome” editing complexes to specific sites. Cis- or trans-acting RNA molecules
facilitate this guiding function but are not native to the sequence regions of the edit
sites, but there are no common sequence motifs that have been identified around the
different C-to-U conversion sites. In positions preceding the edited Cs, a low amount
of G residues has been found. Downstream nucleotides in both mitochondria and
plastids are not involved in the editing site specifications, whereas the upstream
sequences play a significant role. However, in both the organelles, the upstream
region differs in various editing sites, while some sites require only about 5–20
nucleotides and others require around 200 nucleotides. Sequence duplications are
also seen in mitochondria. Here as if enough number of upstream sequences are
present, RNA editing is accurately maintained. By experimenting in vivo with
transgenic plastids, it was proved through upstream and downstream sequence
insertions.
Identification of potential RNA editing intermediates suggests that RNA editing
in plant organelles is a post-transcriptional process. Partially edited transcripts
contain some C bases that have been deaminated to Us. The Cs encoded by the
genome exist, in other potential editing sites. Partially edited RNA molecules do not
follow a particular order of editing. This means that the hypothetical “editosome”
528 M. G. Sharma

Fig. 10.32 Proposed interactions between RNA editing and chloroplast signaling. In well-
functioning photosynthetic cells (left), chlorophyll accumulates in the thylakoid membranes
(green), and chloroplasts perform photosynthesis. The GUN1 protein does not accumulate, and
thus the chloroplast-to-nucleus signaling that depends on GUN1 is not active. MORF2 contributes
to RNA editing (green arrow) and interacts with OPT81, OPT84, and YS1. Light signaling, tissue-
specific signals, and the circadian rhythm (not shown) drive high-level expression of PhANGs
(black arrow), which promotes chloroplast function (Vo et al. 2019)

complex does not linearly scan the RNA molecule and the selection of editing sites
or regions is arbitrary. These partially edited mRNAs are found in minimal amounts
in the plant mitochondria, and they are translated into a family of different proteins.
But this assumption only holds for one type of protein sequence, and it is said to be
present in the protein complexes of the respiratory chain. These sequences generally
are the polypeptide sequences that are best conserved with their respective homologs
in other organisms and are selected by their physiological and biochemical function-
ality. So, it is likely that polypeptides synthesized from unedited RNA molecules
would not function properly and such proteins, for example, would hinder the
efficiency of respiration in mitochondria.
10 RNA Transcription 529

10.8.5 Coediting in Virus

In RNA viruses, during transcription of mRNA, the transcription machinery

incorporates additional nucleotides that are not specific to the viral genome. For
example, in certain paramyxoviruses such as measles, Sendai, parainfluenza, and
mumps, viruses around one to ten G residues are inserted at specific editing sites
(Fig. 10.33). In Ebola viruses, it is observed that additional A residues are
incorporated during transcription.
So, it is observed that co-transcriptional editing of RNA in RNA viruses happens
by insertion of non-templated nucleotides by a mechanism known as “stuttering of
the RNA-dependent RNA polymerase.” Specific sequence motifs are present in the
viral RNA that can induce the RNA polymerase complex to stutter and repeat the last
transcribed nucleotide of the template RNA before resuming transcription.
The replication of Ebola viruses, paramyxoviruses, and other RNA viruses is
facilitated by the transcription of RNA-dependent RNA polymerase that is virus

Fig. 10.33 Certain viruses like the Sendai viruses encode genes to express multiple proteins. They
do this with the help of overlapping open reading frames (ORFs) by RNA editing. In viruses like
these, the RNA polymerase is capable of reading the same template base more than once, creating
insertions that subsequently lead to different mRNAs and generating different types of proteins
530 M. G. Sharma

Fig. 10.34 Negative-strand RNA viruses belonging to paramyxoviridae and ebolaviridae are
known to polyadenylate mRNA during transcription through a polymerase stuttering mechanism.
The viral polymerase acquires a stuttering behavior upon encountering the stop signal present at the
end of each gene comprising a stretch of U bases. After each adenine insertion, the RNA polymer-
ase moves back one nucleotide with the mRNA, copying U hundreds of times at the end of viral
mRNA thereby producing a poly(A) tail and releasing the polyAdenylated mRNA to stop tran-
scription or scan to restart on the next gene

encoded resulting in antisense RNA. Stuttering and pausing mechanisms are mostly
seen in these types of polymerases at certain nucleotide base combinations, mostly
mono- or oligonucleotide tracts (Fig. 10.34). At the nascent mRNA, 30 ends up to
several hundred As are added by the same polymerase, although they are not
templated. These As stabilize the mRNA by using a mechanism similar to
polyadenylation. mRNA polymerase (complex) pauses at these positions, while
the RNA replicase (complex) synthesizes the replication intermediate RNA from
the virion RNA. The same RNA polymerase is influenced in a differential manner by
additional cofactors, and replication takes place in the virion, while transcription
usually occurs in the cytoplasm.
RNA-dependent RNA polymerase encoded by viruses also pauses and
incorporates non-templated nucleotides by the same “stuttering” mechanism around
genomic stop codon of the first open reading frame in the unedited mRNA. The
reading frame is shifted by insertion of one to two Gs or A bases upstream of the
translational stop codon. Upon translation this results in the generation of different
10 RNA Transcription 531

proteins with different carboxy-terminal sequences. Compared to the genomically

predicted first open reading frame, the extra amino acid sequences in the mumps
viruses are known to be double the size.
Transcription termination in E.coli and sequence-specific RNA polymerase paus-
ing and “stuttering” are comparable. Unstable transcription complexes are also
induced similarly by sequences at the template sites. Pyrimidine-rich sequences
are present along with long U stretches preceding Cs can cause the viral RNA
polymerase to slow down, slip back one nucleotide on the template, and incorporate
another G nucleotide opposite the same C again. Due to stuttering progression, a
sequence of three Gs is altered as initially predicted by the genomic RNA to four Gs
in the edited mRNA. RNA viruses facilitate dissociation and realignment of this
polymerase-product complex to the previously transcribed nucleotide on the RNA
template which can be explained by the specific rate constants of dissociation and
RNA-binding protein.

Box 10.1 Scientific Concept: High-Throughput Detection of RNA

Processing in Bacteria: Erin E. Gill et al.
A γ-proteobacterium Pseudomonas aeruginosa is a causative agent of oppor-
tunistic infections in hospitalized immunocompromised patients and chronic
lung infections in patients suffering from cystic fibrosis. P. aeruginosa has
been extensively studied because it has devastatingly contributed to human
morbidity and mortality. P. aeruginosa is of considerable medical importance
due to its metabolic diversity, motility, quorum sensing, ability to produce
biofilm, adaptive responses to evade antibiotic stress, and extreme virulence.
The main P. aeruginosa strain that is implicated in opportunistic infections is
“PAO1.” However, accurate information regarding the transcription start site
(TSS) is unavailable to date. The molecular pathways underlying post-
transcriptional modifications of RNA transcripts remain elusive in Pseudomo-
nas as well as other organisms. E. Gill et al. suggest that understanding the
biochemical makeup of P. aeruginosa, and obtaining the detailed map of TSS
and subsequent RNA processing of transcripts and influences virulence, anti-
microbial resistance, and other essential cellular functions, is crucial to figur-
ing out the mystery behind the regulation of pathogenesis and drug resistance
and the identification of novel drug targets.
Maintaining an inventory of RNA processing sites Transcription Start Sites
is necessary to understand cellular processes. RNA sequence-based analysis
helps map the set of post-transcriptional modifications occurring in the
transcriptomes of organisms. This is a crucial and challenging objective. The
completion of transcription results in the occurrence of a series of tightly
regulated secondary modifications ultimately leading to the maturation of the
RNA transcript. These processes are fundamental to the functionality of many
RNAs and also strongly influence the overall behavior of the RNA molecule.

(continued)
532 M. G. Sharma

Fig. 10.35 RNA transcription and processing. (Erin E. Gill et al.) (a) Initiation of RNA
transcription from a promoter sequence (indicated in red) within the genome and subsequent
polymerization of ribonucleoside triphosphate resulting in a 50 triphosphate at the 50 end of the
nascent mRNA transcript and a 30 hydroxyl at its 30 terminus. (b) mRNAs undergoing cleaving by
endonucleases to giving rise to two fragments of RNA or can undergoing degradation by
exonucleases from 50 or 30 termini. (c) RNA processing events that result in either a 50 triphosphate
(dRNA-Seq) or 50 monophosphate (pRNA-Seq) and that simultaneously contain a terminal 30
hydroxyl

Box 10.1 (continued)

A terminal 50 triphosphate is present in the primary transcript contains. The
conserved pyrophosphatase RppH (YgdP in P. aeruginosa) selectively
removes the 50 triphosphate and leaves a 50 monophosphate in bacteria. This
50 monophosphate causes destabilization of the mRNAs by making them
susceptible to degradation (Fig. 10.35). The multi-subunit degradosome is an
important complex involved in this process. At its core, it contains the 50
phosphate-sensitive exonuclease/endonuclease RNase E. 50 monophosphate is
known to significantly increase RNase E’s endonuclease activity.
Endonucleases that can cleave RNA and leave behind a 50 phosphate can
also result in the production of stable RNAs and activate RNA degradation via
degradosome-mediated pathways required for cellular function.

10.9 Summary

• The process of producing proteins from nucleotides is termed as gene expression.

Genetic information present in the DNA, is first rewritten, generating RNA, the
process of which, is termed as transcription. During transcription, starting with a
DNA template, RNA is essentially synthesized. Every single gene is transcribed
into several copies of mRNA, and each mRNA molecule is capable of generating
identical copies of a single protein.
10 RNA Transcription 533

• The first step in gene expression where the enzyme RNA polymerase converts a
DNA segment into RNA is called transcription. DNA and RNA both make use of
nucleotide base pairing as a complementary language.
• Many different types of RNAs are responsible for various functions. The impor-
tant ones include mRNA, tRNA, and rRNA.
• Prokaryotic transcription has been extensively studied in bacteria such as E. coli.
The transcription of RNA involves three steps: initiation, chain elongation, and
termination. RNA polymerase locates the target DNA by recognizing the pro-
moter region.
• The core enzyme is aided by the σ factor to locate the transcription binding site.
This activity is mediated by specific nucleotide sequences on the DNA known as
the promoter regions. Promoter recognition is crucial for the initiation of
transcription.
• After the transcription cycle is set up, the elongation process has to be stabilized.
The Pol II machinery equips additional factors to stop the premature dissociation
of Pol II. These factors are known as elongation factors, and they associate with
Pol II just after initiation.
• The terminator signal triggers cascades that cause the core enzyme to dissociate
from the template, which releases the newly synthesized RNA transcript and
re-associates with the σ factor so that it can start a new round of transcription.
• Bacterial RNA polymerase is made up of a core complex consisting of multiple
subunits and an initiation factor called the sigma (σ) factor. The core complex has
nonspecific polymerase enzyme activity and can bind to DNA and nicks in a
nonspecific manner and is known as the core enzyme (E).
• At the molecular level, it is understood that the RNA polymerase works by
creating phosphodiester bonds between the incoming ribonucleotide
triphosphates and the growing chain of RNA. This is a thermodynamically
feasible and irreversible reaction. The RNA polymerase adds around 10–100
bases every second.
• The process of transcription is essentially the same in both prokaryotes and
eukaryotes. But more steps are involved in eukaryotic transcription. Bacteria
and species belonging to archaea require only one type of RNA polymerase,
whereas eukaryotes require at least three main enzymes—RNA polymerases I, II,
and III (Pol I, II, III), along with polymerases IV and V which are present in plants
that transcribe different subsets of RNA.
• Transcription factors are DNA-binding proteins that work by repressing or
activating gene transcription. Preferential activity is displayed by some transcrip-
tion factors. These bind to each other, cis-acting DNA sequences as well as to
both DNA and other transcription factors. In order to promote repression or
activation, specific promoters act as binding sites for these transcription factors.
• The degree of supercoiling influences and affects the efficiency of some of the
promoters. Some promoters are also sensitive to the degree of supercoiling, and
some are not; the reason for this lies in the fact that the sequence of some
promoters is easier to melt.
534 M. G. Sharma

• After transcription, the RNA is processed before it is exported to the cytoplasm

for translation. Eukaryotic mRNA is not stable at the ends and is susceptible to
damage thus requiring modification to protect it from ribonucleases. The
pre-mRNA hence undergoes capping at the 50 end immediately after transcription
and is then released by Pol II.
• Polyadenylation is a post-transcriptional mechanism in which the addition of poly
(A) tail to the messenger RNA at the 30 end takes place. The poly(A) tail is around
100–250 residues long.
• RNA splicing is an important post-transcriptional process where the non-coding
intron sequences are removed from the transcript and the exons are subjected to
processing and rejoining. Alternative splicing is a process that allows a messenger
RNA (mRNA) to express different forms of proteins.
• Transfer RNA or the tRNA is the primary molecule that facilitates the process of
translation. It consists of a single RNA strand made up of 75–95 nucleotides.
tRNA is the smallest of the three types of RNA.
• RNA editing involves series of molecular processes where the RNA sequence is
altered to allow the mature RNA to show variance from the RNA that is encoded
by the genomic DNA. Editing includes processes like deletion, insertion, and
substitution of the nucleotides. Conversion of A to I: Adenosine deaminases
convert an A to inosine (I), which is translated in the form of G by the ribosomes.
Conversion of C to U: Cytidine deaminases convert a C base in the RNA to
uracil (U).
• In a mitochondrial system, editing restructures many different mRNA transcripts
and generates a translatable reading frame. This structured re-tailoring is usually
seen in insertion/deletion editing. Mitochondria and plastids in terrestrial plants
show evidence for post-transcriptional editing from C to U as well as U to C in
several transcript sequences.
• In RNA viruses, during transcription of mRNA, the transcription machinery
incorporates additional nucleotides that are not specific to the viral genome.

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Protein Translation
11
Tanushree Banerjee

As we have studied in the previous chapter, DNA is the carrier of genetic informa-
tion. There are four bases, and the permutation and combination of these bases store
this enormous information. The information stored in DNA is transferred to RNA,
and this process is called as transcription. The messenger RNA after being tran-
scribed moves out of the nucleus carrying the genetic information. Hence, it is called
messenger RNA or mRNA. The information present in mRNA is then read and is
translated into proteins. The process of converting the genetic information stored in
mRNA into proteins is called translation. In this chapter we will learn about the
genetic code and how it is being translated to proteins. We will also learn about the
various regulatory mechanisms involved in translation.

11.1 Genetic Code

The information present in the RNA is in the form of ribonucleotide bases organized
in a pattern. In this pattern the bases serve as “letters,” and the combination of three
bases serves as “word.” Each three-letter words made of three bases code for an
amino acid. This three base code is called as triplet code or genetic code or codon.
There are certain features of the genetic code which are universal.

1. Code is unambiguous—Each codon specifies only a single amino acid. More than
one amino acid cannot be coded by the same codon.
2. Code is degenerate—One amino acid can be specified by more than one codon.
Out of 20 amino acids, 18 amino acids are coded by more than one codon.

T. Banerjee (*)
Molecular Neuroscience Research Laboratory, Dr. D. Y. Patil Biotechnology and Bioinformatics
Institute, Dr. D. Y. Patil Vidyapeeth, Pune, India
e-mail: tanushree.banerjee@dpu.edu.in

# The Author(s), under exclusive license to Springer Nature Singapore Pte 537
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_11
538 T. Banerjee

3. Code is contiguous—There are no breaks, no comma, and no punctuation in it. It

is read one after the other.
4. Code is non-overlapping—A ribonucleotide can be a part of only one codon.
5. Start and stop signal: AUG and GUG act as signal to start the translation process,
and UAA (ochre), UAG (amber), and UGA(opal) act as stop signals.
6. The code is linear and universal with minor exceptions.

Initially, it was assumed that DNA directly codes for proteins using ribosomes.
Later, Jacob and Monod postulated the existence of a less stable RNA intermediate,
mRNA, that is translated into proteins.
In the early 1960s, Sydney Brenner proposed that the code must be a triplet since
three bases are the minimal requirement for forming at least 20 combinations coding
for 20 amino acids. If the code would have been a two-letter word, 16 unique
combinations were possible (42). A triplet code gives 64 combinations (43), whereas
4 letter words gives 256 combinations (44), which is far more than the required
number of combinations for generating 20 amino acids.
Existence of triplet code was first experimentally shown by Francis Crick, Leslie
Barnett, Brenner, and R. J. Watts-Tobin. They took bacteriophage T4 which is
known to cause lysis of E. coli strains B and K12. Mutant strain of phage T4, rII,
was unable to infect K12 but could still infect strain B. Crick and colleagues used
acridine dye proflavine which is a DNA-intercalating agent and causes one or more
insertion and deletion mutation during replication. An insertion of a single nucleo-
tide causes the entire reading frame to shift changing all subsequent downstream
codons. These mutations are called frameshift mutations. Crick and colleague
hypothesized that if rII is again treated with proflavine, random frameshift mutations
would occur. That might induce new mutations or could even revert the earlier
mutation by causing some insertion near the original mutation. The revertant could
then be selected by its ability to cause the K12 infection. They observed many
mutants where there was one insertion and one deletion which could cause reversion
to the wild-type behavior. However, when two deletions and two insertions
occurred, wild-type behavior was not restored.

11.1.1 Non-overlapping Nature of the Code

Sydney Brenner imagined a 5-nucleotide sequence, GTACA; parts of the central

codon TAC were shared by the outer codons GTA and ACA. When any central
amino acid is considered, only 16 combinations of tripeptide sequences were
possible. Brenner had actually found many more tripeptide combinations. This
observation supported non-overlapping code.
Another argument against overlapping code was given by Francis Crick in 1957.
He predicted the existence of an adaptor molecule that will have hydrogen-bonding
ability with the nucleotide sequence and form covalent bond with the amino acids. If
genetic code would have been overlapping, various adaptors would have tried to
occupy overlapping sites making the translation process complex and error prone. It
11 Protein Translation 539

was later found that Crick’s prediction about the adaptor molecule was correct.
Transfer RNA (tRNA) plays the role of adaptor molecules.

11.1.2 Degenerate Nature of Code

Crick also proposed that the code is contiguous and lacks any punctuation or breaks.
He hypothesized that out of the 64 codons, only 20 codes for amino acids, and the
rest 44 codes for nothing and hence were referred to as blank or non-sense codons.
However, his experiments later with phage T4 rII point mutants proved that
44 remaining codons were not blank. He observed that cases where wild type
sequence was restored, had mutations like (+) with (
); (++) with (

); and (++
+) with (


). However, in between the deletion and insertions, there were
numerous codons out of frame. It was very likely that out of those wrong codons,
some of them will be among those 44 non-sense codons. In that case the translation
would terminate prematurely, and restoration of wild type would not be possible. As
the mutant combinations were able to infect E. coli K12, Crick and colleague
concluded that remaining 44 codons must not be blank.

11.1.3 Deciphering Genetic Code

In 1968 Nirenberg, Har Gobind Khorana, and Robert Holley won the Nobel Prize in
Physiology or Medicine for their seminal work on the genetic code. Nirenberg
characterized specific coding sequence; Har Gobind Khorana developed the process
for the synthesis of nucleic acids; and Robert Holley discovered the chemical
structure of transfer RNA. Their contribution is recognized for their “interpretation
of the genetic code and its function in protein synthesis.” The experimental model
was a cell-free protein-synthesizing system in a test tube with the enzyme polynu-
cleotide phosphorylase, which allowed the production of synthetic mRNAs.

11.1.4 Synthesizing Polypeptides in a Cell-Free System

The in vitro system is made by adding all the essential factors like ribosomes,
tRNAs, amino acids, and mRNA template for translation. Few of the amino acids
were radioactively labeled. In 1961, mRNA was yet to be isolated. Hence, enzyme,
the polynucleotide phosphorylase, was used to synthesize artificial RNA template
catalyzing the reaction shown in the template.
This enzyme is known to degrade RNA in vivo. However, in vitro in the presence
of high concentration of ribonucleoside diphosphates, the backward reaction rate
increases, leading to RNA synthesis. This enzyme does not require any DNA
template and inserts ribonucleotides depending on their concentration (Fig. 11.1)
(Klug, W.S., et al. Concepts of Genetics, 10th ed. Pearson Education, California,
2012).
540 T. Banerjee

Initially, Nirenberg started synthesizing RNA with only one type of ribonucleo-
tide generating either poly U, poly A, poly C, or poly G. In all their experiments, they
made all amino acids available but radiolabeled only one amino acid per experiment.
When they labeled C14-phenylalanine, they could track its incorporation when the
RNA sequence was poly U. It proved that UUU codon codes for phenylalanine.
Using similar experiments they found AAA codes for lysine and CCC codes for
proline. Poly G could not form a functional template as it gets folded back on itself.
Next, they used RNA heteropolymers for protein synthesis. In their next
experiments, they used combinations of two different nucleotides. The relative
proportion of each ribonucleoside was known to them. Hence, they could predict
the frequency of each of the possible triplet codons. They could ascertain the
percentage of each amino acids in the resulting polypeptide. Upon analyzing the
sequence of the polypeptide, they could predict the composition of the triplet codons
(Tables 11.1 and 11.2).
Let’s assume only A and C are used for the synthesis of RNA in the ratio 1A:5C.
There is 1/6 possibility for A and 5/6 possibility for C to occupy a position. Based on
this assumption, the frequency for AAA will be (1/6)3 or about 0.4%. For AAC,
ACA, and CAA, the frequencies will be same as the number of A is occurring twice

Fig. 11.1 The reaction catalyzed by polynucleotide phosphorylase. The equilibrium favors degra-
dation of RNA, but the reaction can be “forced” towards forming RNA (Adapted from: Klug, W.S.,
et al. Concepts of Genetics, 10th ed. Pearson Education, California, 2012)

Table 11.1 Polynucleotide Phosphorylase Catalyzed Reaction

Possible Probability of occurrence of any
compositions Possible triplets triplet Final %
3A AAA (1/6)3 ¼ 1/216 ¼ 0.4% 0.4
1C:2A AAC, ACA, (5/6)(1/6)2 ¼ 5/216 ¼ 2.3% 3  2.3 ¼ 6.9
CAA
2C:1A ACC CAC (5/6)2(1/6) ¼ 25/216 ¼ 11.6% 3
CCA 11.6 ¼ 34.8
3C CCC (5/6)3 ¼ 125/216 ¼ 57.99% 57.9
Total 100
11 Protein Translation 541

Table 11.2 Mixed Copolymer Experiment

Percentage of amino acids in protein Probable base composition assignments
Lysine <1% AAA
Glutamine 2% 1C:2A
Asparagine 2% 1C:2A
Threonine 12% 2C:1A
Histidine 14% 2C:1A, 1C:2A
Proline 69% CCC, 2C:1A

Fig. 11.2 Mixed copolymer experiment. Results and interpretation of a mixed copolymer experi-
ment in which a ratio of 1A:5C is used (Adapted from: Klug, W.S. et al. Concepts of Genetics, 10th
Edition, Pearson Education, California, 2012)

and C is occurring once. The frequency will be (1/6)2(5/6) or about 2.3% for each
triplet. Similarly, each 1A and 2C will have (1/6)(5/6)2 or 11.6%. CCC will have
(5/6)3 or 57.9%. Because proline appeared 69% of the time, it could be proposed that
proline is encoded by CCC and 2C:1A (11.6%). Histidine appeared 14%, so it is
probably encoded by 2C:1A (11.6%) and 1C:2A (2.3%). Threonine at 12% is likely
to be coded by only one 2C:1A. Asparagine and glutamine each appears to be coded
by one of the 1C:2A and lysine by AAA. Together these form 100% of the amino
acids (Fig. 11.2). So, their theoretical calculation of amino acid composition and
codon assignment was found logical. However, the actual codon sequence for
heterogeneous bases could not be predicted. Subsequently, they used all four
ribonucleotides in various proportions to determine the codon usage. However, the
specific sequences remained unknown.
Har Gobind Khorana synthesized polynucleotides without a template to decipher
the genetic code in the early 1960s. He repeatedly synthesized di, tri, and tetra
nucleotide sequences and then ligated those short stretches enzymatically. This
method was later known as repeating copolymer synthesis. By this approach the
actual sequence of the codons could be deciphered. Let’s understand Khorana’s
experiment: (1) Trinucleotide made of only U and C and then joined together
(UUCUUCUUC) could be read as UUC, UCU, and CUU depending on the initiation
point. In cell-free translation system, it gave rise to polypeptide of three amino acids,
phenylalanine, serine, or leucine. So we know that three triplets codes for these three
amino acids. (2) Then synthesizing dinucleotide sequence of U and C and ligating
them together will create UCUCUC, which can be read in only two ways, UCU and
CUC, generating only leucine and serine. Therefore, from the two experiments, we
542 T. Banerjee

Fig. 11.3 Repeating copolymer synthesis. Synthesis of repeating copolymers from di-, tri, and
tetranucleotides (Adapted from: Klug, W.S. et al. Concepts of Genetics, 10th ed. Pearson Educa-
tion, California, 2012)

can conclude that UCU codon, which is common in both, must be coding for either
leucine or serine but not phenylalanine. (3) For further information tetranucleotide
sequence was created, UUAC, which produces triplets UUA, UAC, ACU, and CUU.
Three amino acids were observed to be incorporated: leucine, threonine, and tyro-
sine. CUU is the common codon in the three experiments, and leucine is the common
amino acids produced. Hence, it could be concluded that CUU codes for leucine
(Fig. 11.3) (Klug, W.S. et al. Concepts of Genetics, 10th ed. Pearson Education,
California, 2012). Therefore, Khorana’s repeating copolymer approach could deci-
pher the actual sequence of the codons. His approach could also prove the degener-
acy of the code. As we can see in the third experiment, there are four unique codons,
but only three amino acids were coded. So, two of the codons must have coded for
the same amino acid.
Another method for deciphering genetic code was developed by Leder, known as
triplet binding assay. In this process the amino acid was radioactively labeled. The
labeled amino acid was then incubated with tRNA to create charged tRNA. By that
time, codon compositions were already known although not the exact codon
sequences. Therefore, it was possible to select a few amino acids that should be
tested for each triplet. The radioactively charged tRNA, the RNA triplet, and
ribosomes were used to carry out the assay. Those were incubated on a nitrocellulose
filter (Fig. 11.4). The nitrocellulose filter retained the ribosomes due to their large
11 Protein Translation 543

Fig. 11.4 Triplet binding Assay. Using UUU triplet in the ribosome to act as a codon, it attracts
AAA anti-codon of the charged tRNAphe. If A is radiolabeled, then the incorporation of A can be
confirmed by the retention of radioactivity in the filter membrane (Adapted from: Klug,
W.S. et al. Concepts of Genetics, 10th ed. Pearson Education, California, 2012)

size. If radioactivity remained in the filter, it indicated that correct amino acid has
been incorporated, and hence the corresponding codon sequence could be predicted.

11.2 Codon: tRNA Interaction

Information stored in mRNA in the language of 4 nucleotide bases is translated into a

polypeptide made of 20 amino acids (Table 11.3). This process of protein synthesis
is therefore called as translation. In every translation process, an adaptor is required
which can understand both the languages. In protein synthesis, this adaptor is tRNA.
It can form chemical bonds with both mRNA and amino acids.
Each amino acid is carried by a particular tRNA which has a specific anti-codon
sequence. The tRNA charged with amino acid and having anti-codon complemen-
tary to the codon present in the RNA base pairs with it. The base pairing between
codon and anti-codon is also anti-parallel like that of DNA. So, if codon is 50 AUG
30 , then anti-codon will be 30 UAC 50 . However, it was observed that tRNAs bind to
several alternative codons apart from the codon to which it is complementary. These
alternative codons have a different base at the third position. The first two bases from
50 end of the codon should form perfect base pairing as per AU: GC rule. At the third
position of the codon, certain amount of flexibility was allowed (Fig. 11.5) (Griffiths,
A. J. F, Introduction to Genetic Analysis. New York, NY :W.H. Freeman &
Company, 2015). Therefore, it was hypothesized that base pairing between codon
and anti-codon at the first two bases (from 50 end in codon and 30 end in anti-codon)
is stringent which ensures accuracy of translation but less stringent at the third
position due to wobbling. Wobble is a situation where there could be more than
one spatial alignment of the base leading to lose base pairing. This was known as
wobble hypothesis (Table 11.4). Wobble hypothesis supported the degeneracy of
the genetic code where codons having same bases at the first two positions and
different bases at the third position could code for the same amino acid.
544 T. Banerjee

Table 11.3 Genetic code tabulation

Second base position
U C A G
First U UUU 1P UCU S UAU Y UGU C U Third
base UUC UCC UAC UGC C base
position UUA L UCA UAA Stop UGA Stop A position
UUG UCG UAG UGG W G
C CUU L CCU P CAU H CGU R U
CUC CCC CAC CGC C
CUA CCA CAA Q CGA A
CUG CCG CAG CGG G
A AUU I ACU T AGU N AGU S U
AUC ACC AGC AGC C
AUA ACA AGA K AGA R A
AUG M ACG AGG AGG G
G GUU V GCU A GAU D GGU G U
GUC GCC GAC GGC C
GUA GCA GAA E GGA A
GUG GCG GAG GGG G

Fig. 11.5 tRNA and codon

interaction. In the third
position (50 end) of the anti-
codon, G can take two
positions due to wobbling.
Thus, it can pair with U and
C. Therefore, single tRNA
species can recognize two
codons—UCU and
UCC (Adapted from:
Griffiths, A. J. F, Introduction
to Genetic Analysis.
New York, NY :W.H.
Freeman & Company, 2015)

Table 11.4 Codon-anti- 50 end of anti-codon 30 end of codon

codon base pairing allowed
G C or U
under wobble rule
C G only
A U only
U A or G
I U, C, or A

Therefore, 20 different tRNAs are present for carrying 20 different amino acids.
Some organisms have extra tRNAs which have different anti-codon regions but gets
charged by the same amino acids. These are called as isoaccepting tRNAs.
11 Protein Translation 545

11.3 Structure of Ribosome

The site of protein synthesis is the ribosome in both prokaryotes and eukaryotes.
These are large ribonucleoprotein complexes present in the cytosol. The complete
ribosome is made of two subunits, the larger subunit and the smaller subunit. The
ribosomal subunits are assembled together only during protein synthesis. At other
times the two subunits exist separately in cytosol. Each subunit of ribosome is
composed of several rRNA types and many proteins. Ribosomal subunits were
isolated and centrifuged in ultracentrifuge. Depending upon their sedimentation
coefficients in Svedberg (S) units which is an indication of molecular size, their
names were derived. In prokaryotes, the small subunit is 30S, and large subunit is
50S, and they associate to form 70S particle. In eukaryotes, the small subunit is 40S
and large subunit is 60S. Small and large subunits together form 80S particle.
Almost two-third of the ribosomes in both prokaryotes and eukaryotes are RNA,
and only one-third are proteins. Although there is difference in the size of ribosome
in prokaryotes and eukaryotes, the overall composition pattern and process of
translation is very similar. It shows that translation is an evolutionarily conserved
process.
In prokaryotes, the large subunit is made of 5S rRNA (120 nucleotides) and
23 SrRNA (2900) nucleotides. The small subunit is made of 16S rRNA (1540
nucleotides). The large subunit has 34 proteins, and small subunit has 21 proteins.
In eukaryotes, the large subunit is made of 5S (120 nucleotides), 5.8S
(160 nucleotides), and 28S rRNA (4700 nucleotides). The small subunit is made
of 18SrRNA (1900 nucleotides). The large subunit has 49 proteins, and small
subunit has 33 proteins (Fig. 11.6).
Ribosome provides binding site for mRNA and tRNA. The mRNA binding site
lies in the small subunit. Ribosome has three tRNA binding sites (A site for acceptor
tRNA, P site occupied by the nascent peptide, and E site occupied by the uncharged
tRNA which has transferred its amino acid to the peptide) in the ribosomes. It spans
both the ribosomal subunits. The anti-codon end is present at the small subunit, and
the amino acylated end is at the large subunit. The small subunit is responsible for
reading the genetic information, while the large subunit is responsible for peptide
bond synthesis, elongation, and protein release. Translocation is brought about by
interplay between both subunits.
The crystal structure of the 50S ribosomal subunit, with the peptidyl transferase
center (PTC) located with the help of model substrates, has been solved by Nissen
et al. in 2000. The PTC consists only of RNA which forms the cavity of PTC.
Another long cavity starts near the PTC and passes through the large ribosomal
subunit and emerges on its back side. This is the ribosomal tunnel which provides
path for the exit of nascent peptides. The inner side of the tunnel is lined with RNA
and nonglobular parts of ribosomal proteins. Proteins like L4, L22, and L39e are also
present in the inner wall of the tunnel. These walls largely consist of hydrophilic
non-charged groups. Hence, it helps the nascent peptide to pass through without
having any strong hydrophobic interactions with the globular proteins lining the
tunnel. Presence of hydrated ions, water molecules, and sugar phosphate backbone
gives negative potential to the tunnel.
546 T. Banerjee

Fig. 11.6 Subunit composition of prokaryotic and eukaryotic ribosome. The large ribosomal
subunits have been shown in light green, and small ribosomal subunits have been shown in dark
green

Crystallographic studies have revealed that the A, P, and E sites are at least 20 Å,
and perhaps as much as 50 Å, wide, thus defining the atomic distance that the tRNA
molecules must shift during each translocation event. This is considered a fairly
large distance relative to the size of the tRNAs themselves. The complete transla-
tional complex of ribosomes with associated mRNA and tRNA was crystallized, and
structure was solved at the atomic level by Ramakrishnan and Noller using
ribosomes from bacterium Thermus thermophilus (Schmeing TM, et al. The crystal
structure of the ribosome bound to EF-Tu and aminoacyl-tRNA. Science.
326, 688-694, 2009;26. Desai N, Brown A, Amunts A, Ramakrishnan V. The struc-
ture of the yeast mitochondrial ribosome. Science. 2017 Feb 3;355(6324):528-531.
27. Desai N, Yang H, Chandrasekaran V, Kazi R, Minczuk M, Ramakrishnan
V. Elongational stalling activates mitoribosome-associated quality control. Science.
2020 Nov 27;370(6520):1105-1110 ivates mitoribosome-associated quality control.
Science. 2020 Nov 27;370(6520):1105-1110). In 2009 Nobel Prize in Chemistry
was awarded to Venkatraman Ramakrishnan, Thomas Steitz, and Ada Yonath “for
studies on the structure and function of the ribosome.” The three groups deciphered
the structure of ribosomes up to a resolution of 3 Å.
Electron microscopic structure of polyribosome was observed in early 1960s
(Fig. 11.7) (Slayter, Henry S. et al. The visualization of polyribosomal structure,
7, 652-657, 1963). Recently, using a unique high-resolution approach—the tech-
nique of time-resolved single-particle cryo-electron microscopy (cryo-EM)—the
70S E. coli ribosome was captured and examined while in the process of translation
11 Protein Translation 547

Fig. 11.7 Electron

microscopic structure of
polyribosomes.
(Adapted from: Slayter, Henry
S. et al. The visualization of
polyribosomal structure. ,
7, 652-657, 1963) (a)
Translational complex of
rabbit reticulocytes
synthesizing
hemoglobin mRNA

at a resolution of 5.5 Å by Niel Fisher’s group. The study demonstrated the

trajectories followed by tRNA during the process of translocation. They also showed
the conformational changes occurring in the ribosomes during the process of trans-
lation using time-resolved single-particle cryo-electron microscopy.
Two additional regions of ribosomes which are important for protein synthesis
are the decoding center in the 30S subunit and the peptidyl transferase center in the
50S subunit. The decoding center ensures that only the correct tRNA having anti-
codon which perfectly base pairs with the codon gets accepted. This site is referred to
as A site. Peptidyl transferase center is formed by a pocket-like symmetrical
23SrRNA dimer that is composed of two RNA units which have L-shaped structure.
The symmetrical association of the two units is important for providing the proper
positioning of the two aminoacyl-tRNAs leading to appropriate stereochemistry. The
size of each symmetrical dimer is approximately the same as that of tRNA. 23S
rRNA interacts with the CCA terminus of peptidyl-tRNA in both the P site and A
site.

11.4 Structure of tRNA

The existence of an adaptor molecule was predicted by Francis Crick in 1957. tRNA
was later discovered. It is a small molecule with a stable compact structure. It is
composed of 75 to 90 nucleotides and is well conserved throughout evolution. In
1965, Robert Holley and colleagues isolated and sequenced tRNA from yeast.
Several unique nucleotides were present in tRNA like inosinic acid (contains purine
hypoxanthine), ribothymidilic acid, pseudouridine, etc. Initially it was difficult to
comprehend the presence of these unique bases. Later it was hypothesized that
presence of these bases increases the probability of hydrogen bonding and helps in
formation of compact conformation of tRNA.
Holley’s analysis of tRNA sequence led him to propose the two-dimensional
cloverleaf model of tRNA (Fig. 11.8) (Griffiths, A. J. F, Introduction to Genetic
Analysis. New York, NY :W.H. Freeman & Company, 2015). tRNA has a
characteristics secondary structure created by hydrogen bonding between base
pairs. His model showed that tRNA sequence is double stranded at certain regions
leaving single-stranded interspersed regions in between them forming a loop
548 T. Banerjee

Fig. 11.8 Structure of tRNA. (a) The schematic structure of yeast alanine tRNA. The tRNA-codon
base pairing has been shown. (b) Diagram of 3D structure of yeast phenylalanine tRNA. The
abbreviations ψ, mG, m2G, mI, and UH2 refer to pseudouridine, methylguanosine,
dimethylguanosine, methylionosine, and dihydrouridine, respectively (Adapted from: Griffiths,
A. J. F, Introduction to Genetic Analysis. New York, NY :W.H. Freeman & Company, 2015)

structure. Hence, it was called as stem-loop structure. Genetic code was already
deciphered, and hence Holley searched for complementary sequence of bases for
each codon. He observed that the complementary bases are present in one of the loop
regions. Hence, it was called anti-codon loop because codons in mRNA are read in
the 50 to 30 direction; anti-codons are oriented and written in the 30 to 50 direction.
Studies on other tRNA species revealed many common features. First, the 30 end
of tRNA molecule has CCA. At this end the amino acid is covalently joined to the
terminal adenosine residue. Second, it has four double helical stems and three single-
stranded loops forming the L-shaped cloverleaf L-shaped flattened two-dimensional
structure. Third is the presence of an anti-codon loop which base pairs with mRNA
codon. Fourth is the presence of a loop rich in dihydrouridine, known as
dihydrouridine loop (DHU). Apart from these, there is a loop whose length varies,
as is known as variable loop. Due to the variation of length of this loop, the length of
tRNA sequence varies. Later X-ray crystal structure of tRNA was solved.

11.5 Charging of tRNA

The freely available tRNA present in the cytosol needs to attach itself to the
appropriate amino acid so that it can carry the amino acid to the small ribosome
subunit and mRNA complex. The class of enzymes known as amino acyl tRNA
synthetases attaches the amino acids to the tRNA. This process is called charging of
11 Protein Translation 549

Fig. 11.9 Charging of tRNA. The tRNA synthetase enzyme has binding site for tRNA and the
amino acid. ATP is hydrolyzed into AMP and 2Pi. This energy is used to synthesize high-energy
ester bond which links the amino acid with the tRNA (Adapted from: Alberts, B. et al. Molecular
Biology of the Cell, 4th edition New York: Garland Science, 2002)

tRNA. There are 20 different aminoacyl-tRNA synthetases recognizing one amino

acid each and joining those to the compatible tRNA. The adenine residue at the 30
end has free 20 and 30 hydroxyl groups. The free amino acid is first activated by ATP,
forming adenylated amino acid by reacting with AMP and releasing two inorganic
phosphates. The adenylated amino acid is then linked with tRNA by forming high-
energy ester bond between the free 20 or 30 hydroxyl of 30 adenine of tRNA and
carboxyl group of amino acid (Alberts, B. et al. Molecular Biology of the Cell, 4th
edition New York: Garland Science, 2002). Adenylation of amino acid and the
linking of adenylated amino acid to tRNA occur as coupled reaction, and the energy
release from phosphoanhydride bond hydrolysis present in ATP is used for the
synthesis of high-energy ester bond. The energy of this bond is subsequently used
for the synthesis of peptide bonds (Fig. 11.9). The aminoacylation reaction proceeds
towards activation of the amino acid due to the hydrolysis of released pyrophos-
phate. In case the tRNA gets charged with the wrong amino acid, it is removed by the
aminoacyl tRNA synthetase enzyme. This enzyme has an amino acid-binding pocket
and an editing pocket. The amino acid-binding pocket allows only the correct amino
acid by creating a spatial constraint. Any amino acid larger or smaller than the
correct amino acid is not able to bind at the binding pocket. However, in cases wrong
amino acid of approximately the same size to that of the correct amino acid binds,
then the enzyme sends it to editing pocket. Here, it is hydrolyzed from tRNA and
released from the enzyme. Hence, these enzymes possess proofreading activity
increasing translational accuracy.
Base pairing between codon and anti-codon leads to incorporation of amino acids
in the nascent peptide chain. This base pairing is specific in the first and second
nucleotide positions of the codon (50 –30 direction). However, at the third base of the
codon, the base pairing may or may not follow Watson-Crick rule of base pairing. If
the first base of the anti-codon from 50 to 30 direction is C, the base pairing is specific.
However, if it is U or G, it may not follow Watson and Crick base pair rules. This
occurs because of a wobble in tRNA positioning. Hence, it is called as Wobble
550 T. Banerjee

hypothesis. It allows a single tRNA to bring more than one amino acid against a
single codon. Hence, it allows codon degeneracy. Therefore, only 32 tRNAs can
recognize 61 different codons.

11.6 Proofreading Activity of Protein and Its Comparison

to RNA and DNA Synthesis

During DNA and RNA synthesis, the fidelity is achieved majorly by two strategies:
correct substrate selection and removal of mismatch called proofreading. In DNA
polymerase (DNAP), the active site selects only dNTPs over rNTPs by the help of
steric gate. Side chains of two amino acids of the DNAP active site exclude the 20 OH
group and hence exclude rNTPs. For RNA polymerase (RNAP), rNTPs are selected
over dNTPs as 20 OH of rNTP interacts with RNAP active site which is important for
entering the active site. Hence, dNTPs are excluded.
In case there is a mismatch, it causes disruption in the active site of the enzyme.
The DNAP slows down, and the wrong incorporation in DNA is removed by 30 to 50
exonucleolytic activity of DNAP. In DNAP I 50 to 30 exonucleolytic activity is also
present which can remove up to ten nucleotides at a time. In RNA synthesis, if
misincorporation occurs, then mismatched nucleotide at position +1 of the RNA is
displaced away from the template. Due to this displacement, RNAP is paused.
RNAP then moves back by one position and takes the mismatched RNA nucleotide
to the proofreading site. The misincorporated nucleotide is cleaved off, and RNA
synthesis resumes.
During protein synthesis, also the fidelity is maintained based on correct selection
of charged tRNA and removal of incorrect tRNA. Both these activities are brought
about by amino-acyl tRNA synthetases (aaTS). These aaTS have two catalytic sites,
one for selection of tRNA and another for editing. Correct amino acids have the
highest affinity for the active site pocket of the synthetase, and based on the affinity
of bonding, the correct amino acid is preferred. As there are specific aaTS for every
amino acid, the active site excludes the amino acids larger than the correct one. In
case of misincorporation, aaTS forces the incorrect amino acid to the editing pocket
where it is removed by hydrolysis from AMP and released from the enzyme. This is
called hydrolytic editing (Beuning, P.J. et al. Hydrolytic editing by class II
aminoacyl-tRNA synthetase. Proc Natl Acad Sci USA. 97, 8916-20, 2000).

11.7 Process of Protein Translation

The process of translation occurs in complex dynamic and continuous process.

However, to understand the step-by-step mechanisms, we will divide the process
into three steps: initiation, elongation, and termination.
11 Protein Translation 551

11.7.1 Initiation

We will discuss the process of translation in prokaryotes. Although the basic steps of
initiation, elongation, and termination remain the same in both prokaryotes and
eukaryotes, the factors involved in them are different. Eukaryotic translation process
will be discussed in Sect. 11.8.
When the translation is not occurring, the ribosomal subunits exist separately in
the cytosol. The small subunit binds to a protein molecule known as initiation factor
3 (IF3). The small ribosomal subunit along with IF3 binds to the mRNA near its
50 end and moves along the mRNA from 50 to 30 direction searching for the start
codon AUG. The search halts at a specific sequence, AGGAGG, known as Shine-
Dalgarno sequence (Fig. 11.10) (Arakawa, K et al. Computational Genome Analy-
sis Using The G-language System. Genes, Genomes and Genomics. 21–13, 2008). It
was discovered by John Shine and Lynn Dalgarno in 1974. The exact sequence
varies slightly from species to species. This sequence is located about three to nine
base pairs upstream of the initiation codon in the 50 UTR of the mRNA. This
sequence base pairs with a pyrimidine-rich sequence, UCCUCC, present at 30 end
of the 16S rRNA of the small ribosomal subunit. This interaction between the small
ribosomal subunit rRNA and mRNA helps the small ribosomal subunit to dock on
the mRNA and form the preinitiation complex. Subsequently, the initiator tRNA
binds the start codon. The amino acid carried by the initiator tRNA in prokaryotes is
N-formylmethionine (fMet). N-formyl methionine is synthesized by the enzyme
methionine tRNA formyltransferase. An initiation factor IF2 with a GTP molecule
facilitates binding of charged initiator tRNA. Then the 50S large subunit of ribosome
is attached to the preinitiation complex with the help of IF1. The assembly of
ribosome is accomplished by the hydrolysis of a GTP molecule to GDP. The
initiation factors IF1, IF2, and IF3 leave once the ribosome assembly is complete.
This completes the formation of 70S initiation complex (Fig. 11.10). When the
ribosome assembly is completed, the A (acceptor), P (peptidyl), and E (exit) sites,
PTC and ribosome tunnel are formed in the ribosome. The initiator tRNA in the
initiation complex occupies the designated P site of the ribosome (Fig. 11.11)

Fig. 11.10 Representation of Shine-Dalgarno sequence. Sequence logo for Shine-Dalgarno

sequence in Escherichia coli. Bases present at each position are represented by stacked alphabets.
Height of each nucleotide corresponds to its contributing frequency (Adapted from: Arakawa, K
et al. Computational Genome Analysis Using The G-language System. Genes, Genomes and
Genomics. 21–13, 2008)
552 T. Banerjee

Fig. 11.11 Initiation of protein translation in prokaryotes. Ribosomes have been shown in light
blue. Shine-Dalgarno sequence has been highlighted in yellow. tRNAs have been shown in green.
The initiation process has been depicted in three steps: (1) formation of preinitiation complex,
(2) formation of 30S initiation complex, and (3) ribosome assembly (Adapted from: Russell, P.J.:
iGenetics: A molecular Approach, 3rd ed. Benjamin Cummings, New York, 2010)
11 Protein Translation 553

(Russell, P.J.: iGenetics: A molecular Approach, 3rd ed. Benjamin Cummings,

New York, 2010).

11.7.2 Elongation

Elongation phase consists of three steps:

1. Bringing the charged tRNAs to join the A site

2. Formation of peptide bond between the incoming amino acid and the most
recently added amino acid to the nascent peptide
3. Translocation of ribosome in the 30 direction

These steps require the help of several elongation factors (EFs) and energy. A
charged tRNA is bound by EF-Tu and GTP. EF-Tu is a monomeric G protein whose
active form (bound to GTP) binds aminoacyl-tRNA. The EF-Tu-GTP-aminoacyl-
tRNA trimeric complex binds to the ribosome A site. Codon-anti-codon recognition
leads to change in ribosome conformation, hydrolysis of GTP and tRNA with the
anti-codon complementary to the codon at the A site enters the A site, and GTP
linked to EF-Tu is hydrolyzed to form EF-Tu-GDP. EF-Tu-GDP complex then gets
released from the tRNA (Lewin, B. Genes VIII. 15th Edition. Upper Saddle
River, NJ: Pearson Prentice Hall, 2004). Released EF-Tu forms a complex with
another protein Ts in the cytosol. Ts releases Tu for replenishing the levels of EF-
TU-GTP (Fig. 11.12).
Then the enzyme peptidyl transferase catalyzes the peptide bond synthesis
between the amino acid of the P site and that of the A site. This leads to addition
of an amino acid to the nascent peptide chain, and the peptide chain now gets
transferred from the P site to the A site. The tRNA at the P site is no longer carrying
the peptide chain. It now occupies the E site from where the uncharged tRNA leaves
the ribosome. The 23SrRNA of the large ribosomal subunit, which forms the
peptidyl transferase center (PTC), catalyzes the peptide bond synthesis. Peptide
bond formation occurs by nucleophilic attack of the α-amino group of the
aminoacyl-tRNA on the carbonyl carbon of peptidyl-tRNA (Nissen, P., Hansen, J.,
Ban, N., Moore, P.B., Steitz, T.A.: The structural basis of ribosome activity in
peptide bond synthesis. Science. 289, 920-930, 2000). The N3 of A2451 of
23SrRNA abstracts a proton from the α-amino group, facilitating the nucleophilic
attack of the nitrogen on the carbonyl carbon of the peptidyl-tRNA. When the ester
bond of the peptidyl-tRNA is cleaved, a proton is delivered from the adjacent 20 -OH
group, which in turn receives a proton from the α-amino group of the aminoacyl-
tRNA. Due to this catalytic ability, 23SrRNA belongs to the category of ribozymes.
In the next step, the elongation factor EF-TG uses the energy released from GTP
hydrolysis to translocate the ribosome by moving it in the 30 direction on mRNA.
This translocation is exactly one codon in length. It places the ribosome over a new
codon which is not occupied by tRNA. This site becomes the new acceptor site. The
tRNA which is attached to the nascent peptide is now occupying the new P site
554 T. Banerjee

Fig. 11.12 Elongation of peptide chain during synthesis in prokaryotes. Ribosomes have been
shown in purple, tRNAs have been indicated in light blue, and nascent peptide chain has been
shown in pink. Tu and Ts proteins present in the cytosol get dissociated, and Tu reacts with
elongation factors (EF) to form EF-Tu. EF-Tu combines with GTP to form EF-Tu-GTP com-
plex (Adapted from: Lewin, B. Genes VIII. 15th Edition. Upper Saddle River, NJ: Pearson Prentice
Hall, 2004)

which was earlier A site. The earlier P site now becomes the E site (Fig. 11.13)
(Kathrin, L. et al. The Role of 23S Ribosomal RNA Residue A2451 in Peptide Bond
Synthesis Revealed by Atomic Mutagenesis, Chemistry & Biology, 15, 485-492,
2008).
The process of elongation and translocation keeps repeating over and over again.
An additional amino acid is added to the nascent peptide chain after each cycle. Once
the nascent polypeptide reaches a certain length (about 30 amino acids), it exits the
ribosome through the ribosome tunnel.

11.7.3 Termination

Termination is the final phase of translation which occurs when the acceptor site
codons are any of these three codons: UAG, UAA, or UGA. These codons do not
specify any amino acids and hence do not invite any tRNA. These are known as stop
codons, or nonsense codons. The termination codon calls upon the GTP-dependent
release factors instead of elongation factors.
11 Protein Translation 555

Fig. 11.13 Catalytic role of 23SrRNA in synthesis of peptide bond. A2451 exists as tautomer. It
can have a negative unprotonated N3 and a neutral protonated N3. (a) N3 of A2451 of 23SrRNA
abstracts a proton from the NH2 group of amino acid of acceptor tRNA. Deprotonated amine group
of amino acid present with acceptor tRNA attacks the carbonyl carbon of the peptidyl-tRNA. (b) A
protonated N3 stabilizes the tetrahedral carbon intermediate by hydrogen bonding to the oxyanion.
(c) The proton from N3 is then transferred to the peptidyl tRNA as the newly formed peptide
deacylates (Kathrin, L. et al. The Role of 23S Ribosomal RNA Residue A2451 in Peptide Bond
Synthesis Revealed by Atomic Mutagenesis, Chemistry & Biology, 15, 485-492, 2008)

In bacteria, two release factors, RF1 and RF2, recognize stop codons. RF1
recognizes UAG and UAA, and RF2 recognizes UAA and UGA. RF1/2 resembles
C-terminal domain of EF-G and tRNA. Hence, it can occupy the A site just like
tRNA. Binding of RRFs causes the PTC to hydrolyze the nascent peptide chains
from tRNA and to leave the ribosome. Subsequently, ribosome release factors
(RRFs) bind to the A site which mimics the anti-codon and acceptor end of tRNA
(Fig. 11.14). EF-G then causes translocation of the ribosome leading to disassembly
of larger and smaller subunits (Russell, P.J.: iGenetics: A molecular Approach, 3rd
ed. Benjamin Cummings, New York, 2010).

11.7.4 Polyribosome

A cell needs to constantly make proteins from hundreds to thousands of mRNA

transcripts. The process of translation must be very fast, accurate, and efficient. A
single bacterial cell might contain 20,000 ribosomes, collectively contributing to
one-fourth of the cell mass. So, a single mRNA is translated by multiple ribosomes
556 T. Banerjee

Fig. 11.14 Termination of peptide synthesis in prokaryotes. (1) Stop codon is at A site. (2) Release
factor binds. (3) Polypeptide chain is released. (4) RF3-GDP binds causing RF1 release. (5) Ribo-
some recycling factor binds to A site. (6) EF-G GTP binds to A site, causing ribosome translocation
and disassembly. (7) Ribosome disassembles, and RRF releases uncharged
tRNA (Adapted from: Russell, P.J.: iGenetics: A molecular Approach, 3rd ed. Benjamin
Cummings, New York, 2010)
11 Protein Translation 557

making multiple copies of the same proteins. Electron micrographs of actively

translating mRNA have revealed beaded necklace kind of a structure of mRNA
being translated by multiple ribosomes. That translation complex is called
polyribosomes or polysomes (Fig. 11.15). Each ribosome independently translates
the complete polypeptide. Hence, polyribosome increases the efficiency of
translation.
In bacteria, since there is no separate nucleus, both transcription and translation
occur in the cytosol. Hence, translation starts in the 50 end of mRNA which is still
being transcribed at the 30 end. Therefore, in prokaryotes, translation and transcrip-
tion are coupled process, whereas in eukaryotes, transcription occurs inside nucleus,
and only the completely synthesized RNA is exported out of nucleus and gets
translated in the cytosol.

11.7.5 Translation of Polycistronic mRNA

Each polypeptide producing gene in eukaryotes produces monocistronic mRNA,

meaning mRNA that directs the synthesis of a single kind of polypeptide. In
eukaryotes, a single start codon is identified in mRNA to initiate the synthesis of

Transcription
DNA

Ribosomes
Growing
mRNAs of
polypeptide
increasing length
chains
b Translation

Fig. 11.15 Electron micrograph of a polyribosome. (a) Multiple ribosomes translating a single
mRNA. The ribosomes closest to the stop codon have the longest polypeptide [Adapted from O. L.
Miller et al., 1970, Science; 169:392–5]. (b) Schematic rendition of the polyribosome electron
micrograph (Adapted from: Sanders, M.F., Bowman, J.L.: Genetic Analysis: An Integrated
Approach, 2nd ed. Pearson Education, New Jersey, 2015)
558 T. Banerjee

one kind of polypeptide chain. In contrast, bacterial and archaeal genes often share a
single promoter, and the resulting mRNA transcript can lead to the synthesis of
multiple polypeptide chains. In bacteria, genes participating in a single metabolic
pathway are part of a single operon, and one operon produces single polycistronic
mRNA. Each cistron contains sequence information for translation initiation. In
between the start and stop codons of the adjacent cistrons, there are intercistronic
spacer sequence that is not translated.

11.8 Prokaryotic vs Eukaryotic Protein Translation

Although the overall steps of translation are very similar in prokaryotes and
eukaryotes, there are differences in the factors involved; the sequences that act as
signals for translation initiation are different. In this section we will discuss eukary-
otic translation and how it is different from prokaryotic translation process.

11.8.1 Eukaryotic Translation Initiation

For translation initiation in prokaryotes, Shine-Dalgarno sequence acts as the

docking site for initiator tRNA, and the preinitiation complex forms there. However,
in certain prokaryotes like proteobacteria and mitochondrial genome, Shine-
Dalgarno sequence is absent. In them the small subunit of ribosomes scans the
mRNA from the 50 end, and depending upon RNA, unfoldedness and accessibility of
the initiation codon protein translation can start. If recognition site is different from
consensus recognition sequence, then it will sometimes ignore the first AUG to
produce two or more proteins differing in N terminus. This process is called as leaky
scanning. Hence, a single mRNA can lead to synthesis of different proteins having
variable length at their 50 end. Apart from that, there are structural and sequence
motifs of mRNA, which also contribute to Shine-Dalgarno independent initiation.
The size of eukaryotic ribosomes is larger than that of the prokaryotes, and the
mRNA has expansion sequences apart from the core sequence required for transla-
tion. There are other major differences in the translation process. Eukaryotic mRNA
contains a purine-rich sequence (A and G), three bases upstream from that of the
AUG initiator codon which is followed by a G (A/GNNAUGG) where N can be any
nucleotide base. This was discovered by Marilyn Kozak and hence is called Kozak
sequence (Fig. 11.16). It is analogous to the Shine-Dalgarno sequence of
prokaryotes.

11.8.1.1 50 mRNA Capping

50 end of mRNA has a 7-methylguanosine cap (7-mG). Capping occurs while
transcription is occurring. The enzymes Cet1, Ceg1, and Abd1, an RNA
50 triphosphatase, a GTP-mRNA guanylyltransferase, and an RNA guanine-7-
methyltransferase, bring about the process of capping. One of the terminal phosphate
groups is removed by RNA triphosphatase. A bisphosphate group (i.e., 50 (ppN)[pN]
11 Protein Translation 559

Fig. 11.16 Representation of Kozak sequence. A sequence logo showing the most conserved
bases around the initiation codon from 10,000 human mRNAs. Height of each nucleotide
corresponds to frequency of its occurrence. More conserved residues have more height

n) remains at the terminus. GTP is then added to mRNA by guanylyltransferase. In

this process the GTP loses the pyrophosphate. This results in the 50 –50 triphosphate
linkage, producing 50 (Gp)(ppN)[pN]n. The methylation of the 7-nitrogen of guanine
is then carried out by mRNA (guanine-N7-)-methyltransferase, leading to demeth-
ylation of S-adenosyl-L-methionine.

11.8.1.2 Circularization of mRNA During Translation

During eukaryotic translation, the 50 and 30 ends of the mRNA come together with
the help of eukaryotic initiation factors. Circularization may serve a regulatory role
to ensure translation of only intact (capped and polyadenylated) mRNAs. eIF4F
complex is composed of eIF4E, eIF4A, and eIF4G proteins. This eIF4F binds to the
50 -cap of an mRNA and prepares an mRNA for recruitment of a 40S subunit. The
small subunit of ribosome binds at the 50 UTR. The eukaryotic initiation factor
(eIF2) along with GTP brings the methionine charged initiator tRNA to the 50 cap.
Small subunit along with eIF2-GTP, mettRNA, eIF1, eIF1A, eIF5B with bound GTP,
eIF3 large subunit, and 40S ribosomal subunit makes 43S preinitiation complex.
eIF4B promotes the ATP-dependent RNA helicase activity of eIF4A, and eIF4F is
needed to unwind secondary structure present in a 50 -UTR that would otherwise
impede scanning of the 40S subunit during initiation. 43S preinitiation complex
starts scanning along 50 to 30 direction in search of start codon. The scanning process
is dependent on ATP-powered helicases but independent of mRNA secondary
structure or sequence. 43S complex is recruited to the Kozak sequence by eIF4F.
The poly(A) binding protein (PABP), which binds the poly(A) tail, interacts with
eIF4G and eIF4B which are present at the 50 cap. This leads to circularization of
mRNA (Gallie, D.R.: The role of polyA binding protein in the assembly of cap
binding complex during translation initiation in plants. Translation (Austin).
2, e959378, 2014). When 43S complex binds the mRNA at the start site, it forms
the 48S preinitiation complex. IF2 and eIF5B are structurally similar GTPases that
catalyze joining of the large subunit to the preinitiation complex.
There are certain eukaryotic mRNAs which are not capped like plus-strand RNA
virus mRNAs (poliovirus, human rhinovirus, and hepatitis A and hepatitis C
viruses); growth factors VEGF, FGF2, and PDGF; transcription factors c-myc;
560 T. Banerjee

apoptosis-associated gene products Apaf-1; the potassium channel Kv1.4; etc. In

addition, many plant messengers are uncapped. Their 50 UTRs are mostly 200 bases
long or have more than one AUG codon. They are translated by cap-independent
internal initiation. The ribosome binds to an internal RNA sequence near the
initiator AUG. Such sequences are known as the internal ribosome entry sites
(IRES). IRES structure has conserved structural motifs but less conserved sequence
motifs. IRES have been categorized into different types based on their requirement.
Type I requires eIF4G and associated factors, Type II requires eIF2 and associated
factors but not eIF4G, Type III doesn’t require either eIF2 or GTP, and Type IV
seems to interact directly with rRNA. For example, hepatitis C virus can bind to
mRNA at an IRES without any initiation factors. Another major difference is that
eukaryotes have methionine as initiator amino acid and not formylated methionine.
The eukaryotic mRNA has stretches of multiple adenine residues at its 30 end, known
as poly(A) tail which is unique to eukaryotes.
Archaeal translation shares features of both eukaryotic and prokaryotic transla-
tion with certain differences from both the systems. Archaeal ribosomal subunit
composition is similar to that of bacteria than of eukaryotes. The process of transla-
tion initiation is similar to that of eukaryotes. It uses methionine as first amino acid
and not N-formylated methionine. Unlike bacteria, most of archaeal mRNA is
leaderless, lacking 50 UTR and lacking Shine-Dalgarno sequence. The process of
mRNA start site recognition in leaderless sequences is not yet known. Both bacteria
and eukaryotes have 50 UTR unlike most archaea. Therefore, it is hypothesized
based on observations, and in vitro experiments that last common ancestor (LUCA)
of bacteria, archaea, and eukaryotes had leaderless sequence lacking 50 UTR. 50 UTR
is a more recent development. Though in a few archaeal species Shine-Dalgarno
sequence have been found. Archaeal initiation factors (aIFs) are homologous in
structure and function to eukaryotic initiation factors (eIFs).

11.8.1.3 Eukaryotic Translation Elongation

The biochemistry of polypeptide elongation is highly conserved in all three domains
of life. They all use two elongation factors: one for bringing the charged tRNA to the
acceptor site and the other for translocation of ribosome to the 30 direction. The
names of archaeal and eukaryotic homologs of EF-Tu and EF-G present in bacteria
are given in the table below (Table 11.5). Based on sequence comparison, archaeal
elongation factors are more similar to that of eukaryotes; hence eukaryotes are
evolutionary closer to archaea, as proposed by Carl Woese. Elongation rate in
bacteria is 20 amino acids per second, and in eukaryotes, it is 15 amino acids per
second.

Table 11.5 Translation elongation factor homologs

Function Bacterial homolog Archaeal homolog Eukaryotic homolog
Adjusts tRNA in A site EFT aEF1 eEF1
Promotes translocation EFG aEF2 eEF2
11 Protein Translation 561

11.8.1.4 Eukaryotic Translation Termination

The elongation cycle continues until one of the three stop codons UAA, UAG, or
UGA is encountered at the A site. Instead of charged tRNA, the release factors
(RF) bind and catalyze the release of polypeptide bound to tRNA at the P site. The
eukaryotic release factor, eRF3, is a GTP-binding protein. The eRF3-GTP acts in
concert with eRF1 to promote cleavage of the peptidyl-tRNA, thus releasing the
completed protein. The uncharged tRNA now occupies the P site. After the release of
polypeptide, ribosome release factors bind and cause translocation of ribosome. As
there is no peptide, the tRNA occupying the earlier P site now exits the ribosome
from the E site, and ribosome disassembles.

11.9 Regulation of Protein Translation

Translation process is majorly regulation at the initiation step by various

mechanisms which we are going to discuss in this section. The rate of assembly
of multifactor preinitiation complex and its interaction with eIF4F complex
interacting with the mRNA cap form the first step in the regulation of translation
initiation (Sonenberg, N., Hinnebusch, A.G.: Regulation of translation initiation in
eukaryotes: mechanisms and biological targets. Cell. 136, 731-745, 2009). The
rate of selection of the start codon by the scanning mechanism is the next step
which can regulate the process of eukaryotic translation. The scanning process is
in turn dependent upon the activity of helicases. One of the principal mechanisms
of translation control is by the phosphorylation of eIF2α on Ser51. There are four
different eIF2α kinases in mammals activated by different stresses, PKR (double-
stranded RNA in virus infection), PERK (unfolded proteins in the ER), HRI (heme
deprivation), and GCN2 (amino acid starvation), that phosphorylate the same
residue in eIF2α. eIF2α-GTP brings initiator mettRNA. Once tRNA base pairs
with mRNA, eIF2α-GTP is hydrolyzed to eIF2α-GDP which then leaves the PIC.
The guanine exchange factor eIF2B then removes GDP and replenishes eIF2-
α-GTP for the next round of initiation. However, during cellular stress, the eIF2
kinases phosphorylate eIF2α which remain tightly bound to eIF2B. Hence, eIF2B
is not able to exchange GDP to GTP. eIF2α-GTP is not replenished, delivery of
imettRNA is stopped, and hence translation initiation stops. Repression of transla-
tion initiation is an efficient mechanism to conserve energy and nutrients during
stress.
Recently it has been shown that during stress conditions when the cells need to
produce specific proteins related to stress management and reduce the production of
other proteins that are involved in cell growth and proliferation, the abundance of
tRNA pool varies. The tRNA pool gets skewed towards those which can recognize
the rare codons. Thus, tRNA pool adjustment is an important mechanism for
regulating the translation of stress-related proteins.
Another important mechanism for translation regulation is by controlling the
50 7mGcap. Binding of eIF4F can be hindered by eIF4E homolog, 4E-HP. The
562 T. Banerjee

interaction between eIF4G and eIF4E in the eIF4F complex is hindered by eIF4E-
binding proteins (4E-BPs). Hence, eIF4E-BPs can inhibit cap-dependent translation.
miRNAs (approximately 22 nt long oligonucleotides) are another significant factor
for controlling protein synthesis. miRNA can destabilize mRNA or inhibit transla-
tion by being a part of protein complex known as RNA-induced silencing complex
(RISC). miRNA-loaded RISC base pairs with complementary sites located at the 30
UTR of many mRNA, regulating their translation.

11.9.1 Translation Pause

During translation, it is observed that at certain points of translation, complex

ribosomes are close together as if they halted. This process is called as ribosome
stalling which leads to a pause in the process of translation. This process is known to
occur in both prokaryotes and eukaryotes. The stalling process is required for
ensuring the fidelity of translation process. Hence, it is a part of ribosome-associated
quality control process. Ribosome stalling promotes ribosome disassembly and
releases the mRNA for degradation.

Box 11.1 Scientific Concept: Protein Synthesis by Single Ribosomes:

Francesco Vanzi et al.
We have learned that the energy required for peptide bond comes from the
high-energy ester bond which links the amino acid to the 30 adenine residue of
tRNA. Energy required for ribosome translocation, while maintaining the
fidelity of amino acid selection and the correct reading frame, comes from
the GTPase activities of two G-protein elongation factors, EF-Tu and EF-G.
To understand the production of force and displacement of ribosome, single-
molecule technique was needed.
In 1999, Sytnik et al. showed that ribosomes adsorbed on the mica surface
are able to bind amino-acyl-tRNA at the peptidyl transfer site (P site) of the
ribosome. Francesco et al. extended their work by measuring
polyphenylalanine polypeptide synthesis on poly(U)-programmed mica-
bound ribosomes. Ribosomes were adsorbed on mica surface at different
concentrations of 70S ribosome. Surface images of ribosomes adsorbed on
mica were obtained by tapping mode atomic force microscopy. Synthesis of
poly(Phe) was observed to be dependent on elongation factor and temperature
but not on the concentration of charged tRNA.
Changes in the range of motion of 30 end of mRNA was done by micro-
scopic techniques called tethered particle motion (TPM). Ribosomes (1 μM),
N-Ac[14C]-Phe-tRNAPhe (1 μM), and biotinylated long-chain poly
(U) (2.6 μg/μL) were preincubated at 30  C for 15 min before being applied
to a mica flow chamber (10–15 μL) for 5 min. The chamber was then washed,

(continued)
11 Protein Translation 563

Box 11.1 (continued)

and blocking solution (15 μL) was added. Thereafter, neutravidin-labeled
fluorescent beads were added and incubated for 30–60 min. The 30 biotinylated
ends of mRNA were then fluorescently labeled by neutravidin fluorospheres
(Fig. 11.17). After the removal of excess beads by washing, three different
groups of beads were visualized by epi-fluorescence with an inverted micro-
scope: one freely diffusing under Brownian motion; second, immobilized
beads; and third group with diffusive motion with restricted volume (i.e., the
root means squared horizontal displacement from the average position of the
bead, Drms >100 nm). The restricted Brownian motion was around a central
nodal point, mostly in a radial pattern. They observed a time-dependent
reduction in the Drms (Vanzi, F. et al. Protein synthesis by single ribosomes.
RNA. 9, 1174-1179, 2003).
TPM data pooled from six beads displaying a reduction of Drms with time
and applying the physical properties of poly(U) led to calculation of peptide
synthesis rate by a single ribosome.

Fig. 11.17 Principle of the tethered particle method. (a) A microsphere (shown in pink) is attached
to the 30 end of immobilized ribosome bound mRNA molecule (curvy black line). End-to-end
length of the tether is shown as a black dotted line. Nascent peptide is shown in orange. The
microsphere can diffuse only in the radial area marked in green. (b) As peptide synthesis proceeds,
the ribosome pulls the 30 end of the mRNA towards itself, reducing the range of restricted diffusion
564 T. Banerjee

Box 11.2 Scientific Concept: Errors in Protein Synthesis Increase

the Level of Saturated Fatty Acids and Affect the Overall Lipid Profiles
of Yeast: Ana Rita D et al.
Protein synthesis is a highly accurate mechanism like DNA replication and
transcription. Under normal conditions, the error rate of amino acid
misincorporation is only 10
3 to 10
4. Cell has its protein quality control
mechanism like molecular chaperones, proteasomes, autophagy, etc. which
ensures the normal functioning of the cell. However, there exist certain
mutations which lead to increase in error rate causing significant deleterious
effect on the cell.
In yeast, codon misreading decreases growth rate, alters cell morphology,
increases reactive oxygen species, and causes deregulation of gene expression,
proteotoxic stress, etc. Lipids play significant role in cell physiology by
controlling membrane fluidity and participating in signaling cascades and
energy metabolism. They are also important for protein functions and
protein-protein interactions. Therefore, it is important to understand that errors
in protein synthesis can have significant effect on lipid profiles and metabo-
lism. Ana Rita D et al. constructed yeast strains to express a recombinant
Ser-tRNA that decodes Ala and Gly codons (Araújo, A.R.D. et al. Errors in
protein synthesis increase the level of saturated fatty acids and affect the
overall lipid profiles of yeast. Plos One. 13, e0202402, 2018). The profiles
of phospholipid (PL), triglyceride (TG), and fatty acids (FA) were then
evaluated by various techniques. Fatty acid profiles were assessed by
analyzing fatty acid methyl ester by gas chromatography. The analysis showed
reduction in unsaturated fatty acids and increase in saturated fatty acids. PL
classes were separated by thin layer chromatography (TLC). Phosphatidylcho-
line was the most abundant PL in the growth phase. The profile of triglycerides
was analyzed by LC-MS (liquid chromatography-mass spectrometry). The
most abundant TG species had either three C16 or two C16 and one C18
fatty acyl chains. The mechanism by which amino acid misincorporation leads
to changes in lipid profile remains to be understood.

Box 11.3 Scientific Concept: Protein Synthesis by Single Ribosomes:

Francesco Vanzi et al.
We have learned that the energy required for peptide bond comes from the
high-energy ester bond which links the amino acid to the 30 adenine residue of
tRNA. Energy required for ribosome translocation, while maintaining the
fidelity of amino acid selection and the correct reading frame, comes from
the GTPase activities of two G-protein elongation factors, EF-Tu and EF-G.

(continued)
11 Protein Translation 565

Box 11.3 (continued)

To understand the production of force and displacement of ribosome, single-
molecule technique was needed.
In 1999, Sytnik et al. showed that ribosomes adsorbed on the mica surface
are able to bind amino-acyl-tRNA at the peptidyl transfer site (P site) of the
ribosome. Francesco et al. extended their work by measuring
polyphenylalanine polypeptide synthesis on poly(U)-programmed mica-
bound ribosomes. Ribosomes were adsorbed on mica surface at different
concentrations of 70S ribosome. Surface images of ribosomes adsorbed on
mica were obtained by tapping mode atomic force microscopy. Synthesis of
poly(Phe) was observed to be dependent on elongation factor and temperature
but not on the concentration of charged tRNA.
Changes in the range of motion of 30 end of mRNA was done by micro-
scopic techniques called tethered particle motion (TPM). Ribosomes (1 μM),
N-Ac[14C]-Phe-tRNAPhe (1 μM), and biotinylated long-chain poly
(U) (2.6 μg/μL) were preincubated at 30  C for 15 min before being applied
to a mica flow chamber (10–15 μL) for 5 min. The chamber was then washed,
and blocking solution (15 μL) was added. Thereafter, neutravidin-labeled
fluorescent beads were added and incubated for 30–60 min. The 30 biotinylated
ends of mRNA were then fluorescently labeled by neutravidin fluorospheres.
After the removal of excess beads by washing, three different groups of beads
were visualized by epi-fluorescence with an inverted microscope: one freely
diffusing under Brownian motion; second, immobilized beads; and third group
with diffusive motion with restricted volume (i.e., the root means squared
horizontal displacement from the average position of the bead, Drms
>100 nm). The restricted Brownian motion was around a central nodal
point, mostly in a radial pattern. They observed a time-dependent reduction
in the Drms.
TPM data pooled from six beads displaying a reduction of Drms with time
and applying the physical properties of poly(U) led to calculation of peptide
synthesis rate by a single ribosome.

11.10 Summary

• Genetic code was deciphered by Nirenberg, Khorana, and Holley. Har Gobind
Khorana synthesized polynucleotides without a template to decipher the
genetic code.
• Genetic code is triplet code formed by three nucleotides specifying an amino acid.
Genetic code is universal, degenerate that is one amino acid can be coded by more
than one codon, unambiguous that is one codon can code for only one amino acid,
and contiguous as there are no gaps and no overlapping.
566 T. Banerjee

• Triplet codon gives 64 combinations (43) to be recognized by 20tRNA, coding for

20 amino acids. The reading frame is marked by an initiation codon that is either
AUG or GUG. There are three stop codons: UAA, UAG, and UGA.
• Protein synthesis comprises of four steps: (1) binding of amino acids by appro-
priate tRNAs, (2) initiation, (3) elongation, and (4) termination.
• Amino acyl tRNA synthetase enzymes bring about charging of tRNA with
appropriate amino acids. Charged tRNA interacts with the codon of the mRNA
with the help of its anti-codon loop. This binding allows flexibility at 30 end of
codon which is called wobble hypothesis. Wobble rule allows one tRNA to
recognize more than one codon.
• Protein synthesis occurs in the ribosomes; ribosomes have two subunits: small
and large. Prokaryotes have 70S ribosome, and eukaryotes have 80S ribosome.
• Amino acid is linked to the 30 end adenine by high-energy ester bond. The high-
energy ester bond is used for peptide bond synthesis.
• Process of translation is divided into three steps: initiation, elongation, and
termination. In bacterial translation initiation, the small subunit of ribosome
gets positioned over initiation codon. The first tRNA brings in N-
formylmethionine in bacterial cells, and subsequently the large subunit of ribo-
some binds with the help of initiation factors and GTP.
• In prokaryotes, Shine-Dalgarno sequence is recognized by the small ribosome
subunit during scanning, and in eukaryotes, mRNA has Kozak sequence which is
recognized by small ribosomal subunit. Some eukaryotic mRNAs lack consensus
initiation sequence but have internal ribosome entry site.
• 7-methylguanosine cap (7-mG) is present in eukaryotic mRNA. Circularization of
mRNA occurs during translation in eukaryotes.
• Elongation step is dependent on two GTP-bound translation elongation factors.
During elongation, charged tRNA enters A site, and peptide bond is formed
between amino acid at A site and P site. The ribosome moves to the next
codon, and earlier P site becomes the E site; A site becomes the new P site.
• Termination occurs when either of the three stop codons occupies the A site. The
stop codons are recognized by release factors, and ribosome is disassembled by
ribosome release factors.
• Translation is tightly regulated in the initiation step by phosphorylation of eIF2α
by eIF2α kinase.

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Jurado AS, Costa V, Domingues P, Domingues MRM, Santos MAS, Witt SN (2018) Errors in
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Regulation of Gene Expression
in Prokaryotes 12
Tanushree Banerjee

We have learned how genetic information is encoded in DNA and how it is

organized into genes. We have also learned that the information encoded in genes
gets translated into proteins. We must understand that not all proteins are required at
equal quantity at all times. Some are required more, and some are required less in
amount. Depending upon environmental conditions, stress factors, growth phase,
and the requirement of a cell keep changing. Therefore, the protein synthesis rates
also keep changing. The process of producing gene products is called as gene
expression, and it is a tightly regulated process so that it can be modified depending
upon the requirement of the cell. In this chapter, we will learn about the mechanism
of gene regulation specifically in prokaryotes.
Bacteria serves as an important model organism for understanding gene regula-
tion. It has short life cycle, multiple generations can be studied easily over a short
span of time, and hence it is easy to identify mutants. Therefore, we now discuss the
changes in gene expression in response to environmental changes.

12.1 Concept of Gene Regulation

Escherichia coli is an excellent model organism for studying gene regulation. They
can switch on and switch off expression of certain genes depending upon environ-
ment or phase of life cycle like gene replication, cell division, etc.
It was observed that bacteria synthesize lactose-metabolizing enzymes only when
lactose was present in the medium. These enzymes were therefore called as adaptive
or facultative enzymes. Later, that terminology was replaced with inducible enzyme

T. Banerjee (*)
Molecular Neuroscience Research Laboratory, Dr. D. Y. Patil Biotechnology and Bioinformatics
Institute, Dr. D. Y. Patil Vidyapeeth, Pune, India
e-mail: tanushree.banerjee@dpu.edu.in

# The Author(s), under exclusive license to Springer Nature Singapore Pte 569
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_12
570 T. Banerjee

as the production gets induced only when the inducer-like lactose is present. The
pathway is then said to be inducible. The other enzymes which are always present
are called constitutive enzymes.
A contrasting system also exists, where the presence of gene product inhibits the
gene expression. Tryptophan is an amino acid which gets synthesized by the cell. If
tryptophan is present in sufficient amount, the cell does not need to synthesize it
anymore. Therefore, it inhibits the anabolic pathway which leads to tryptophan
synthesis. Therefore, the end product of the pathway tryptophan acts as a repressor.
Therefore, the pathway is then said to be repressible.
Inducible and repressible pathways can be controlled by positive and negative
regulation. In positive regulation mode, the gene expression continues when a
regulator molecule directly stimulates RNA production. In this case, the regulator
molecule is called as activator. In negative regulation mode, the RNA production
continues as default unless it is being shut off by a regulator molecule. That regulator
molecule is called as repressor. Sometimes, another small molecule binds to the
repressor and enables it to bind to DNA causing repression of gene expression
(Fig. 12.1). This small molecule is called as corepressor. Other mechanism for
gene repression is an inhibitor binding to an activator so that positive regulation
by activator does not occur. Both corepressor and inhibitor lead to gene repression.
Therefore, it can be said that negative regulation is important where default pathway
of expression is “on” and positive regulation occurs where default pathway is “off.”
Both inducible and repressible systems could be controlled by the combination of
positive and negative control. Both lactose metabolism pathway and tryptophan
synthesis pathway are under negative control mechanism.

12.2 Concept of Operon

It was at the turn of the twentieth century that Francois Jacob and Jaques Monod
gave the concept of adaptive enzymes. Regulation of the lac operon was first
described by François Jacob and Jacques Monod. Their research demonstrated
how enzyme quantities can be controlled directly at the level of transcription.
They hypothesized that certain metabolic enzymes are expressed only when cell is
exposed to their respective substrate. When a bacterium does not have the substrate,
it does not need to synthesize the enzyme which metabolizes it. They further
investigated this hypothesis by studying lactose metabolism in E. coli. They
observed that presence of lactose increased the enzymatic activity of the lactose-
metabolizing enzymes by 1000–10,000 times. They also observed that lactose-
metabolizing enzyme activity increased as there was enhancement in the expression
of the enzyme. Removal of lactose leads to immediate lowering in the gene expres-
sion of the entire lactose metabolism pathway. Hence, it became evident that all the
genes involved in lactose metabolism are being controlled for transcription together.
The group of two or more genes which get transcribed from a single promoter are
called as operon. Therefore, operon could be called as a DNA which gets transcribed
by a single promoter synthesizing polycistronic mRNA (two or more genes). An
12 Regulation of Gene Expression in Prokaryotes 571

Fig. 12.1 Binding sites on a genetic regulatory protein. In these examples, a regulatory protein has
two binding sites: one for DNA and the other for a small effector molecule. The conformational
changes in the regulatory protein are brought about by the binding of small effector molecule,
leading to changes in its DNA-binding site. a) Regulation of operon by repressor in presence and
absence of inducer b) Regulation of operon by activator in presence and absence of inducer
c) Regulation of operon by repressor in presence and absence of co-repressor d) Regulation of
operon by activator in presence and absence of inhibitor (Adapted from: Brooker, R.J.: Genetics:
Analysis and Principle, 6th ed. pp 336–360. McGraw-Hill Education, New York, 2018)

operon is flanked by a promoter for initiating mRNA synthesis and a terminator

indicating end of mRNA synthesis (Fig. 12.2).
572 T. Banerjee

Fig. 12.2 Overview of gene expression in bacteria. The binding of regulatory proteins can either
activate or block transcription (Adapted from: Griffiths, A. J. F. Introduction to Genetic Analysis.
New York, NY :W.H. Freeman & Company, 2015)

Fig. 12.3 Structural organization of lac operon. Regulatory lacI genes has its own lacI promoter.
Lac operon has CAP site (purple), lac promoter (lacP, light orange), lac operator (lacO, green),
structural genes (lacZYA, blue), and lac terminator (gray) (Adapted from: Brooker, R.J.: Genetics:
Analysis and Principle, 6th ed. pp 336–360. McGraw-Hill Education, New York, 2018)

12.3 Metabolism of Lactose in E. coli

Lactose-metabolizing enzymes are present in two distinct transcriptional units: lac

operon and lacI. lacI is not the part of lac operon. First, we will discuss about the lac
operon. It has a catabolite activator protein (CAP) site, promoter (lacP), operator
(lacO), and three protein-encoding genes lacZ, lacY, and lacA. lacZ encodes for
β-galactosidase (Fig. 12.3). This enzyme cleaves lactose into glucose and galactose.
lacY encodes for lactose permease. This protein helps in active uptake of lactose.
lacA encodes galactoside transacetylase which modifies lactose and other lactose
analogues by attaching hydrophobic acetyl groups. Attachment of acetyl group helps
lactose to diffuse out of the cell, preventing toxicity caused by excessive lactose.
CAP site is regulatory DNA sequence present in lac operon. Catabolite activator
protein (CAP) binds to this site, hence called CAP site. Operator site is another DNA
regulatory element where the lac repressor binds (Beckwith 1967).
lacI has its own promoter which is constitutively expressed at a low level. It
encodes for lac repressor which binds to the operator of the lac operon. It is a
homotetrameric protein which is required in small amounts to repress lac operon
(Fig. 12.4). Negative supercoiling of DNA enhances lacI binding to the operator;
12 Regulation of Gene Expression in Prokaryotes 573

Fig. 12.4 LacI binds to two operator sequences in tetrameric form leading to looping of DNA.
There are two dimeric lacI functional subunits (red+blue and green+orange). Each subunit binds to
two DNA operator sequences (labeled). Tetrameric lacI thus induces DNA looping (Rutkauskas D.
et al. Proc Natl Acad Sci U S A. 106, 16627–16632, 2009)

hence, it is known to act as a topological barrier which is superhelically modulated to

control the expression of lacZYA under various growth conditions.

12.3.1 Lactose Uptake in E. coli

Lactose is taken up by lactose permease. It is encoded by lacY gene. It is a

transmembrane protein which actively takes up lactose from the environment. It
cotransports lactose with H+. Since bacteria maintains a high proton gradient across
its membrane, it actively accumulates lactose in the cytosol against its concentration
gradient by utilizing the free energy released from downhill translocation of H+.
LacY also catalyzes the converse reaction, utilizing free energy released from
downhill translocation of sugar to drive uphill translocation of H+. It has 12 helices
that forms a hydrophilic cavity creating a single binding site that is alternatively
574 T. Banerjee

Fig. 12.5 A possible lactose/H+ symport mechanism. The key residues involved in changing the
symporter conformation are indicated. H-bonds are shown in black dotted lines. The protons are
shown in red, and the substrates are shown in green (Kaback, H.R. C R Biol. 328, 557–67, 2005)

Table 12.1 lac operon genes and regulatory sequences

accessible to either side of the membrane. Binding of substrate leads to major

conformational shift in the enzyme known as inward and outward conformations.
Influx consists of six steps: starting from the outward-facing conformation (A),
protonation of LacY (B), binding of lactose (C), a conformational change that results
in the inward conformation (D), release of substrate (E), release of the H+ (F), and
return to the outward conformation (Fig. 12.5).
12 Regulation of Gene Expression in Prokaryotes 575

Fig. 12.6 Schematic summarizing of the roles of β-galactosidase in the cell. The enzyme
β-galactosidase can cause hydrolysis of lactose to galactose plus glucose and transgalactosylate
lactose to form allolactose, and it can hydrolyze allolactose. The presence of lactose results in the
synthesis of allolactose which binds to the lac repressor and reduces its affinity for the lac operon.
This in turn allows the synthesis of β-galactosidase, the product of the lacZ gene (Adapted from:
Juers, D.H. et al. Protein Sci. 21, 1792–807, 2012)

12.3.2 Structural Gene

There are three structural genes present in the lac operon, lacZ, lacY, and lacA. lacZ
codes for the enzyme β-galactosidase (Table 12.1). β-galactosidase is a tetramer of
four identical polypeptide chains, each of 1023 amino acids. This enzyme has three
catalytic activity: It can cleave the disaccharide lactose to form glucose and
576 T. Banerjee

galactose, which can then enter glycolysis; the enzyme can catalyze the
transgalactosylation of lactose to allolactose, and the allolactose can be cleaved to
the monosaccharides. It is allolactose that binds to lacZ repressor and creates the
positive feedback loop that regulates the amount of β-galactosidase in the cell. It
hydrolyzes X-gal (5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside), a soluble
colorless compound. X-Gal consists of galactose linked to a substituted indole
(Fig. 12.6). Its hydrolysis releases the substituted indole. Indole then spontaneously
dimerizes to give an insoluble, intensely blue product.
lacY gene codes for an integral cytoplasmic membrane protein, lactose permease.
It belongs to a conserved transporter family known as Major Facilitator Superfamily.
LacY protein is made of 417 amino acid residues. It has 12 helices. It acts as
symporter of H+ and lactose. Helices are connected with hydrophilic loops with
both N and C termini facing the cytosol.
lacA gene codes for galactoside transacetylase. It acetylates lactose, and
acetylated lactose can diffuse out of the cell membrane reducing lactose-induced
toxicity. Unlike β-galactosidase and lactose permease, the function of galactoside
transacetylase remains debatable. The action of acetylation of lactose was extended
to other galactoside molecules like isopropyl-1-thio-βD-galactoside (IPTG), with the
identification of 6-O acetyl-IPTG as the chemically altered compound. However,
acetylated-IPTG did not revert to the original IPTG and did not act as an inducer of
the lac operon or as a substrate of lactose permease.

12.3.3 Regulatory Mutation

In the 1950s, Jacob, Monod, and Pardee identified rare mutant strains of bacteria
which had abnormal lactose metabolism. One type of mutant-designated lacI

resulted in the constitutive expression of lac operon. Even when the lactose was
absent, lac operon in these mutants continued to express. The exact mode of action of
lac repressor was not known at that time. It was thought to produce an activator for
the operon which kept it transcriptionally active throughout. To understand the
nature of this mutation, they applied a genetic approach in 1961. It involved bacterial
mating by conjugation. The circular segments of DNA are called F factors. Some-
times, the F factors carry the genes that were originally present in the chromosome.
Then they are called as F0 factors. They identified F0 factors which carried the lacI
gene and lac operon. These F0 factors can get transferred from one cell to another by
conjugation. The strain of bacteria containing F0 factors are called as merozygote or
partial diploid.
Mutations in Regulator Gene: Lac repressor encoded by the lacI gene has two
binding sites: one for binding the DNA at the operator site and other for binding
allolactose. There are lacI
mutants which either fail to produce lac repressor or the
repressor is unable to bind to the operator site. Hence, the lac operon remains
constitutively active both in presence and absence of lactose. There are other
regulatory mutations known for the lacI gene. lacIs mutation produces repressor
that cannot bind allolactose. Hence, it always remains bound to the operator
12 Regulation of Gene Expression in Prokaryotes 577

Table 12.2 Synthesis of beta-galactosidase and permease by haploids and partial diploids with
regulatory gene mutation

Table 12.3 Synthesis of beta-galactosidase and permease by haploids and partial diploids with
structural gene mutation

(Table 12.2). Even if lactose is present, the operon cannot be induced creating a
super-repressed state.
Mutation in Structural Genes: Mutant strains which had lost the ability to
synthesize β-galactosidase or permease were identified and were mapped to lacZ
and lacY structural genes, respectively. These mutations led to changes in the amino
acid sequence and structure of these proteins which caused loss of function in most
cases. From the study of partial diploids, they could infer that lacZ and lacy
mutations are independent of each other. Partial diploids with lacZ + lacY- on
plasmid and lacZ-lacY+ on bacterial chromosome produced normal enzymes
(Table 12.3). Hence, they could conclude that single functional gene either in
chromosome or in plasmid is capable of producing normal lac operon
phenotype (Juers et al. 2012).
Mutation at DNA-Binding Site: Jacob and Monod could identify mutants
having defective promoter regions. Those were called lacP- mutations. RNA poly-
merase is not able to bind to the defective promoter. These mutant strains don’t
produce any protein of the lac operon either in the absence or presence of lactose.
lacP- mutations are cis acting and hence inhibit the synthesis of genes present in the
578 T. Banerjee

same DNA molecule. Therefore, lacI+lacP-lacZ+/lacI+lacP+lacZ- will not produce

functional β-galactosidase.
Mutations in the Operator Site: They identified certain mutations where
β-galactosidase was produced even in the absence of lactose. The mutation was
mapped to the operator region. It was called constitutive operator or lacOc. Such
mutation doesn’t allow the binding of repressor. Hence, RNA polymerase can
constitutively bind to the promoter, and transcription continues even in absence of
lactose. A partial diploid lacI + lacOclacZ+/lacI + lacO + lacZ+ shows constitutive
production of β-galactosidase. It showed that constitutive mutation lacOc is domi-
nant over lacO+. lacI + lacO + lacZ+/lacI + lacOclacZ- did not show constitutive
β-galactosidase production. Therefore, the operator mutations affect genes which are
present on the same molecules.

12.3.4 Positive and Negative Control

In positive control mode, the presence of an activator directly stimulates the operon
transcription. For lac operon, presence of lactose acts as an activator, and hence, it is
a positive regulator of the operon. Lactose gets converted to allolactose by
β-galactosidase. Each of the four subunits of the lac repressor has single bonding
site for allolactose. Allolactose then binds to the repressor, induces a major confor-
mational change in the repressor, and reduces the affinity of the repressor for the
operator. Hence, when allolactose is present, the repressor can no longer bind to the
operator site, and the operon is actively transcribed. The action of a small effector
molecule such as allolactose is called as allosteric regulation. Repressor molecule
acts as the allosteric protein, and the binding sites for allosteric molecule is called
allosteric site.
In negative control, the operon stays in actively transcribing mode unless bound
by the repressor. For lac operon, repressor is transcribed by lacI gene in a separate
transcriptional unit. This repressor binds to the operator site and keeps the operon in
transcriptionally inactive state (off state). When the bacteria are not having any
lactose in its system, it does not need to synthesize the lactose metabolizing
enzymes. Hence, the operon is kept switched off by the repressor. Binding of
repressor to the lac operator to keep it in default off state is considered to be the
negative control of the operon.

12.3.5 Genetic Evidence for Operon Model

Generation of merozygotes described in the previous section could lead to identifi-

cation of lacI gene function. There are two main points in their experiment: First, two
lacI genes may be different alleles. For example, the chromosomal DNA might be
carrying the mutant lacI
allele, and the F factor might carry the normal lacI allele.
Second, the genes on F factor and chromosomal DNA are not physically adjacent to
each other. Their group used the lacI
mutant strain which constitutively expresses
12 Regulation of Gene Expression in Prokaryotes 579

lac operon and compared it with various merozygotes. The merozygote had a lacI

mutant gene on the chromosome and normal lacI allele on the F0 factor. Each strain
was grown and divided into two tubes. One tube did not contain lactose, and the
other tube contained lactose. Cells were sonicated which led to release of the
enzymes present inside the cell. If lac operon was expressed, then among the
released enzymes β-galactosidase would have been present. β-galactosidase was
known to breakdown the substrate β-o-nitrophenylgalactoside (β-ONPG). β-ONPG
is a colorless molecule, but upon being cleaved by β-galactosidase, it gives yellow
color. Therefore, the amount of yellow color generated was the indication of
presence of β-galactosidase and expression of lac operon.
In the lacI
mutant strain, yellow color due to β-ONPG breakdown was obtained
whether or not lactose was present. It was expected as the mutant lacks the lac
repressor, the operon is constitutively expressed, and β-galactosidase gets produced
even in the absence of lactose. It means that lacI
mutant loses the inducible nature
of lac operon. However, for the merozygote, β-galactosidase was observed to be
expressed only in the presence of lactose as evident from the yellow color produced
by the breakdown of β-ONPG. Therefore, it could be concluded that one normal
copy of lacI gene is present in the F0 factor although the chromosome contained the
mutant lacI. lacI therefore was predicted to encode repressor protein which could
diffuse throughout the cell and bind to lac operon. It was also inferred that lacI gene
need not be physically adjacent to the lac operon or present within the operon.
Repressor, once present inside the cell, can bind to the operator either on chromo-
some lac operon or F0 factor lac operon. Therefore, the repressor is known as trans-
acting factor, and this effect brought about by the repressor is called trans effect
(Fig. 12.7).
In contrast, when a normal lac operon and lacI gene containing F0 factor is
introduced into a cell with defective operator site on the chromosome, the lac operon
on the chromosome continues to be expressed without lactose. This occurs because
repressor cannot bind to the defective operator. Having a normal operator site in the
F0 factor did not lead to inducible nature of chromosomal lac operon. Hence,
operator did not show any trans effect. It, therefore, is known to have cis-effect or
in other words, operator is a cis-acting element.

12.4 Mechanism of Lac Operon

As we learned in the previous sections, lac operon has the organization as CAP site,
promoter site, operator site, lacZ gene, lacY gene, lacA gene, and the terminator site.
LacI is not part of the operon and has a separate promoter site. We know that lac
operon needs to get expressed only when lactose is present. In the absence of lactose,
its analog allolactose is also absent. In the absence of allolactose, Lac repressor
remains bound to the operator, preventing operon activation. However, in presence
of lactose, allolactose becomes available. It binds to the repressor, changing its
conformation so that it can no longer bind to the operator (Beckwith 1967). This
leads to induction of lac operon expression (Fig. 12.8).
580 T. Banerjee

Fig. 12.7 Evidence that the lacI gene encodes a diffusible repressor protein. Starting material: The
genotype of the mutant strain was lacI
lacZ+ lacY+ lacA+. The merozygote strain had an F0 factor
that was lacI+ lacZ+ lacY+ lacA+. The F0 factor had been introduced into the mutant strain via
conjugation (Adapted from: Brooker, R.J.: Genetics: Analysis and Principle, 6th ed. pp 336–360.
McGraw-Hill Education, New York, 2018).
12 Regulation of Gene Expression in Prokaryotes 581

Fig. 12.8 The cycle of lac operon induction and repression. Lac operon codes for genes that
metabolize lactose. When lactose is present, genes of the lac operon is induced, and proteins
involved in lactose uptake and metabolism are synthesized. When the lactose is absent, the lac
operon is repressed, blocking the transcription of lactose-metabolizing genes (Adapted from:
Brooker, R.J.: Genetics: Analysis and Principle, 6th ed. pp 336-360. McGraw-Hill Education,
New York, 2018)

In reality, the repressor is not able to inhibit the transcription of the lac operon
completely. Basal levels of all three enzymes encoded by the lac operon are present
although the level is too low and is not enough to enable the bacterium to utilize
minimal lactose present in the environment. However, when lactose concentration
rises in the environment, it needs lactose permease to go inside. At that point, the
basal level of lactose permease present before the actual induction of lac operon
helps the cell to take up lactose. Availability of lactose inside the cell then induces
lac operon expression. Therefore, it is important that the repressor does not
completely inhibit transcription of lac operon.
When lactose gets depleted in the environment, the concentration of allolactose
inside the cell also keeps reducing due to the action of metabolic enzymes. After a
certain point, allolactose concentration decreases below the minimal level required
for repressor binding. Therefore, despite the high affinity of allolactose for the
repressor, it is not able to bind to it.
582 T. Banerjee

Fig. 12.9 CAP-cAMP

complex. CAP-cAMP
complex binding bends the
DNA at an approximate angle
of 90 . The bending enables
the RNA polymerase to bind
and activate transcription of
lac operon (Chakerian, A.E.,
Matthews, K.S. Mol.
Microbiol. 6, 963–968, 1992,
Fulcrand, G. et al. Sci Rep. 14,
19243, 2016)(Adapted from:
Griffiths, A. J. F, Introduction
to Genetic Analysis. New
York, NY :W.H. Freeman &
Company, 2015)

Table 12.4 Transcription condition for the lac operon

12.5 Regulation of Lac Operon

Apart from regulation of lac operon by lactose and repressor protein, it is also
regulated by another way that is known as catabolite repression. It is brought
about by the presence of glucose, which is a catabolite. We all know that glucose is
the more readily available form of sugar which is easily catabolized to produce more
energy in the form of adenosine triphosphate (ATP) than lactose. Considering the
efficiency of energy production, the bacteria need not utilize lactose in the presence
of glucose. Therefore, presence of glucose represses the transcriptional activation of
12 Regulation of Gene Expression in Prokaryotes 583

Fig. 12.10 Regulation of lac operon by catabolite repression. (a) High rate of transcription when
lactose is present and glucose is absent. (b) Low rate of transcription in absence of lactose and
glucose as CAP is active but repressor is bound to operator. (c) Both lactose and glucose are present.
584 T. Banerjee

lac operon. Lactose gets utilized only when glucose is depleted in the environment.
The use of one sugar after the other by the bacteria is called diauxic growth.
However, glucose is not able to bind to the operon but brings about its action by
the help of an effector molecule called cyclic-AMP (cAMP). cAMP is produced
from ATP by the enzyme adenylyl cyclase. Transport of glucose into the cell inhibits
adenylyl cyclase. Hence, availability of glucose reduces cAMP level. The effect of
cAMP on lac operon is mediated by catabolite activator protein (CAP). CAP is
composed of two subunits, each of which binds to one molecule of cAMP. When
cAMP binds to CAP, the CAP-cAMP complex bends the DNA at an approximate
angle of 90 . This bending enables the RNA polymerase to bind and activate
transcription of lac operon (Fig. 12.9). When only lactose is present, allolactose
and cAMP levels are high. Allolactose binds to repressor and does not allow it to
bind to the operator. cAMP binds to the CAP. cAMP-bound CAP interacts with CAP
site. CAP interacts with RNA polymerase and helps it to bind to the promoter.
Therefore, in presence of lactose, lac operon is highly active. In presence of lactose
and glucose, allolactose is high but cAMP is low. As cAMP is not available to bind
to the CAP, CAP is not able to bind to the CAP site. Hence, transcription of lac
operon continues at low rate. When only glucose is present, cAMP levels are very
low and allolactose levels are also low (Table 12.4). So the repressor is bound to the
operator site, and CAP site is not occupied by the CAP protein. Hence, transcription
of lac operon continues at very low basal rate (Fig. 12.10).
The term catabolite repression may be confusing as it involves action of an
inducer cAMP and an activator CAP. When the term was coined, it was observed
that glucose represses transcription of lac operon. The mode of interaction of cAMP
and CAP was not known. Hence, it was called catabolite repression.

12.6 Effect of Mutation on Lac Operon

In the previous section, we have learned about the lac operon mutation, lacI
. It was
the study on lacI
mutant that proved lac repressor is a diffusible trans-acting
protein whereas operator is a cis-acting element. Later, molecular understanding of
lac operon was gained by genetic and crystallographic studies. The operator site was
identified due to mutations that prevented repressor binding. These mutations, called
lacO
or lacOc mutations located at the operator site, resulted in constitutive operon
expression even if the repressor is normal. It was shown to localize at operator site,
which is now called as O1. In the late 1970s, two additional operator sites were
identified, O2 and O3.

Fig. 12.10 (continued) Transcription is low due to the lack of CAP binding. (d) Glucose is present
and lactose is absent. Transcription rate is very low due to lack of CAP binding and binding of
repressor (Adapted from: Brooker, R.J.: Genetics: Analysis and Principle, 6th ed. pp 336–360.
McGraw-Hill Education, New York, 2018)
12 Regulation of Gene Expression in Prokaryotes 585

Fig. 12.11 Overview of trp operon. In absence of tryptophan, operon transcription occurs. In
presence of high tryptophan, operon is repressed (Adapted from: https://en.wikipedia.org/wiki/Trp_
operon)

12.7 Trp Operon in E. coli

We will now learn about another important operon called the trp (pronounced as
“trip”) operon. The genes present in this operon are responsible for encoding
enzymes involved in the synthesis of tryptophan. This operon contains five
enzyme-encoding genes called as trpE, trpD, trpC, trpB, and trpA, which are
involved in tryptophan biosynthesis. Apart from these, there are two genes, trpR
and trpL, that play a role in regulation of trp operon. TrpL is part of trp operon, and
trpR acts as a separate transcriptional unit having its own promoter. trpR encodes for
trp repressor protein. When tryptophan levels in the cell are very low, trp repressor
cannot bind to the operator site and its transcription continues. When the tryptophan
levels in the cell becomes high, it acts as a corepressor and binds to the repressor
(Fig. 12.11). Binding of corepressor to the repressor brings a conformational change
in the repressor, and hence it can bind to the operator. Binding of repressor hinders
the binding of RNA polymerase to the promoter, and hence trp operon is shut off
(Fig. 12.12). The anabolic end product of the trp operon, tryptophan, itself acts as the
repressor for the operon (Crawford and Stauffer et al. 1980).
586 T. Banerjee

Fig. 12.12 Regulation of trp operon by end product tryptophan. (a) When tryptophan levels are
low, it cannot bind to trp repressor protein, repressor is not able to bind to the operator site, and
transcription continues. (b) When tryptophan levels are high, it binds to the repressor. The repressor
12 Regulation of Gene Expression in Prokaryotes 587

In a gram-positive bacteria Bacillus subtilis, the trp operon is present within a

supraoperon. There are six genes of trp operon which are flanked by three genes of
supraoperon on each side.

12.7.1 Trp Operon Structural Genes

The full-length polycistronic trp mRNA is about 6800 nucleotides in length. It codes
for five trp polypeptides. The operon codes for two bifunctional polypeptides, that is,
trpD and trpC. TrpD consists of glutamine amidotransferase domain (designated as
trpG domain as the analogous monofuctional trpG protein is present in other
organisms). The other domain is an anthranilate phosphoribosyltransferase,
designated as trpD domain. Similarly, trpC polypeptide also has two domains, and
the amino terminal has trpC domain. This domain is responsible for the indole-3-
glycerol phosphate synthetase reaction. The distal half is responsible for isomeriza-
tion of phosphoribosylanthranilate. TrpE and TrpD polypeptides form a tetrameric
functional complex (Yanofsky 1971). These complexes catalyze the reaction of
chorismate + glutamine + anthranilate and anthranilate + PRPP +
phosphoribosylanthranilate. TrpB and Trp A polypeptides form a complex that
catalyzes the reaction of indole-3-glycerol phosphate + L-serine leading to synthesis
of L-tryptophan. The structural genes are organized in the same order as the
functions of their corresponding polypeptide domains in tryptophan synthesis. The
trpC region preceding the trpF region and trpB preceding trpA are the only
exceptions.

12.7.2 Concept of Attenuation

We learned that trp repressor binds to the operator site only in the presence of
corepressor tryptophan. Hence, in the presence of tryptophan, the operon for trypto-
phan synthesis is shut off. In 1970s, Yanofsky and colleagues observed mutant
strains of bacteria which lacked trp repressor but could still inhibit trp operon in the
presence of high tryptophan concentration. They also found mutations where trpL
gene which codes for the leader sequence was missing and high expression of trp
operon was observed. Studies on these mutant strains led to the understanding of
another mode of trp operon regulation called attenuation.
During attenuation, transcription begins but is terminated before the complete
mRNA is made. A short stretch of mRNA is transcribed, and it terminates shortly
past the trpL gene. The transcription is terminated before the transcription of

⁄


Fig. 12.12 (continued) then binds to the operator to inhibit transcription (Adapted from: Brooker,
R.J.: Genetics: Analysis and Principle, 6th ed. pp 336–360. McGraw-Hill Education, New York,
2018)
588 T. Banerjee

Fig. 12.13 Sequence of the trpL mRNA produced during attenuation. trpL mRNA has self-
complementary regions which base-pairs with each other. Regions 1 and 2, 2 and 3, and 3 and
4 have possible base pairing. One region can base-pair with the other only once (Adapted from:
Brooker, R.J.: Genetics: Analysis and Principle, 6th ed. pp 336–360. McGraw-Hill Education,
New York, 2018). So if 2 has hydrogen bonded with 1, it cannot base-pair with 3. Similarly, if
region 3 has hydrogen bonded with 4, it cannot base-pair with 2. The last U in the purple attenuator
sequence is the last nucleotide that would be transcribed if attenuation is occurring

structural genes; hence, attenuation stops production of tryptophan. The attenuation

occurs due to the presence of attenuator sequence. This sequence is present
immediately downstream from the operator site. The first gene in trp operon is
trpL gene. The mRNA of trpL gene codes for 14 amino acids that form the leader
peptide. The leader peptide contains two tryptophans. These two tryptophan acts as
sensor for tryptophan levels in the cell. If tryptophan levels are high, then only the
tRNA gets charged with tryptophan, and leader peptide is synthesized. In case
tryptophan is less, the tRNA doesn’t get charged with tryptophan, and the leader
peptide synthesis slows down.
Another important feature of leader mRNA is that it has self-complementary
regions which base-pairs with each other to form stem loops. Region 2 is comple-
mentary to region 1 and also to region 3. Region 3 is complementary to region 2 as
well as to region 4. Therefore, three stem loops are possible: 1–2, 2–3, and 3–4. One
region can take part in only one stem loop formation. So if region 2 pairs with 1, it
cannot base-pair with region 3. Alternatively, if region 2 base-pairs with 3, then
region 3 cannot base-pair with region 4 (Fig. 12.13). The 3–4 stem loop together
with U-rich attenuator forms an intrinsic terminator and leads to ρ-independent
termination.
The formation of 3–4 stem loop with U-rich attenuator needs to form for attenua-
tion. The amount of tryptophan regulates the formation of 3–4 stem loop. In bacteria,
generally, translation and transcription occur together. As the transcription of trpL
gene continues, region 1 rapidly hydrogen bond with region 2, and region 3 is not
able to base-pair with region 2 and can therefore hydrogen bond with region
12 Regulation of Gene Expression in Prokaryotes 589

Fig. 12.14 Mechanism of

attenuation of trp operon by
stem loop formation of trpL
mRNA. Attenuation occurs in
part (a) and (c) due to
formation of a 3–4 stem
loop. (a) No Translation, 1-2
and 3-4 stem loops form
b) Low Tryptophan Levels,
2-3 stem-loop forms c) High
Tryptophan levels, 3-4 stem-
loop form) (Adapted from:
Brooker, R.J.: Genetics:
Analysis and Principle, 6th ed.
pp 336–360. McGraw-Hill
Education, New York, 2018)
590 T. Banerjee

4. Therefore, the attenuation occurs just after the transcription of trpL gene. When
tryptophan concentration is low, trp-charged tRNA is not formed and is enough
amount to support translation. Ribosome halts at Trp codons of leader trpL gene as it
waits for charged tRNAtrp. When this occurs, region 1 is covered by ribosome and
cannot base-pair with region 2. Therefore, region 2 base-pairs with region 3. As
region 3 cannot base-pair with region 4, 3–4 stem loop is not formed and attenuation
does not occur. Therefore, the tryptophan operon is successfully transcribed
(Fig. 12.14).
When cells have just the sufficient amount of tryptophan, the mRNA translation
occurs till ribosome reaches the stop codon of trpL gene. The pausing of ribosome at
the stop codon inhibits region 2 from base pairing with region 3, allowing region 3 to
form a stem loop with region 4. Since 3–4 stem loop gets formed, the transcription of
the rest of the tryptophan operon is not transcribed (Fig. 12.14).
In Bacillus subtilis, Trp operon is regulated by attenuation where two alternative
RNA secondary structures are formed: anti-terminator and terminator. A tryptophan-
activated RNA-binding protein, TRAP, can exist in either of these alternative
structures. When tryptophan availability is less, TRAP is inactive, and anti-
terminator RNA structure persists leading to transcription of tryptophan structural
genes.
Attenuation regulates transcription of many other amino acid-synthesizing
operon. In all these operons, the leader peptide contains the amino acid which is
synthesized by the enzymes coded by the operon. For example, histidine operon has
seven histidine codons in the leader sequence.

12.7.3 Mechanism of Trp Operon

We learned till now that operons can be regulated by either positive and negative
regulation and sometimes both. Catabolic enzymes are mostly inducible, and ana-
bolic enzymes are repressible. Trp operon is repressible as it is an anabolic operon
and the end product, and tryptophan acts as a corepressor which binds to the
repressor molecule. Binding of repressor-corepressor complex to the operator
shuts off the operon transcription. Tryptophan availability inside the cell controls
the formation of charged tRNAtrp which in turn regulates attenuation process. Hence,
genetic regulation provides bacteria with an energy-efficient mechanism for
preventing the overproduction of any anabolic product more than the cell’s
requirement.

12.8 Regulation of Gene Expression in Lambda Phage

Bacteriophages are the class of viruses which uses bacteria as their host. We know
that viruses can invade host cells and utilize host cell machinery to produce viral
gene products. Viruses can then exist within the host cell (lysogeny) by integrating
its DNA into host chromosome or can form progeny viruses, lyse the host cell, and
12 Regulation of Gene Expression in Prokaryotes 591

Fig. 12.15 A map of phage, showing the major genes. PL promoter for leftward transcription of
the left early operon, PR promoter for rightward transcription of the right early operon, PRE
promoter for repressor establishment, and PRM promoter for repressor maintenance (Adapted
from: Russell, P.J.: iGenetics: A molecular Approach, 3rd ed. pp 36–59. Benjamin Cummings,
New York, 2010)

invade new host cells (lysis). In the lysogenic phase, the expression of the viral
genome is repressed, and the virus is said to exist as prophage.
Bacteriophage λ has emerged as important genetic model as it shares gene
regulatory mechanism with both prokaryotes and eukaryotes. Once it adsorbs onto
the bacterium and inserts its DNA into host cytoplasm, the phage DNA circularizes.
At that point, the decision is made whether to follow lysogenic or lytic cycle. The
decision leads a highly regulated genetic switch. There are two promoters, PL and
PR. PL leads to transcription toward the left-hand side, and PR leads to transcription
in the right-hand side (Fig. 12.15). In rightward transcription, cro (control of
repressor and other) is the first gene to be transcribed. Cro protein plays an important
role in initiating the lytic phase. In the leftward transcription, N is the first gene to be
transcribed. The resulting N protein is a transcription anti-terminator. It helps
transcription to proceed past the termination sites. As a result of N protein synthesis,
other genes are transcribed; those are cII, O, P, and Q. cII protein can turn on
592 T. Banerjee

Fig. 12.16 Regulation of phage lambda genes for deciding lysogeny and lysis. Expression of
genes after infection of E. coli and the transcriptional events that occur when either the lysogenic or
lytic pathway is followed. In the figure, stimulation of transcription is indicated by green arrows,
and repression of transcription by red arrows (Adapted from: Russell, P.J.: iGenetics: A molecular
Approach, 3rd ed. pp 36–59. Benjamin Cummings, New York, 2010)

production of cI protein (repressor) and int protein (product of integrase gene,

required for inserting phage DNA into host chromosome). O and P genes code for
DNA replication proteins. Protein Q turns on late genes involved in lytic phase.
12 Regulation of Gene Expression in Prokaryotes 593

Fig. 12.17 Hypothetical contributions to local repressor concentration at a bacterial operator

(O) include contributions because of free repressor and contributions because of DNA-bound
repressor at auxiliary operators (Oaux) through DNA looping (Becker, N.A. et al. Nucleic Acids
Res. 41, 156–166, 2013)

12.8.1 Lysogenic Pathway

The establishment of lysogenic pathway requires cII protein (translated from cII
gene of early right operon) and cIII (translated from cIII gene of early left operon).
cII is stabilized by interacting with cIII. The stabilized cII protein then activates the
transcription of cI. The product of cI gene is called the λ repressor. It binds to
operator regions OL and OR. The binding of λ repressor inhibits further transcription
of early operons under the control of PL and PR. N and cro proteins translated from
the operons under PL and PR can no longer take place. N and cro are highly unstable
proteins, and hence their cellular levels come down quickly. There is another
promoter known as promoter for repressor maintenance, PRM. Repressor gene
is transcribed from that promoter to maintain the repressor levels. Thus, if enough
repressor is available, then it binds to OL and OR, shutting down the transcription
from early promoters. It also leads to production of int protein by the action of cII,
establishing lysogeny.

12.8.2 Lytic Pathway

The lytic pathway is induced by DNA damage caused by ultraviolet light. Bacteria
has RecA proteins which functions in DNA recombination and repair. When DNA is
damaged, RecA stimulates the repressor polypeptides to cleave themselves into two
and gets inactivated. In the absence of repressor, cro gene is transcribed. Cro protein
reduces RNA synthesis from PL and PR. This leads to reduction of cII protein
synthesis. Reduction in cII blocks λ repressor synthesis from PRM. Synthesis from
PR is also reduced, but if enough Q protein is available, the synthesis of late genes
continues for establishing the lytic phase (Fig. 12.16). Therefore, to summarize, if
repressor dominates, lysogeny ensues, and if cro dominates, lysis occurs.
594 T. Banerjee

Box 12.1: Scientific Concept: Mechanism of Promoter Repression by Lac

Repressor-DNA Loops – Nicole A. Becker et al.
We have learned that lac repressor is both positively and negatively regulated.
It stays repressed when the operator is bound by the repressor. The repressor
binding becomes weak in presence of allolactose, and as a result, RNA
polymerase can access the promoter transcribing the lac operon genes. Apart
from the promoter proximal operator, there are two more auxiliary operators
present just upstream and far downstream of the promoter proximal operator.
The authors of this paper studied the role of these auxiliary operators in
regulation of lac operon. The authors of this paper hypothesized that local
repressor concentration at regulatory promoter proximal operator is necessary
for lac operon repression. The auxiliary operators help in increasing the local
repressor concentration at the regulatory operator in distance-dependent man-
ner (Fig. 12.17).
The authors created many artificial promoter constructs in bacterial cells
with artificial auxiliary operators at varied distances from the regulatory
operator. They observed that repressor binding increases in a distance-
dependent manner by strong auxiliary operators. They also observed that
DNA looping occurring in the repressor traps the promoter and occludes
RNA polymerase binding. They also observed that downstream auxiliary
operator increased local repressor concentration but did not trap the promoter
in the repressor loops. Hence, they concluded that promoter repression is
equivalent to DNA loops of comparable size whether the promoter lies within
the loop or adjacent to the loop.
Their results also showed that DNA looping can repress T7 transcription
initiation but not T7 transcription elongation. It could be possible that RNA
polymerase cannot initiate from the tightly bent DNA due to lack of sufficient
energy required for untwisting the DNA.

12.9 Summary

• Gene expression is tightly regulated in prokaryotes depending upon the require-

ment of the cell. For anabolic pathways, genes are expressed only when the final
product of the pathway is either absent or very low than the required amount. For
proteins involved in catabolism, the corresponding genes are expressed only
when the required catabolite is present in the system.
• The genes participating in a metabolic pathway are organized together which are
called as operons. They are mostly organized as promoter, operator, structural
genes, and terminator. Structural genes are transcribed as single mRNA.
• For inducible operons, transcription is normally shut off, unless the inducer is
present. In repressible operons, transcription is normally on unless shut off by the
12 Regulation of Gene Expression in Prokaryotes 595

presence of repressor. When repressor binds and inhibits transcription, it is called

negative control. When inducer/activator binds and initiates transcription, it is
called positive control.
• Repressor may or may not be coded by the operon. Therefore, it might act as trans
element. Repressor protein often act in trans, but operators are mostly cis-acting
elements.
• The lac operon is an inducible operon. It gets induced by the presence of lactose.
Lactose gets converted to allolactose, which then binds to repressor and
inactivates it. Once the repressor becomes inactive, it leaves the operator and
RNA polymerase can then bind to the promoter to transcribe its structural genes.
• The lac operon is also controlled by positive regulation through catabolite
repression. cAMP is the catabolite activator protein for lac operon. Its levels are
inversely related to glucose. Therefore, when glucose is low, cAMP is high and it
activates the transcription of lac operon.
• lac operon has three operator sites: O1 is the regulatory operator, and O2 and O3
are the auxiliary operator. Auxiliary operators increase the local concentration of
repressor and hence facilitate binding of repressor to the regulatory operator.
• The trp operon of E. coli is a repressible operon. It normally transcribes the
structural genes unless tryptophan is present in high amount in the cell. Trypto-
phan acts as corepressor. Hence, in presence of corepressor, the repressor binds to
the operator and shuts off transcription.
• The trp operon is also regulated by attenuation. The first gene of trp operon is the
leader sequence. It has four self-complementary regions. Stem loop formed
between regions 3 and 4 leading to attenuation of transcription. Tryptophan
residues present in the leader sequence also regulates trp operon by attenuation
process.
• Jacob, Monod, and Pardee’s experiment on partial diploid or merozygotes
showed that lac repressor is a diffusible trans-acting element, whereas operator
only acts in cis form.
• Viruses can integrate its DNA into host chromosome and undergo lysogenic life
cyle or can form progeny viruses, lyse the host cell, and invade new host cells,
which is called lytic life cycle. Bacteriophage λ can choose between lysogenic
and lytic life cycle by the help of genetic switches.
• λ-phage has two principal promoters, PL and PR, for transcription of early
operons. PL leads to transcription toward the left-hand side, and PR leads to
transcription in the right-hand side. In rightward transcription, cro (control of
repressor and other) is the first gene to be transcribed. In the leftward transcrip-
tion, N is the first gene to be transcribed. The resulting N protein is a transcription
anti-terminator.
• The establishment of lysogenic pathway requires cII protein and cIII. cII is
stabilized by interacting with cIII. The stabilized cII protein then activates the
transcription of cI.
• cI codes for λ repressor which binds to operator regions OL and OR. The binding
of λ repressor inhibits further transcription of early operons under the control of
596 T. Banerjee

PL and PR. N and cro proteins translated from the operons under PL and PR can no
longer take place.
• There are two more promoters, known as PRE and PRM, known for their function
of repressor establishment and repressor maintenance, respectively. If repressor
dominates, lysogeny ensues, and if cro dominates, lysis occurs.

References
Becker NA, Peters JP, Lionberger TA, Maher LJ III (2013) Mechanism of promoter repression by
Lac repressor–DNA loops. Nucleic Acids Res 41:156–166
Beckwith JR (1967) Regulation of the lac operon. Recent studies on the regulation of lactose
metabolism in Escherichia coli support the operon model. Science 156:597–604
Brooker RJ (2018) Genetics: analysis and principle, 6th edn. McGraw-Hill Education, New York,
pp 336–360
Chakerian AE, Matthews KS (1992) Effect of lac repressor oligomerization on regulatory outcome.
Mol Microbiol 6:963–968
Crawford IP, Stauffer GV (1980) Regulation of tryptophan biosynthesis. Annu Rev Biochem 49:
163–195
Fulcrand G, Dages S, Zhi X, Chapagain P, Gerstman BS, Dunlap D, Leng F (2016) DNA
supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia
coli. Sci Rep 14:19243
Griffiths AJF, Wessler SR, Carroll SB, Doebley J (2015) Introduction to genetic analysis. Freeman
& Company, New York, NY :W.H
Juers DH, Matthews BW, Huber RE (2012) LacZ β-galactosidase: structure and function of an
enzyme of historical and molecular biological importance. Protein Sci 21:1792–1807
Kaback HR (2005) Structure and mechanism of lactose permease. C R Biol 328:557–567
Russell PJ (2010) iGenetics: a molecular approach, 3rd edn. Benjamin Cummings, New York, pp
36–59
Rutkauskas D et al (2009) Proc Natl Acad Sci U S A 106(39):16627–16632
Yanofsky C (1971) Tryptophan biosynthesis in Escherichia coli. Genetic determination of the
proteins involved. JAMA 218:1026–1035
Regulation of Gene Expression
in Eukaryotes 13
Aathmaja Anandhi Rangarajan

Similar to prokaryotes, eukaryotes have also evolved with complex gene regulation
mechanisms to control the expression of variety of genes in different cell types.
Thousands of proteins orchestrate in a timely manner to ensure the activation and
repression of genes in response to internal and external environment. This spatial and
temporal precision of gene expression is critical in all biological processes from cell
differentiation to cell death, while deregulation of gene expression often leads to
disease. The regulation of gene expression happens in several stages including
transcription, posttranscription, translation, and posttranslation. This complex regu-
lation involves both cis-acting and trans-acting elements. Cis-acting elements are
present in the coding and noncoding regions of the DNA itself involving remodeling
or modifications of the DNA. Trans-acting elements are proteins such as transcrip-
tion factors and other DNA-binding proteins that enhance or suppress gene
expression.
Most of the gene regulation in eukaryotes happens at the level of transcription
initiation. Transcription is mainly performed by RNA polymerase II along with
several cis- and trans-acting factors. The eukaryotic transcriptions dictated by two
distinct sets are cis-acting DNA elements: (i) proximal regulatory elements which
contain the core promoter and (ii) distal regulatory element which includes
enhancers, silencers, insulators, and locus control regions (Fig. 13.1). Trans-acting
factors are usually regulatory proteins like transcription factors that binds to
cis-acting elements to activate or repress transcription. In humans, the transcription
factors are present far less in numbers (~1850) when compared to the number of
genes (~20,000 to 25,000) (Venter et al., 2001). To compensate for this skewed
proportion, the cell has evolved with multiple cis-acting elements that can work
along in various combinations with trans-acting factors to control gene expression.

A. A. Rangarajan (*)
University of Michigan, Ann Arbor, MI, USA
e-mail: anandhir@msu.edu

# The Author(s), under exclusive license to Springer Nature Singapore Pte 597
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_13
598 A. A. Rangarajan

Distal regulatory elements

Locus control
region
Distal
Insulator
Silencer
Enhancer

TSS
r
ce

Downstream
an
m
ea
h

MTE DPE Enhancer

En

INR
str

BRE TATA
Up

CpG

Core promoter
Proximal promoter
elements

Fig. 13.1 Figure depicting several cis-acting elements involved in the regulation of gene expres-
sion during transcription. The core promoter which spans about 1 kb is surrounded by promoter
elements such as CpG islands and upstream and downstream enhancers. The core promoter contains
initiator element (INR) which also possesses transcription start site (TSS) surrounded by TFIIB
recognition element (BRE), TATA box (TATA), motif ten element (MTE), and downstream
promoter element (DPE). Distal regulatory elements such as insulator, silencer, and locus control
region are located proximal or distal to the promoter region

During transcription initiation, as a first step, RNA polymerase II binds to the core
promoter region together with some transcription factors. The first identified core
promoter region was TATA box which possesses consensus sequence of TATAa
(t)AAg(a) located 25 to 30 bp upstream of the transcription start site. Later, it was
found that TATA box only consists of 32% of human promoters. Apart from TATA
box, core promoter elements can contain other elements such as initiator element
(Inr), downstream promoter element (DPE), downstream core element (DCE), TFIIB
recognition element (BRE), and motif ten element (MTE) (Maston et al., 2006).
These core promoter elements are present at specific distance from the transcription
initiation site, and they possess distinct consensus sequences, which are described in
Fig. 13.2. These elements in combination or alone recruits specific transcription
factors to initiate or repress transcription.
Core promoter elements are surrounded by proximal promoter elements that
spans from
50 to
500 region of the transcription start site and alters the rate
and level of transcription. These regions typically contain multiple sites for binding
activators. An example for the proximal promoter elements is the CpG islands which
are 500 bp–2 Kb in length and are highly GC rich. 60% of human genes promoter
region contains CpG islands. Most of the CpG dinucleotides present in the genome
are methylated at the cytosine base whose fifth carbon is methylated, whereas the
one in the CpG island is unmethylated. These are involved in regulation of many
housekeeping genes and other regulated genes. When methylation proteins such as
MeCP2 bind the cytosine of CpG island and also recruit histone-modifying
13 Regulation of Gene Expression in Eukaryotes 599

Fig. 13.2 RNA polymerase II core promoter elements. The core promoter elements are represented
in colored boxes. TFIIB recognition element (BRE), TATA box, initiator element (INR), motif ten
element (MTE), downstream promoter element (DPE), and downstream core element (DCE) are
represented with respect to the transcription start site (+1). Consensus sequence of these elements
are shown with the corresponding color of each element

complexes, it results in the repressive chromatin structure leading to silencing of

transcription.
Apart from core and proximal promoter elements, there are several distal
elements such as enhancers, insulators, silencers, and locus control regions that
repress or activate the genes. Enhancers typically contain multiple binding sites for
the binding of specific transcription factors that work together to enhance transcrip-
tion. Enhancers are independent of orientation and distance and independent and can
be present several Kb away from the transcription start site. It is proposed that by
“looping out,” the intervening DNA sequence enhancer region comes in contact with
the core promoter region. In contrast to enhancers, silencers repress the transcrip-
tional activity. The function of enhancers is independent of distance and orientation
from the promoter region. Silencers provide binding sites for repressors which
sometimes act in the presence of corepressor. These repressors and corepressors
may block the activator from binding to the enhancer sites or block the binding of
RNA polymerase II to start the transcription. In some cases, repressors can also
recruit chromatin remodeling complexes which perturbs the binding of transcription
complexes. Insulators or boundary elements limit the activity of transcriptional
regulatory elements. They are of typically 0.5–3 Kb in length and possess roles
such as blocking promoter-enhancer activity or block the repressive chromatin
region (Recillas-Targa et al., 2002). They function in position-dependent and
orientation-independent manner. Locus control regions (LCR) consists of groups
of several regulatory elements which collectively work to control the gene expres-
sion. LCRs consist of several cis-acting elements such as insulators, enhancers,
silencers, chromosome scaffold attachment regions, and nuclear matrix. They are
general bound by transcription factors, activators, and repressors. Each of the
component affects the gene expression differentially, and the collective action of
these results in the overall effect on the gene expression. These LCRs confer spatial
and differential gene expression depending on the stimuli (Li et al., 2002).
600 A. A. Rangarajan

13.1 Control of Transcription by Protein

Apart from the cis-acting elements, several trans-acting proteins affect the transcrip-
tion. The trans-acting proteins involved in the initiation of transcription can be
categorized into three, namely, general transcription factors and promoter specific
activators and coactivators. General transcription factors include RNA polymerase II
itself and transcription factors such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and
TFIIH. The general transcription factors assemble in the core promoter in the
orchestrated order to enable the formation of pre-initiation complex (PIC) which
enables the RNA polymerase II to initiate the transcription at the transcription start
site. In the first step, TFIID comprised of multi-subunit TATA box binding proteins
(TBPs) and tightly bound TBP-associated factors (TAF). TBP makes direct contact
with RNA polymerase along with the transcription factors such as TFIIA and TFIIB.
TBP also provides binding sites for certain activators which stimulate transcription at
the stage of pre-initiation complex. The binding of TFIIA further enhances the
binding of TBP to the TATA box. TFIIB interacts with multiple proteins TFIID,
TFIIF, and RNA polymerase II along with BRE elements downstream of TATA
box. Binding of TFIIB is the rate-limiting step in the recruitment of RNA polymer-
ase II which is stimulated by activators. In the next step, once the pre-initiation
complex is formed, TFIIE binds to RNA polymerase II and TFIIH. TFIIF then binds
to this complex and interacts with RNA polymerase II and TFIIB. It helps in
nonspecific binding of DNA sequences and helps stabilizing pre-initiation complex.
In the last step, a multi-subunit complex TFIIH binds and initiates the transcription.
This complex contains ATPase, protein kinase, and helicase activity which unwinds
the DNA around the initiation site (Orphanides et al., 1996).
This entire assembly of pre-initiation complex and the activation in the core
promoter region allow merely basal level of transcription from the transcription
start site. Transcriptional activity from the promoter is enhanced severalfold by other
special class of proteins named activators.

13.1.1 By Activators

Transcriptional activity of many genes is greatly stimulated by a set of promoter-

specific factors known as activators. Activator proteins contains two domains,
namely, DNA-binding domain (DBD) and activator domain (AD) which is crucial
for the activation of transcription. The important role of activators is to bring the
RNA polymerase II along with the general transcription factors to the core promoter.
It does that by binding to the general transcription factors and changes its confirma-
tion which activates the pre-initiation complex formation. Activators can also help in
the subsequent transcription process such as initiation and elongation after the
formation of pre-initiation complex by helping in the clearance of pre-initiation
complex and releasing paused RNA polymerase II (Green, 2005). Examples of
activator proteins include zinc finger proteins, homeobox, fork head, helix-loop-
helix (HLH), ETS, basic leucine zipper (bZIP), and PIT-Oct-Unc (POU)
13 Regulation of Gene Expression in Eukaryotes 601

DNA-binding domain (Pabo, 1992). DNA-binding sites of activators are generally

small of about 6–12 bp. Most of the activators form hetero- or homodimers; hence,
their binding regions usually comprise of two identical half sites. Activators undergo
numerous posttranslational modifications including phosphorylation, acetylation,
and glycosylation. In many cases, these posttranslational modifications have positive
role in the transcriptional activation. For example, acetylation of p53 enhances its
binding to DNA and thereby positively affect the transcription. Transcriptional
activators which interact with regulatory proteins to enhance transcription are
referred to as coactivators. Coactivators generally cannot bind DNA and needs the
help of other DNA-binding proteins. Coactivators act similar to activators in
stimulating pre-initiation complex or by remodeling repressive chromatin
complexes. Since most of the histone-modifying enzymes and chromatin remodeling
enzymes do not have sequence specificity, these enzymes are brought to the target
gene sites by the coactivators. The most important effect of the activators is the
synergistic activation of transcription. Synergy refers to the higher activation level
by the combination of multiple regulatory factors due to the action of several factors
acting together. In some specialized scenario, multiple activators bind with each
other to form a stable nucleoprotein complex on the DNA called enhanceosome.
These multiple activators cooperatively activate the target promoter. One of the best
studied examples of enhanceosome is the IFN-β promoter in which all the activator
comes in contact with the cofactor p300. The recruitment of the cofactor p300 is only
possible when all of the activation domains are present. Enhanceosomes are gener-
ally found in tightly regulated genes such as those that are involved in wound healing
or pathogen defense (Ma, 2011).

13.1.2 By Repressors

Similar to activation, repression is also an important gene regulatory mechanism

which is mediated by specific set of proteins called as repressors. The mechanism of
repression is important to avoid nonessential transcription from several genes which
are detrimental and costly to the cell if not regulated. There are two types of
repression observed in eukaryotes, general or global repression or gene-specific
repression. Transcriptional repression occurs when repressors sequester regions or
proteins involved in pre-initiation complex such as RNA polymerase II which results
in overall decrease in the transcription. Gene-specific repression takes place when
specific gene or gene sets is targeted by repressor or corepressor. Gene-specific
repression is mediated by repressor or corepressor which binds to activator or
coactivator and inhibits their activity or decreases their level. In some cases, it
could also bind to promoter and does not allow the formation of pre-initiation
complex. In short-range repression, repressor acts locally by decreasing the activity
of activators or members of the pre-initiation complex that is present proximally. On
the other hand, in the long-range repression, distally located repressor proteins affect
the activity of the promoters by remodeling chromatin or by looping out the
intervening DNA sequence to access the activators or coactivators. These
602 A. A. Rangarajan

short- and long-range repressions are not mutually exclusive, and both can occur at
the same time. For example, hairy protein can perform both short-range and long-
range interactions at the same time.
Repressor proteins are categorized into three categories: Class I, Class II, and
Other types. Class I repressor proteins all possess DNA-binding activity. They could
be sequence specific or non-sequence specific. Class II repressor proteins cannot
bind to DNA themselves but act with other proteins to enable repression. They are
also termed as corepressors. Some proteins of Class II can also have dual role which
act as both activators and repressors at different contexts. Class III proteins does not
bind DNA directly or indirectly but usually act on activators, coactivators, and
pre-initiation complex to reduce the levels of these proteins (Gaston and Jayaraman
2003).
In some cases, relatively small amount of variation in the gene expression might
significantly change the cell fate. Some corepressors are involved in the fine of such
genes. In yeast, it was found that such corepressors act as chromatin-modifying
enzymes and reduce the transcriptional noise while not shutting off the transcrip-
tional machinery.

13.1.3 By Steroid Hormones

Regulation of gene expression can also be regulated by steroid hormones which act
on intracellular transcription factors which positively or negatively regulate specific
sets of gene. Steroid hormones are synthesized from cholesterol and are lipophilic in
nature. They are produced in several regions of the body such as adrenal cortex
(glucocorticoids, mineralocorticoids, adrenal androgens), testes (testicular androgen,
estrogen), and ovary (estrogen and progestins). Steroid hormones reach the target
cell via blood, cross the membrane by diffusion because of the innate lipophilic
property, and bind to steroid hormone receptors (SHR). Usually, steroid hormone
receptors are bound by chaperons which prevent them from folding and aggregation.
Binding of steroid hormones to the steroid hormone receptor relives it from the
chaperone, changes its confirmation to active form, enters the nucleus where it binds
to the target genes (Fig. 13.3). These active forms of steroid hormone receptors can
regulate gene transcription in several ways: (i) by binding to the hormonal-
responsive elements (HRE) and initiating chromatin remodeling which activates or
represses the target gene machinery, (ii) by binding to other transcription factors that
can modulate the activity of other genes, and (iii) by cross-talking with signal
transduction pathway to activate nuclear transcription factors which in turn regulate
the target genes. SHRs are characterized by DNA-binding domain which helps in
binding to HRE and ligand-binding domain (LBD) that binds to ligand.
Androgens (testosterone and dihydrotestosterone) are male sex steroid hormones
that are responsible for the proper functioning of male reproductive system. Testos-
terone and dihydrotestosterone bind to androgen receptors which mediate transcrip-
tion. Androgen receptors are present in male reproductive tract, in female
reproductive tract, and in various other diverse tissues (Davey and Grossman,
2016). Estrogens influence many processes including reproduction, cardiovascular
13 Regulation of Gene Expression in Eukaryotes 603

Fig. 13.3 General mode of activation by steroid hormones. Steroid hormones (blue) enters the
cytoplasm and binds to monomeric steroid hormone receptor (SHR) (green) which leads to the
release of hsp70/hsp90 chaperone complex (red). SHR dimerizes and translocates into the nucleus
followed by binding to hormone-responsive element (HRE). This recruits activator proteins
(brown) which enable chromosome remodeling to enable the RNA polymerase binding (magenta)
with general transcription factors (pink) to activate the transcription

health, bone integrity, behaviors, and cognition. Estrogen binds to estrogen receptor
in the nucleus. This complex further binds to sequences containing estrogen
response element along with activator protein SP1 in the promoter region. This
binding results in the recruitment of co-regulatory proteins that results in increased
or decreased levels of regulatory proteins which results in response to particular
stimuli. Estrogen receptors exist in two different forms: ERα and ERβ. ERα is
expressed in the liver, mammary gland, pituitary, hypothalamus, uterus, and vagina.
ERβ is highly expressed in prostrate, ovary, colonic epithelium, and lung (Deroo and
Korach, 2006). The activation of glucocorticoid hormones is mediated by cortisol
that is secreted from the adrenal gland which in turn binds to glucocorticoid
receptors. After the glucocorticoid receptor is activated by the hormone, it undergoes
homodimerization, translocates into nucleus in which it binds to specific
DNA-responsive elements, and activate transcription. Activated glucocorticoid
receptor can also bind to transcription factors such as NF-κB or AP-1 and repress
them from acting as transcription factors. Thus, it plays a dual role in activating the
anti-inflammatory protein in the nucleus and repressing pro-inflammatory protein
such as NF-κB or AP-1 in the cytosol. Glucocorticoid receptors are expressed in
most of the cell types in the body which takes place in regulation of several
biological processes such as development, metabolism, and immune
response (Vandevyver et al., 2014). Mineralocorticoid receptors possess similar
affinity for both mineralocorticoids such as deoxycorticosterone and glucocorticoids
such as cortisol. Upon activation by the binding of ligand, it translocates inside the
nucleus, homodimerizes, and further binds to hormone-responsive elements present
in the promoter, thereby activating transcription. Mineralocorticoid receptors are
expressed in several types of tissues such as heart, central nervous system, brown
adipose tissue, kidney, sweat glands, colon, and immune cells. It helps in
maintaining the normal salt concentration in the body by activating the proteins
604 A. A. Rangarajan

involved in regulating ionic transports such as Na+/K+ pump (Funder, 2005).

Progesterone receptors are activated by the steroid hormone progesterone. After
binding of progesterone to the progesterone receptor, the receptor undergoes dimer-
ization and enters the nucleus to induce transcription of specific genes. Progesterone
receptors exist in two isoforms, namely, PR-A and PR-B. PR-A is required for
reproductive function and uterine development of whereas PR-B is required for
development of mammary gland. Most PR-positive cells express both the isoforms
including uterus, mammary gland, brain, ovary, testes, pancreas, and tissues in lower
urinary tract (Daniel et al., 2011).

13.2 Regulation of Transcription by Chromatin

Nearly 99% of the DNA in the eukaryotes is packed into highly complex, condensed
structure known as chromatin. Chromatin dynamics plays a crucial role in the
transcriptional regulation and chromosome function. The basic structural subunit
of chromatin consists of nucleosome. Each nucleosomes consists of octamer of
histones made up of two copies of four core histones H2A, H2B, H3 and H4 that
is wrapped around the DNA as shown in the Fig. 13.4. The core octamer with the
linker DNA and the histone H1 comprises the basic repeating subunit of chromatin
complexes. Histone octamers help in maintaining the nucleosomal stability by
making numerous contacts with the nucleosomal DNA. The N-terminal region of
the histones protrudes outside of this core complex since it undergoes several
covalent modifications which regulate the chromatin structure and function. Chains
of nucleosomes are placed in zigzag confirmation which forms complex, highly
organized, condensed structure.
Chromatin dynamics is intertwined with the gene regulation. This condensed
state of chromatin itself poses structural barrier for the binding of transcription
factors and other proteins to bind and initiate transcription. In another level of
regulation, the histones are removed or modified by the chromatin-modifying
complexes which render the chromatin in actively transcribing state of inactive
state. These chromatin-modifying complexes fall under two categories based on

Fig. 13.4 Organization of histone core proteins. Core proteins of histones are depicted in the figure
as H2A, H2B, H3, and H4. Each of the histone is present in two copies forming an octamer which
wraps the DNA (black)
13 Regulation of Gene Expression in Eukaryotes 605

their type of regulation: (i) ATP-dependent chromatin-modifying complexes which

use energy from ATP hydrolysis to disrupt or alter the histones, and
(ii) histone-modifying enzymes which are responsible of posttranslational modifica-
tion of histone molecules thereby regulating the gene expression (Li et al 2007).

13.2.1 Chromatin Remodeling

Chromatin dynamics is shaped by several ATP-dependent chromatin remodeling

enzymes. These remodeling enzymes take part in several processes of chromosomal
dynamics. They enable the formation of properly spaced nucleosomes, move or eject
histones facilitating transcription factor binding, and participate in exchanging the
canonical histones with histone variants in specific chromosomal regions.
All of the ATP-dependent chromatin remodeling enzymes are categorized into
four types depending on the type of ATPase subunit they possess: (i) imitation
switch (ISWI), (ii) chromodomain helicase DNA binding (CHD), (iii) switch/
sucrose non-fermentable (SWI/SNF), and (iv) INO80. ISWI and CHD complexes
help in initial DNA histone complexes to mature into nucleosomes. The important
role of ISWI is to create arrays of nucleosomes by placing nucleosomes at specific
distance from each other, thereby limiting chromosome accessibility and gene
expression. SWI/SNF family of proteins is involved in the ejection or sliding of
histones from the DNA, thereby promoting the binding of transcription factors and
other specific proteins. INO80 subfamily is involved in replacement of canonical
histone with variant histones. For example, they are involved in replacing canonical
histones H2A and H3 with other histone variants mediated by Snf2-related CBP
activator protein (SRCAP) and p400. This inclusion of variant histone may affect the
transcription factor recruitment, exclusion of proteins, and activity (Fig. 13.5).
Nucleosomal density has been found to be lower in the regions of functional
transcriptional factor binding sites. Once activators bind to the promoters, they
recruit other coactivators and general transcription factors to assemble
pre-initiation complex (PIC). Histone acetylation has also been important for gene
activation at the promoter region, and histone acetylases are also recruited by
activator proteins. Activators and coactivators also interact with chromatin
remodelers SWI/SNF complex to free the DNA from histones for the recruitment
of pre-initiation complex. Histones which are bound at the promoter region that are
active are lost after pre-initiation complex assembly which reassemble in the pro-
moter region once the genes are turned off. In large number of genes where partial
pre-initiation complex is present, the nucleosomes were found to be intact, whereas
when the RNA polymerase II binds and subsequently transcription is activated,
nucleosomes are removed from the DNA indicating the role of RNA polymerase
II itself as chromatin remodeler.
During transcription elongation, RNA polymerase is released from the general
transcription factors and travels into the coding region of the genome where it
encounters nucleosomal barrier. These barriers are overcome by transcription
machinery along with the help of chromatin remodeling complexes. During
606 A. A. Rangarajan

Fig. 13.5 Different functions of chromatin remodeling complexes. The ATPase-translocase sub-
unit of all remodelers is depicted in pink; additional subunits are depicted in green (imitation switch
(ISWI) and chromodomain helicase DNA binding (CHD)), brown (switch/sucrose non-fermentable
(SWI/SNF)), and blue (INO80). Nucleosome assembly: Particular ISWI and CHD subfamily of
chromatin remodeling complex participates in the deposition of histones, nucleosome maturation,
and spacing. Chromatin access: SWI/SNF subfamily remodelers alter chromatin by repositioning
nucleosomes, ejecting octamers, or evicting histone dimers. Nucleosome editing: Remodelers of the
INO80 subfamily (INO80C) change nucleosome composition by exchanging canonical and variant
histones, for example, and installing H2A.Z variants (yellow) (Figure taken with permission from
Clapier et al. 2017)

elongation, RNA polymerase II C-terminal domain undergoes two important phos-

phorylation events in which Ser5 and Ser2 are phosphorylated. These phosphoryla-
tion are important for recruitment of elongation complex along with chromatin
remodeling enzymes. PAF/RTF complex with the help of Spt4/5 binds
phosphorylated Ser5 and recruits chromatin remodeling complexes. PAF helps in
recruiting H3K4 methyltransferase Set1 complex (COMPASS) for elongating RNA
polymerase II. PAF complex associated with phosphorylated Ser5 is also important
for recruiting other chromatin regulators Chd1 and histone chaperone-like factors
Spt6 and FACT. These chromatin regulators help in clearance and sequestering
histones downstream of the elongating RNA polymerase II complex paving way for
the elongation.

13.2.2 Histone Modification

Histones are not only involved in the formation of nucleosomes, but histone
modifications play a crucial role in the regulation of gene expression. These
modifications are done by histone-modifying enzymes which bind to N-terminal
region of histone molecule that are exposed from the nucleosome. There are seven
major posttranslational modifications that are described to take place on tails of
13 Regulation of Gene Expression in Eukaryotes 607

histone: (1) acetylation, (2) methylation, (3) phosphorylation, (4) ADP ribosylation,
(5) glycosylation, (6) sumoylation, and (7) ubiquitylation. Each of these modifica-
tion results in distinct effect on gene regulation (Bannister and Kouzarides, 2011).

13.2.2.1 Histone Acetylation

Histone acetylation is one of the most well studied and first discovered posttransla-
tional modification in histone. All of the histone proteins (H2A, H2B, H3, and H4)
can be acetylated at several lysine residues. Acetylation cancels the positive charge
of histone molecules, thereby reducing the binding of histones to the DNA, which
helps in exposing the DNA for the binding of transcription factors and other
DNA-binding proteins. Thus, histone acetylation helps in activation of transcription.
The acetylation of several lysine residues results in combinatorial enhanced effect on
the structure of the nucleosome when compared to the single residue acetylation.
These are performed by histone acetyltransferase (HAT). Humans possess five
groups of HATs: HAT1, CBP/p300, GNAT, MYST, and other HATs. In contrast
to HATs, histone deacetyl transferase (HDAT) deacetylates the histone core and
restores the positive charge and binding of histone, resulting in transcriptional
repression. There are four classes of HDATs (class I, class II, class III, class IV).
Different classes of HDATs are associated with multiple complexes and have
functional redundancy (Luo and Dean, 1999).

13.2.2.2 Histone Methylation

Unlike histone acetylation, histone methylation leaves the charge of the histone
molecule unchanged and therefore does not affect the binding of histones to the
DNA directly. Histone methylation affects the binding of other chromatin
remodeling enzymes to the DNA which in turn has a positive or negative effect on
transcription. Histones are usually methylated at lysine or arginine residues. At
lysine residues, mono-, di-, or trimethylation can occur, and at arginine residues,
mono- and dimethylation can occur. Methylation has diverse effect on the status of
the transcription. H3K4 and H3K79 mono-methylation leads to transcription activa-
tion, whereas H3K9 and H3K27 dimethylations are associated with transcription
repression. Like acetylation, methylation of histones is regulated by histone
methyltransferase (HMTs) and histone demethylases (HDMs). HMTs can be
categorized into two categories: (i) lysine methyltransferases (KMTs) and protein
arginine methyltransferases (PAMTs). HDMs are further categorized into two
groups: Jumonji domain containing HDMs and lysine-specific demethylase 1 or
2. The former removes the methyl group using Jumonji domain, while the latter
removes methyl group with other complexes. Methylation regulates chromatin
structure by interacting with other chromatin remodelers. It was shown that
trimethylation of H3K4 recruits chromatin remodeling complexes such as ING
protein and CHD protein which in turn activates the transcription (Zentner and
Henikoff, 2013).
608 A. A. Rangarajan

13.2.2.3 Histone Phosphorylation

Histone phosphorylation leads to reduction of positive charge of histones eventually
leading to destabilization of histone-DNA binding, thereby enabling DNA access to
the protein complexes. Histone phosphorylation has been found to impact
nucleosomal structure, open the chromatin, and enable the binding of transcription
factors and DNA repair proteins. After the double-stranded break, the histones H2A
and H2A.X variant are phosphorylated which recruits the DNA repair machinery
and increases the accessibility of double-stranded break to the repair proteins. In
some cases, the phosphorylated histone protein can decrease the binding of proximal
methylated lysine with its binding partner. For example, phosphorylated H3S10
blocks the binding of H3K9me3 and K3K27 with HP1 and S28, respec-
tively (Banarjee and Chakravarti 2011).

13.2.2.4 Histone ADP Ribosylation

Histones can be mono- or poly-ADP ribosylated on glutamate and arginine residues.
ADP ribosylation adds negative charge to the histones, thereby loosening the contact
of histones with DNA leading to loosened chromatin state. Mono- and poly-ADP
ribosylation is mediated by ADP ribosyl transferases (ART) and ADP-ribose-protein
hydrolases (ARHs) or poly-ADP-ribose glycohydrolases removes the ADP ribose
subunit by hydrolysis. All four core histone proteins (H2A, H2B, H3, and H4) along
with the linker H1 can be ADP ribosylated. Poly-ADP ribosylation of H3, H2B, H4,
and H1 is induced by DNA damage, indicating their possible involvement in DNA
damage response mechanism (Messner and Hottiger, 2011).

13.2.2.5 Histone Sumoylation and Ubiquitylation

Unlike other posttranslational modifications in histones which result in smaller
modifications in the amino acid side chains, sumoylation or ubiquitylation leads to
large covalent modification by the addition of large peptides. In histone sumoylation
or ubiquitylation modifications, small ubiquitin-like molecules (SUMO) (100 or
fewer amino acids) or ubiquitin (76 amino acids) are added to the lysine residue of
the histones by the consecutive action of three enzymes: E1-activating enzyme,
E2-conjugating enzyme, and E3-ligating enzymes. Ubiquitylation can be reversed
by the action of isopeptidases. Histone ubiquitylation is usually observed on H2A
and H2B tail lysine residues. H2AK119 mono-ubiquitylation was shown to repress
transcription by its interaction with polycomb complexes. DNA double-stranded
breaks induce H2BK123 mono-ubiquitylation which plays an important role in
recruiting DNA repair proteins. All the core histone proteins undergo sumoylation
which antagonizes acetylation or ubiquitylation that can take place at the same lysine
residue. The molecular mechanism by which sumoylation affects the chromatin has
yet to be elucidated.

13.2.2.6 Other Histone Modifications

Other noncanonical histone tail modifications are histone deimination, addition of
histone O-GlcNAcylation, tail clipping, and protein isomerization. In histone
deimination, PADI4, which is a peptidyl arginine deiminase, converts arginine to
13 Regulation of Gene Expression in Eukaryotes 609

citrulline that cancels the positive charge of arginine. In histone O-GlcNAcylation,

some histones like H2A, H2B, and H4 undergoes modifications by β-linked
N-acetylglucosamine (O-GlcNAc) at serine or threonine residues. O-GlcNAc
catalyzes this reaction, whereas β-N-acetylglucosaminidase removes the monosac-
charide from the histone. In tail clipping, certain residues at the N-terminal tail
region of the histone are clipped. For example, the mouse enzyme Cathepsin L was
shown to remove the H3 histone N-terminal region during embryonic state cell
differentiation. Histone proline isomerization is mediated by proline isomerases
which catalyze the interconversion of cis and trans conformations in the peptide
bonds of proline. One of the known examples of proline isomerase is scFpr4 from
yeast which isomerizes H3P38. This modification is shown to be linked to H3K36
methylation which could probably affect the recognition of methyltransferase.

13.3 Gene Silencing by DNA Methylation

DNA methylation is a major epigenetic modification of eukaryotic genome, which

has profound effect on eukaryotic genome. DNA methylation usually results in
silencing of transcription. DNA methylation also plays a crucial role in important
processes involved in cell differentiation and developmental processes such as
genomic imprinting, spermatogenesis, and X-chromosome inactivation. DNA meth-
ylation also plays a role in silencing retrotransposons by preventing deleterious
recombination during embryonic development. DNA methylation is prominent in
lower eukaryotes to higher mammals, and the enzymes involved in the process are
highly conserved during evolution. DNA methylation is mediated by DNA
methyltransferases (Dnmt) which catalyze the transfer of methyl group from
S-adenyl methionine to the fifth carbon of cytosine residue to form 5-methyl
cytosine. There are two classes of DNMTs described according to their role:
(i) Dnmt1 and (ii) Dnmt3a and Dnmt3b. Dnmt1 takes part in DNA replication,
and methylation pattern from the parent DNA is copied to the daughter DNA strand
(Fig. 13.6a). Dnmt3a and Dnmt3b are involved in the formation of new methylation
pattern in the genome; thus, they are termed as de novo Dnmts. (Fig. 13.6b).

13.3.1 Mechanism of Gene Silencing by DNA Methylation

Nearly 4% of the human genome is methylated. The majority of DNA methylation

happens in the cytosine residue that precedes a guanidine or CpG island. CpG refers
to cytosine and guanine residue separated by a phosphate group. CpG islands are
found primarily near the promoter regions than at the other regions of the genome.
However, most of the CpG sites in the early embryonic stage and the somatic cells
involved in the cell differentiation are unmethylated to allow the expression of those
genes. The methyl group of methyl cytosine protrudes into the major groove of the
DNA, thereby directly repressing transcription by affecting the binding of several
DNA-binding proteins and transcription factors. Methyl-CpG binding proteins
610 A. A. Rangarajan

Fig. 13.6 Types of DNA methylation by DNA methyltransferases (Dnmts). (a) Dnmt1 maintains
the methylation pattern in the daughter strand during replication. Dnmt1 duplicates the methylation
pattern (gray) of the parent strand and makes the copy of methylation (red) onto the CpG sites in the
daughter strand (green). (b) Dnmt3a and Dnmt3b are the de novo methyltransferases that transfers
methyl group to the genomic DNA on the CpG sites

(MBDs) bind to methyl groups in the CpG-binding domains and recruit repressor
complexes resulting in transcriptional repression. This family of proteins includes
MeCP2, MBD1, MBD2, MBD3, and MBD4. Three of the MBD proteins, MBD1,
MBD2, and MeCP2, involved inhibition of transcription in methylation-dependent
manner. All the four MBD proteins associate with different corepressor complexes.
MeCp2 binds to mSin3a corepressor complex to achieve repression. MBD2 is a part
of MeCp1 repressor complex, and it is involved in DNA binding. MeCp1 is a
multiprotein complex that contains Mi-2-NuRD (nucleosome remodeling histone
deacetylase) repressor, comprising of histone deacetylases and chromatin
remodeling protein Mi-2. MBD3 also interacts with Mi2-NuRD repressor com-
plex (Newell-Price et al., 2000 and Moore et al. 2013)

13.3.2 DNA Methylation and Its Interaction with Other Chromatin

Modifiers

However, the binding of this complex needs deacetylation of histones. DNA meth-
ylation and the associated factors also act together with the histone modification
system. Histone acetylation usually results in activation of transcription, and DNA
methylation pattern is negatively correlated with histone acetylation. Dnmt1 and
Dnmt3b binds to histone deacetylases and deacetylating histones which results in
13 Regulation of Gene Expression in Eukaryotes 611

condensed chromatin and transcriptional silencing. Dnmt1 and Dnmt3a bind to

histone methyltransferase SUV39H1 which inhibits transcription by methylating
H3K9. H3K9me-2-specific methyltransferases G9a/GLP binds to Dnmt1 enzyme
and enables de novo methylation of retrotransposons in embryonic stem cells. MBD
proteins also interact with several histone deacetylases (HDACs) to achieve
corepression. UHRF1 (ubiquitin-like PHD and RING finger domain containing 1)
protein binds to methylated H3K9 and maintains the activity of Dnmt1 enzyme.
H3K4 methylation inhibits the CGI methylation, while most of the CpG sites are
subjected to methylation by Dnmts. H3K4 methyltransferase Setd1 is recruited to
CGI sites though its interaction with Cfp1 that binds to unmethylated CpG sites. This
recruitment enhances the H3K4 methylation at these sites in CpG sites that are
unmethylated. Apart from histone-modifying enzymes, DNA methylation also plays
a role with ATP-dependent chromatin-remodeling enzymes. MBD3 protein interacts
with Mi2-NuRD repressor complex. This complex also contains Mi2β which is a
part of SWI/SNF family of chromatin remodelers that destabilize histone DNA
interaction.

13.3.3 Demethylation of DNA

Although DNA methylation patterns can be transferred from mother cell to daughter
cells, the methylation pattern changes during different processes of development. It
changes due to physiological cues during development or as a pathological response
to cancer and aging processes. DNA demethylation can take place via active and
passive mechanisms. Active demethylation involves Ten-eleven translocation
enzymes (tet) that add a hydroxyl group to methyl group of 5-methyl cytosine
which converts it to 5-methylhydroxycytosine. Tet enzyme can also mediate oxida-
tion of 5-methylcytosine to form 5-formylcytosine or 5-carboxylcytosine. In passive
mechanism, Dnmt1 enzyme is either absent or inhibited, thereby inhibiting the
methylation of cytosine during cell division. Active DNA demethylation takes
place in germ cell during development and also in somatic cells in gene-specific
manner. During embryonic development at stage E7.5, posterior epiblast cells are set
to be formed as primordial germ cells (PGCs). During migration to the genital ridge,
it has a similar epigenetic pattern to that of epiblast cells. Incidentally, by the time it
reaches the genital ridge at stage E11.5, most of the epigenetic markers are changed
including the deletion of DNA methylation pattern which induces transcription of
several genes. In somatic cells, demethylation happens in locus-specific manner at
brain-derived neurotrophic factor (Bdnf) when the neurons are depolarized. When T
cells are active and when there is no DNA replication, the enhancer region of
interleukin-2 gets demethylated.
612 A. A. Rangarajan

13.3.4 DNA Methylation and Diseases

DNA methylation is crucial for regulation of gene expression in several physiologi-

cal and developmental processes. Alteration in the DNA methylation pattern results
in several diseases including cancer since methylation is an epigenetic modification
and 5-methylcytosine, in which cytosine can be deaminated to thymine, is passed to
the daughter cells. Thus, methylated CpG island acts as a hotspot for mutations to
occur. MBD4 contains thymine DNA glycosylase that recognizes T-G mismatch and
corrects 5mc deamination. Methylation of the CG1 sites at the FMRI gene 50 end
causes fragile X syndrome. Fragile X syndrome patients have 200 copies of CGG
sites at their 50 end, whereas normal individual possesses 50 sites. Because of the
presence of 200 CGG sites, these regions are silenced by the de novo
methyltransferases resulting in silencing of FMRI gene. Mutation of Dnmt1 is
associated with T-cell lymphomas, whereas Dnmt3 mutation results in chromosomal
aberrations including chromosomal fusions and aneuploidy. Mutations in MECP2
results in Rett syndrome which is a neurological disorder. Rett syndrome is
associated with females who are heterozygous for mutations in the X-linked
MECP2 gene. Rett syndrome is characterized by impaired motor skills, microceph-
aly, abnormal breathing, and other associated neurological complications. Global
DNA hypermethylation is also involved in the development and progression of
cancers. When DNA methylation is reduced with DNA methyltransferase inhibitors,
it resulted in suppression of some forms of cancer. On the other hand,
hypomethylation could also result in some forms of cancer such as cervical cancer,
ovarian cancer, colon cancer, and hepatocellular carcinomas (Li and Zhang, 2014).

13.4 Alternative Splicing of mRNA

In eukaryotes, single gene can code for multiple proteins, and the eukaryotic mRNA
contains both introns (noncoding region) and exons (coding region). However,
before translation into proteins, the noncoding introns should be removed from the
mRNA, whereas the coding exon must be joined. This process of trimming introns
and combining several combinations of exon regions of mRNA to generate different
proteins is called splicing. This process increases the functional diversity of a gene
by the formation of several proteins and adds to another level of gene regulation.
When splicing process is tampered, it results in disruption of cellular functions
leading to disease. RNA splicing was first discovered in adenovirus and later was
discovered in other eukaryotes. The mechanism and the proteins involved in splicing
reaction are highly conserved among different genera of eukaryotes.

13.4.1 Molecular Mechanism of Splicing

The process of splicing is mediated by dynamic macromolecular ribonucleoprotein

(RNP) complex known as the spliceosome. There are two types of spliceosome
13 Regulation of Gene Expression in Eukaryotes 613

Fig. 13.7 Schematic representation of esterification reaction of pre-mRNA splicing. E1 and E2

represent exons. The solid line represents introns. The branch site adenosine is represented by letter
A. The phosphate group is represented as (p) in 50 and 30 splice sites. (Figure adapted with
permission from Will and Lührmann 2011)

complex prevalent in eukaryotes: U2-dependent spliceosome that catalyzes the

eviction of U2 type of introns and the less common U-12-type spliceosome. There
are several cis-acting and trans-acting factors which act together to ensure precise
splicing reaction. Intron is recognized by 30 splice site, branch site, and 50 splice site.
The 50 splice site is characterized by GU at the 50 end of intron which is followed by
the branch site. Branch site is located usually 18–40 nucleotides upstream of 30 splice
site which is usually characterized by A nucleotide. The branch site is succeeded by
several pyrimidine residues called polypyrimidine tract. This is flowed by 30 splice
site at the end which is characterized by AG nucleotide. Additionally, there exist
other cis-acting regulatory elements such as exonic and intronic splicing enhancers
(ESE and ISE) and exonic and intronic splicing silencers (ESS and ISS). They
facilitate the binding of regulatory proteins which enhances or represses the action
of the spliceosome complex at the nearby splice site. The most prominent splicing
repressors consist of heterogeneous ribonucleoproteins (hnRNPs) family and
polypyrimidine tract-binding protein (PTB) family. The introns are generally
cleaved by two esterification reaction. In the first reaction, the 20 OH located at the
branch site adenosine performs nucleophilic attack on the 50 splice site and cuts
them. The 50 end region of the phosphate is ligated to the branch adenosine which
forms a lariat structure. In the second step, the 30 OH of the 30 splice site is cleaved
which leads to ligation of 50 and 30 exons forming the mature mRNA (Fig. 13.7).
Spliceosome is assembled in the ordered fashion by snRNPS (small nuclear
ribonucleoproteins) and other associated proteins to precisely recognize the splice
sites and perform the splicing reaction. In the first reaction, U1 snRNP recognizes
and binds the 50 splice site forming complex E. In the next step, SF1 and U2AF
proteins interact with branch site and polypyrimidine tract. This is succeeded by the
binding of U2snRNP binding to branch site forming A complex. The preassembled
U4/U6.U5 tri-snRNP is recruited which generates precatalytic B complex. After this,
protein and RNA rearrangements lead to dissociation of U1 and U4 snRNPs which
leads to activated B complex. Prp2 which is an RNA helicase catalytically activates
B* complex which performs the first step of splicing reaction. Finally, the process
leads to the formation of C complex which performs the next catalytic step. Finally,
the spliceosome complex disassembles and further performs next rounds of splicing
reaction (Fig. 13.8).
614 A. A. Rangarajan

Fig. 13.8 Steps involved and assembly and disassembly of spliceosome complex. Various
snRNPS are indicated in circles. Exons are indicated by colored boxes, and introns are represented
by solid lines. The other associated proteins which facilitate conformational changes in various
complexes, such as Prp5, Sub2/UAP56, Prp28, Brr2, Prp2, Prp16, Prp22, Prp43, and Snu114, are
indicated. (Figure adapted with permission from Will and Lührmann 2011)

13.4.2 Modes of Alternative Splicing

In many cases, the mRNA can be alternatively spliced by including or excluding

specific introns or exons leading to wide variety of proteins from single mRNA. This
mechanism is known as alternative splicing. More than 95% of human mRNA
undergoes alternative splicing in tissue-, development-, or signal transduction-
specific manner. This accounts for the formation of more than 90,000 proteins
from approximately 25,000 coding region.
There are five different modes of alternative splicing that were observed. (a) Exon
skipping: In exon skipping, coding exon is spliced out from the primary transcript.
Exon skipping is the usual type of alternative splicing that occurs in mammalian
cells. (b) Mutually exclusive exons: In this type, one of the two exons is retained
while the other is skipped. (c) Alternative 50 splice site: In this type, noncanonical 50
splice site is used which modifies the 30 boundary of upstream exon. (d) Alternative
13 Regulation of Gene Expression in Eukaryotes 615

Fig. 13.9 Types of alternative splicing: In this figure, introns are indicated by solid black line
between the exons. Exons are represented in different colored boxes, and the splicing events are
represented in blue

30 splice site: In this type, noncanonical 30 splice site is used which modifies the 50
boundary of downstream exon. (e) Intron retention: In intron retention, some introns
are retained without being spliced in the messenger RNA which gets translated to
proteins along with other exons (Wang et al., 2015) (Fig. 13.9).

13.4.3 Alternative Splicing and Disease

When splicing machinery is disrupted, it leads to mis-splicing of several transcripts

leading to mutant gene transcripts and aberrant proteins. Thus, changes in the
splicing mechanism lead to several diseases including cancer. Many mutations
associated with human in disease conditions are found in splicing components.
Spinal muscular atrophy, a neurodegenerative disease, is caused due to the mutation
of SMN1 gene-coding SMN protein which regulates snRNP assembly. Hutchinson-
Gilford progeria is a genetic disorder whose symptoms include premature aging.
This is caused by the mutation in the intronic splice site which leads to the formation
of cryptic splice site. Thalassemia is caused by abnormality in the hemoglobin gene
which leads to abnormal levels of red blood cells. This is caused due to the mutation
at the 50 splice site of hemoglobin gene. Apart from these, alterations in the splicing
616 A. A. Rangarajan

causes several forms of cancer. Changes in splicing was observed in cancer cells in
CD44, which is a transmembrane glycoprotein. Nonsense mutation in the exon 18 of
the BRCA1 gene which interrupts the exonic splicing enhancer causes breast and
ovarian cancer. Mutation of 659 codon of MlH1 gene which results in exon skipping
is responsible for hereditary nonpolyposis colorectal cancer (Tazi et al., 2009).

13.5 RNA Interference

RNA interference is a phenomenon in which 20–30 nt noncoding RNA inhibits

translation by binding to target mRNA molecule. RNA interference was initially
discovered in C. elegans for which Nobel Prize was awarded in the year 2006 to
Andrew Fire and Craig C. Mello. Both microRNA (miRNA) and short interfering
RNA (siRNA) play a key role in RNA interference. Although RNA interference was
initially discovered in C. elegans, the phenomenon was also later discovered in
plants, fungi, Drosophila, and other mammals.

13.5.1 MicroRNA

MicroRNAs (miRNAs) are genetically encoded noncoding RNA that involves in

regulation of genes. Approximately 5% of human genome produce more than 1000
miRNAs which regulate at least 30% of the genes. MicroRNAs regulate several
processes including cell growth, differentiation, heterochromatin regulation, and cell
proliferation. The dysfunction of this process is implicated in neurological disorders,
cardiovascular diseases, and cancer. A miRNA is processed from primary miRNA
(pri-mRNA) in the nucleus. It is typically 1000 nt long consisting of single or
clustered double-stranded hairpins. This pri-mRNA is further processed by the
microprocessor complex that comprises of Drosha and dsRNA-binding protein
DGCR8. After processing 65–70 nt, stem loop structure containing miRNA is
produced.

13.5.2 Small Interfering RNA

Small interfering RNA (siRNA) is generally exogenously produced: synthetic- or

viral-induced interfering RNA. siRNAs have well-defined structure containing
20–24 nt long dsRNA phosphorylated at 50 ends and hydroxylated at 30 end with
two overhanging nucleotides. In the next step, Dicer enzyme catalyzes the formation
of siRNAs from long dsRNAs. siRNAs are of immense important for studying gene
function by knocking out specific genes. siRNA can be easily delivered into the cell
by transfection.
13 Regulation of Gene Expression in Eukaryotes 617

13.5.3 Molecular Mechanism of RNA Interference

RNA interference on mRNA targets by exogenous (siRNA) and endogenous

(miRNA) origin is regulated by RNA-induced silencing complex (RISC). The first
step of RNA interference involves processing of RNAs which can be loaded onto the
RISC complex. Endogenous miRNA is processed from pre-miRNA which leads to
formation of characteristic stem loop structure in the nucleus, and further is
transported to cytoplasm. Exogenous siRNA (of viral origin or synthetically made)
is transported to cytoplasm. At cytoplasm, it is cleaved into short RNA fragments by
Dicer enzyme. Dicers are large endonucleases containing a helicase domain,
dimerized pair of RNase III domain that cleaves the RNA and PAZ domains (from
the names of the proteins PIWI, Argonaute, and Zwille proteins), which specifically
binds to 30 overhangs of RNA. After processing with Dicer, dsRNA is formed which
is a duplex of 20 to 24 nt long with 2 nt overhang at 30 and a phosphate group at the
50 end.
In the subsequent step, Dicer is aided by dsRNA-binding protein (dsRBP) to load
the dsRNA complex to Argonaute protein of RISC. Dicer, Argonaute, and TRBP
constitute minimum RISC loading which is responsible for generating double-
stranded RNA and loading onto the Argonaute protein. After presenting the
dsRNA to the Argonaute, the 30 and 50 end of the guide strand is bound by the
PAZ proteins and MID domain of the protein forming the RISC. While the complex
is loaded onto RISC, only one strand of RNA is attached to which is necessary
silencing while the other strand is removed. The former is known as guide strand,
and the latter is known as passenger stand, respectively. In the case of miRNA, the
loaded strand from the duplex is referred to as miRNA, and the opposite strand is
known as miRNA*. In the next step, RISC performs searching for the target mRNAs
and binds to single-stranded mRNA which is complementary to the guide strand.
The seed sequence constituting 2–6 bp of the guide strand initializes the binding to
the target. Once it is bound to the target, RISC cuts the mRNA between 10 and 11 nts
in a non-ATP-dependent manner. The target mRNA is further degraded preventing
translation of it into proteins. In case of miRNAs, rather cleaving miRNAs bind to
the 30 untranslated regions of the target mRNA with imperfect complementarity
which blocks the binding of ribosome preventing translation (Wilson and Doudna,
2013) (Fig. 13.10).

13.5.4 Applications of RNA Interference

RNA interference is a very useful biological tool to study the function of genes.
siRNAs are designed for gene of interest and transfected into the cells, thereby
decreasing the level of gene expression. The siRNAs may not completely abolish the
expression of gene but can decrease it to a significant level. The length and type of
siRNA used vary according to the species. In mammals, short RNAs are used since
long RNAs elicit immune response. Functional genomics applications of RNA
interference have been extensively used in studying the gene functions of
618 A. A. Rangarajan

Fig. 13.10 Molecular mechanism of RNA interference. Double-stranded RNA (dsRNA) is

cleaved by Dicer (green) and dsRBP-like TRBP (magenta). The resulting RNA duplex is bound
by Argonaute protein. In the subsequent step, the passenger strand is removed, and the guide strand
attached to Argonaute protein which forms the RISC. The RISC binds to complementary mRNA
(blue) and degrades them by the action of Argonaute protein
13 Regulation of Gene Expression in Eukaryotes 619

C. elegans and Drosophila melanogaster since characteristic phenotype generated

by RNA interference is heritable, and simple delivery of siRNA makes it attractive
tool for studying gene functions in C. elegans. RNA interference has also been
extensively used for mapping genome and annotation. RNA interference has also
been used in functional genomic studies in plants such as wheat, Arabidopsis, and
maize.
RNA interference technology also has many therapeutic applications. Many
preclinical studies have shown efficient suppression of target genes, making it an
attractive tool in clinical therapy. RNA interference treatment was initially proposed
as antiviral strategy targeting several viral RNAs such as hPV, HIV, hepatitis B,
hepatitis A, influenza virus, SARS coronavirus, and Adenovirus. Many clinical trials
have shown successful deregulation of oncogenes (cancer-causing genes), making it
as an attractive option for treatment of various cancer. Since RNA interference can
target-specific genes, it does not affect the function of other genes, thereby increas-
ing the target specificity of cancer treatments which is a major roadblock with
chemotherapeutic approach. RNA interference has also shown potential in treating
neurological diseases such as Alzheimer’s, Parkinson’s, and neuroglutamine
diseases. RNA interference has also played a significant role in biotechnology in
the development of wide variety of crops. It has resulted in the invention of unique
type of crops such as tobacco free from nicotine, coffee plants free from caffeine, and
nutrient-enhanced plants. By targeting polyphenol oxidase gene by RNA interfer-
ence in apples, browning of apples after cutting was abolished, since polyphenol
oxidase-silenced apples cannot convert chlorogenic acid into quinone product which
causes browning of apples (Agrawal et al., 2003).

13.6 Posttranscriptional Regulation of Gene Expression

Posttranscriptional control plays a crucial role in the control of gene expression. It

usually represents control of RNA level between the stages of protein transcription
and translation. The stability, the distribution, and the level of mRNA determine the
quality and level of various proteins. Thus, the stability and maturation of mRNA is
highly regulated, and it varies during development or different environmental
conditions.

13.6.1 Control of mRNA Degradation

The mRNA molecules are usually synthesized by RNA polymerase II and are
relatively less stable molecules. The typical structure of mRNA contains cap struc-
ture at the 50 region and polyadenylation site at the 30 region which protects them
from degradation. However, many mRNAs do not result in the translation into
proteins, and after the translation of proteins, the mRNA has to be degraded;
otherwise, it would result in overproduction of proteins. The mRNA degradation
pathway in eukaryotes begins with the process of deadenylation of 30 end. The
620 A. A. Rangarajan

Fig. 13.11 Depiction of different routes of mRNA degradation. Poly-A-binding protein (blue) is
bound to 30 polyadenylation region, and 50 region is bound by cap. Enzyme Dcp-1 (red) and Dcp-2
(orange) complex cleaves the 50 cap region leaving the monophosphate at 50 end. This is attacked by
Xrn-1 endonuclease which cleaves the mRNA in 50 to 30 direction. Deadenylases (yellow) cleave
the polyadenine residues at the 30 end. This is then targeted by multiprotein exosomal degradation
complex (yellow) which cleaves the mRNA in 30 to 50 direction

process deadenylation is the most critical step in the mRNA degradation. Later, the
50 cap will be removed by the process of decapping, and the RNA will be degraded in
50 to 30 direction by Xrn-1 endonuclease and 30 to 50 direction by exosomal complex
(Fig. 13.11).

13.6.1.1 30 Deadenylation
The poly-A tail length at the 30 region of the mRNA determines the stability and
mRNA transport and translation. The poly-A tail region is usually bound by poly-A-
binding proteins (PABP) which facilitate translation and shortening of the poly-A
tails enabling 30 to 50 exonuclease activity. Poly-A tail reduction sends signal for
mRNA degradation to several pathways such as nonsense-mediated mRNA decay
(degradation because of premature stop codon), ARE-mediated decay (mRNAs
possessing AU-rich elements), and miRNA-mediated decay. In eukaryotes, PAN2-
PAN3 and CCR4-NOT are two important protein complexes involved in
deadenylation.
13 Regulation of Gene Expression in Eukaryotes 621

13.6.1.2 Decapping
Another way of destabilizing and getting access to the mRNA is by decapping the 50
end of the mRNA. The process of decapping is performed by Dcp-2 enzyme
belonging to Nudix hydrolase family which is conserved among the eukaryotes.
Dcp-2 cleaves the mRNA using the Nudix motif, which is triggered by Dcp-1
enzyme, resulting in m7GDP and 50 monophosphate RNA. Dcp-2 enzyme prefers
enzyme that are at least 25 nt in length.

13.6.1.3 50 to 30 mRNA Degradation by Xrn-1 Endonuclease

Decapped mRNA with the exposed 50 phosphate end is amenable to degradation by
Xrn-1 endonuclease. Xrn-1 processively cleaves the mRNA from 50 to 30 direction.
Decapping and Xrn-1 endonuclease activity are coupled, in which Xrn-1 interacts
with one of the decapping complex protein Dcp-1 or its homologues.

13.6.1.4 30 to 50 Degradation by Exosomes

In some cases, mRNAs can also be degraded in 30 to 50 direction. Typically, this type
of 30 to 50 mRNA degradation is usually mediated by exosome machinery. The 30
end of the RNA is targeted by multi-subunit enzyme complex exosome for degrada-
tion and processing. The components of exosome are highly conserved in
eukaryotes. Six subunits, Rrp41, Rrp42, Rrp43, Rrp45, Rrp46, and Mtr3, forms
the hexameric core ring. Rpr4, Rpr40, and Csl4 bind to RNA though their KH motif-
binding domain, and Rpr44 contains the PIN domain which attaches the exosome
ring and performs 30 to 50 endonucleolytic cleavage. After the degradation by
exosome complex, the remaining m7GpppN is hydrolyzed by m7G-specific
pyrophosphatase DcpS. DcpS is called scavenger decapping enzyme which has
specificity for small RNA fragments hydrolyzing the remaining cap region.

13.6.1.5 Other Types of Aberrant mRNA Degradation

When mRNA was aberrantly synthesized, it results in the formation of aberrant
proteins; therefore, such mRNAs have to be identified and degraded. One way of
removing these aberrant transcripts is nonsense-mediated mRNA decay (NMD) in
which mRNAs containing premature termination codon is identified and degraded.
NMD also degrades RNA with long 30 untranslated region (UTR), mRNAs
possessing an upstream ORF at the 50 UTR, and mRNAs containing introns at the
30 UTR. The effect of NMD is dependent on many cis-acting elements in the mRNA
and the exon junction complex (EJC) components. The NMD factors are highly
conserved among the eukaryotes. The key protein of NMD pathway is UPF1 which
is an ATP-dependent RNA helicase. UPF1 interacts with UPF2, an acidic protein
which in turn interacts with UPF3, an RNA-binding protein. UPF1 with UPF2 and
UPF3 enhances degradation of mRNA by endonucleolytic cleavage, 50 to 30 or 30 to
5 mRNA degradation.
In some cases, error in the mRNA causes stalling of the ribosome blocking further
translation. It may lead to No-go decay (NGD), nonstop decay (NSD), or nonfunc-
tional 18srRNA decay (NRD). Dom34-Hbs1 protein takes part in all of these
pathways. When elongation is blocked by secondary mRNA structures or while
622 A. A. Rangarajan

translating several positively charged amino acids, it results in No-go decay (NGD).
Consequently, the aberrantly formed protein is degraded by ubiquitin-based protein
degradation. When transcripts lack in-frame stop codon, it results in continues
translation, and the nonstop decay (NSD) is triggered by cryptic polyadenylation
signal in which ribosome is stalled. Defects in the ribosomal 18srRNA caused by
mutations or by improper biogenesis can lead to translational stalls which lead to
nonfunctional 18srRNA decay (NRD) (Newbury, 2006 and Siwaszek, et al., 2014).

13.6.2 Control of Protein Degradation

Protein degradation is one of the important cellular machinery which determines the
half-life of proteins. The half-life of protein varies between different kinds of
proteins ranging from minutes to hours. The proteins that are turned over rapidly
act as regulatory protein in other processes such as transcription factors. This rapid
turning-over process enables to increase or decrease the levels of the protein in
accordance with the external stimuli. Mistranslated and aggregate proteins are also
recognized and degraded rapidly by the protein control machinery, the accumulation
of which otherwise is detrimental to the cellular processes (Glick et al, 2010). Protein
degradation in eukaryotes takes place by two well-studied systems, namely,
lysosome-mediated autophagy and ubiquitin proteasome pathway.

13.6.2.1 Autophagy
Autophagy plays an important role in removal of misfolded and aggregate proteins;
degrades damaged organelles such as peroxisomes, mitochondria, and endoplasmic
reticulum; and targets intracellular pathogens. The process of autophagy involves
lysosomal-mediated protein degradation by several autophagy-related proteins
(ATG). Autophagy is classified into three types, namely, macro-autophagy
(autophagy), micro-autophagy, and chaperone-mediated autophagy. In all of these
types, proteolytic degradation is mediated by cytosolic machineries of the lysosome.
(i) In macro-autophagy (autophagy), the degradation of cytosolic membrane takes
place by sequestering into membrane-bound vesicle called autophagosome which is
then fused to the lysosome. (ii) In micro-autophagy, the lysosomal components are
directly taken into the lysosome by lysosomal invaginations. In chaperone-mediated
autophagy, chaperone proteins such as Hsc-70 help in translocating the proteins
across the lysosomal membrane. These chaperone proteins are recognized by the
lysosomal-associated protein 2A (LAMP-2A) which binds to target protein and
degrades them. The macro-autophagy (also referred to as autophagy) is a complex
process orchestrated by several proteins on consequential manner. It involves
(i) formation of phagophore, (ii) autophagosome formation, and (iii) lysosome
fusion and degradation (Fig. 13.12).
(i) Formation of phagophore: The formation of phagophore involves activation by
conserved nutrient sensors such as mTOR and AMP-activated kinase (AMPK)
in which mTOR acts as an inhibitor and AMPK acts as an activator. It activates
the two assembly complexes, ALGI/PLKI and P13K complex, which help in
the formation of phagophore.
13 Regulation of Gene Expression in Eukaryotes 623

Fig. 13.12 Molecular mechanism of autophagy. Autophagy is controlled by signals from mTOR
and AMPK pathway which depends on the activity of ATG-1/ILK-1 and P13K complexes. In the
subsequent steps, ATG5-ATG12:ATG16L conjugates LC3 to lipid phosphatidylethanolamine to
form LC3-II which enables the anchoring to autophagosomal membrane. It fuses to the lysosome
via LAMPs and RAB7, and hydrolases of lysosome degrades the molecules and releases the
products for further recycling

(ii) Autophagosome formation: Autophagosome formation consists of two conju-

gation steps. In the first step, ATG7 and ATG10 mediate the conjugation of
ATG5, and ATG12 proteins interact and bind to ATG16L. In the next step,
ATG7 and ATG3 along with ATG5-ATG12:ATG16L complex conjugate LC3
to lipid phosphatidylethanolamine (PE) to form LC3-II that allows the binding
of the complex to the autophagosomal membrane.
(iii) Lysosome fusion and degradation: The mature autophagosome then fuses with
the lysosome mediated by lysosomal proteins such as LAMPs and RAB7. Once
it is fused, the targeted protein or organelle is digested by several lysosomal
hydrolases into final products consisting of amino acids, lipids, and nucleotides
which translocate to the cytoplasm and get incorporated into the cellular
processes (Fig. 13.12).

13.6.2.2 Ubiquitin-Proteasome Pathway

Selective protein hydrolysis in eukaryotic cells takes place via ubiquitin-proteasome
pathway in which cytosolic and nuclear proteins are marked with ubiquitin which is
targeted for rapid proteolysis. Ubiquitin is an 8.6 kDA regulatory protein that is
highly conserved among the eukaryotes. Ubiquitin attaches to the lysine residues of
the protein via an isopeptide bond. Additional ubiquitin residues are added to the
ubiquitin residue forming polyubiquitin chain. Such polyubiquitylated chains are
recognized by the 26S proteasome complex that degrades the proteins and releases
the ubiquitins which are used in the next cycle.
624 A. A. Rangarajan

Fig. 13.13 The ubiquitin-proteasome pathway: The ubiquitin residues are activated by
ATP-dependent manner by E1 ubiquitin-activating enzyme. It is then conjugated to E2 via E2
ubiquitin-conjugating enzyme. This complex interacts with E3 which interacts with E2 and target
protein and transfers the ubiquitin to the target protein. After polyubiquitylation by addition of
ubiquitin chains, the substrate is targeted to 26S proteasome where the proteins are deubiquitylated
and the target protein is degraded and the released ubiquitin is recycled for the next round

The process of ubiquitination consists of three steps: (i) activation,

(ii) conjugation, and (iii) ligation.

(i) Activation: This process involves two steps and is catalyzed by E1 ubiquitin-
activating enzyme. E1 enzyme binds to both ATP and ubiquitin and catalyzes
the reaction in which acyl adenyl group is transferred to C-terminus of
ubiquitin. In the next step, ubiquitin is transferred to cysteine residue with the
release of AMP.
(ii) Conjugation: This step is catalyzed by the E2 ubiquitin-conjugating enzymes.
In this process, the E2 enzyme catalyzes trans(thio)esterification reaction in
which ubiquitin is transferred from E1 to E2.
(iii) Ligation: This step is catalyzed by E3 ubiquitin ligases. In this step, E3 ligase
interacts with the E2 and target protein. It then mediates isopeptide bond
formation between lysine residue of the target protein and C-terminal glycine
residue of ubiquitin. There are several hundreds to ubiquitin ligases present.
Polyubiquitylation takes place by the addition of additional ubiquitin residues
via one of the seven lysines that is present in the ubiquitin.
13 Regulation of Gene Expression in Eukaryotes 625

The 26 s proteasome is about 2000 kDa consisting of one 20S protein subunit and
two regulatory cap subunits. The hollow core in the central region enables forming
cavity in which protein degradation takes place. Each end of the proteasome contains
19S regulatory cap proteins which possess multiple ubiquitin-binding sites. These
cap proteins recognize the ubiquitin-tagged proteins that enable transferring them to
the catalytic core where the protein hydrolysis takes place (Fig. 13.13).

13.6.2.3 Mechanism of Protein Degradation by Ubiquitin Proteosome

Polyubiquitylated substrate is recognized by the intrinsic ubiquitin receptors proteins
of 19S cap proteins. The ubiquitin receptor proteins contain N-terminal ubiquitin-
like domain (UBL) and few or more ubiquitin-associated domain (UBA). A 19S
proteasome cap recognizes the UBL domains, and the UBA domains bind ubiquitin
through three helix bundles. The ubiquitin chain is removed from the substrate by
deubiquitylase enzyme, Rpn11, and the ubiquitin chains are recycled. The substrate
is further extended by the Rpt1–6 ATPases and transferred to the 20S proteasomal
complex for degradation. The 20S core particle mediates proteolysis by the
β-subunits via threonine-dependent nucleophilic attack. Degradation of protein
takes place in the central chamber by the action of two β rings and degrades the
substrate to short polypeptides of 7–9 residues long. In some cases, it forms
biologically active and functional molecule such as NF-κB which plays a crucial
role in immune response.

13.6.2.4 Ubiquitination and Diseases

Ubiquitination process is an important posttranslational modification which is
involved in processes such as cell cycle progression, proliferation, and development.
When ubiquitin pathway is disrupted, it results in several diseases. High levels of
ubiquitin in the brains cause decreased level of amyloid precursor protein which
plays a key role in triggering Alzheimer’s disease. Mutation in ubiquitin B resulting
in truncated peptides have also been observed in Alzheimer’s disease and
tauopathies. Mutations in ubiquitin E3 ligase cause several diseases such as
Angelman syndrome, Fanconi anemia, and 3-M syndrome. Mutation of E3 ligase
is also implicated in several types of cancer such as renal cell carcinoma, breast
cancer, colorectal cancer, and glioblastoma (Dikic, 2017 and Saeki, 2017).

Box 13.1: Scientific Concept: Quantifying Site-Specific Chromatin

Mechanics and DNA Damage Response
DNA is wound around the histones forming highly condensed structure called
chromatin. The chromatin is found to be highly dynamic with several conden-
sation states rather than more rigidly defined euchromatin and heterochromatin
region. Whitefield et al. (2018) studied the dynamics of transcriptionally active
and inactive region using single particle tracking and also analyzed the
chromosome dynamics during DNA damage. They used 96 tetracycline

(continued)
626 A. A. Rangarajan

Box 13.1 (continued)

response elements (TRE) named U2OS TRE that allows controlling gene
expression be the binding of transcription activator (TA) or transcription
repressor (TetR) tagged with mCherry. GFP-tagged fibrillarin was transfected
to visualize bulk chromatin motion. The tracking revealed that transcription-
ally active regions were indistinguishable from chromosomal bulk dynamics
motion, whereas transcriptionally repressed regions showed decreased mobil-
ity compared to transcriptionally active region and chromosomal bulk dynam-
ics (Fig. 13.14a and b).
During DNA damage such as double-stranded breaks, the nucleosome must
reorganize itself for DNA, and histones have to be displaced in order to splice
back the DNA by repair proteins. Whitefield et al. (2018) studied the effect of
DNA damage by using KillerRed-labeled cells to induce DNA damage with
transcriptional activator (TA) or repressor (TetR). In transcriptionally
repressed regions, induction of DNA damage increased the chromosomal
mobility similar to that of the bulk chromosome dynamics (Fig. 13.14d). In
contrast, upon DNA damage, transcriptionally active regions undergo
dynamic changes in a time-dependent manner which results in chromatin
relaxation and reduction in propagation at these regions (Fig. 13.14c). The
overall effect of it decreases the large-scale chromatin motions, thereby reduc-
ing improper repair and translocations in the chromosome. These conclusions
give us the concept that the human genome is present in variety of chromatin
states rather than previously determined two states of euchromatin and
heterochromatin.

Box 13.2: Scientific Concept: Transcriptional Gene Silencing in Humans

Transcriptional gene silencing by small noncoding RNAs was extensively
studied in yeast (Saccharomyces pombe), plants (Arabidopsis thaliana), and
worms (C. elegans) and was later found to also control gene transcription and
epigenetic states in human cells. Transcriptional gene silencing (TGS) can
cause long-term epigenetic effect on the genes that can be transferred to the
daughter cells in contrast to RNA interference. Endogenous transcriptional
gene silencing was shown to be facilitated by microRNAs (miRNAs). In 2005,
FANTOME and ENCODE consortia revealed that human genome encodes
large number of long noncoding RNAs (lncRNAs) which are more than 200 nt
in length, and many of them are antisense to the protein coding counterparts.
Later, the biological role of lncRNA was revealed in the X-chromosome
inactivation and regulation of p15 and p21 tumor-suppressor genes and several
other genes such as BDNF, MYCN, DHRS4, KCNQ1, NBAT, and HIV-1.

(continued)
13 Regulation of Gene Expression in Eukaryotes 627

Box 13.2 (continued)

Small RNA mediates epigenetic transcriptional gene silencing (TGS) by
making use of template mRNA at the specific promoter and further recruits
epigenetic silencing complex which consists of DNMT3a, Ago1, EZH2, and
HDAC1. Long noncoding RNAs also work with similar epigenetic silencing
complexes which are targeted to the specific loci for epigenetic silencing. In
human cells, both small and long noncoding RNAs mediate its action via
DNMT3a which in turn regulates the epigenetic state of the target genes
(Fig. 13.15). This is of particular interest because of its potential to be long-
lasting and heritable which could play a significant role in therapeutics. These
new findings provide us the hope of using small and long noncoding RNAs in
therapeutic applications. It is a promising therapeutic approach because of
strand-specific targeting of gene, stability of small RNAs, and its prolonged
effect on the gene expression that can be used for a specific gene of interest and
possess high efficiency because endogenous pathway of RNA-directed tran-
scriptional gene silencing overlaps with the small and long noncoding RNA
silencing mechanism.

13.7 Chapter Summary

• Regulation of eukaryotic gene takes place at the level of transcription,

posttranscription, translation, and posttranslation levels. The transcription levels
are regulated by cis- and trans-acting regulatory elements. Cis-acting elements
include enhancers, silencers, and locus control regions, and trans-acting factors
consist of activators, repressors, and specific transcription factors.
• Chromatin complexes consisting of nucleosomes also play an important role in
controlling transcription. Nucleosomes consist of octamer of histones around
which the DNA is wrapped. Chromatin remodeling complexes and histone-
modifying enzymes positively or negatively influence the nucleosomal structure
to activate or repress transcription.
• DNA methylation is a major epigenetic modification resulting in silencing of
transcription. The majority of DNA methylation happens in the CpG islands that
are found near the promoter elements.
• Splicing is an important posttranscriptional modification in which introns are
spliced out to produce mature mRNA with exons for translation into proteins.
Splicing is mediated by macromolecular complex called proteasome. In alterna-
tive splicing, combinations of exons are used to get different proteins.
• RNA interference is a phenomenon in which noncoding consisting of 20–30 nt
inhibits translation by binding to target mRNA. This includes endogenous
microRNA (miRNAs), long noncoding RNA (lncRNA), and exogenous or viral
origin small interfering RNA (siRNA).
628 A. A. Rangarajan

Fig. 13.14 Mean square displacement (MSD) comparing mobility of the bulk chromatin motion to
the four tracers. Bulk chromatin motion measured as cotransfected GFP fibrillarin. (a) TA-mCherry
(TAmCh) is denoted by red circles, and cotransfected GFP fibrillarin (Fib. (TAmCh)) is denoted by
green line. (b) TetR-mCherry (TetRmCh) is denoted by purple circles, and cotransfected GFP
fibrillarin (Fib. (TetRmCh)) is denoted by green line. (c) TA-KillerRed (TAKR) is denoted by red
diamonds, and cotransfected GFP fibrillarin (Fib. (TAKR)) is denoted by green line. (d) TetR-
KillerRed (TetRKR) is denoted by purple diamonds, and cotransfected GFP fibrillarin (Fib.
(TetRKR)) is denoted by green. Error bars are SEM. (Figure taken from Whitefield et al. 2018)

• The stability of mRNA is determined by the action of several mRNA degrading

enzymes that play an important role in posttranscriptional regulation. The
mRNAs can be decapped at the 50 end and deanylated at the 30 end. It can then
be attacked by the Xrn-1 endonuclease or degraded by the exosome.
• Posttranslational control involves the regulation of protein stability and degrada-
tion. Along with the bulk proteins, mistranslated and aggregated proteins are also
rapidly degraded. This is performed by the process of autophagy in which
lysosomal hydrolases perform protein degradation. In ubiquitin-proteasome
13 Regulation of Gene Expression in Eukaryotes 629

Fig. 13.15 Antisense RNA-directed transcriptional gene silencing (TGS). Small antisense non-
coding RNAs can be (A) introduced into the nucleus and (B) interact with and recruit epigenetic
silencing complexes consisting of DNMT3a, Ago1, EZH2, and HDAC1 to homology containing
targeted loci by interactions with low-copy promoter-associated transcripts resulting in (C) epige-
netic silencing consisting of histone and DNA methylation and ultimately chromatin compaction of
the targeted locus. (D) Long antisense noncoding RNAs have also been observed to interact with
similar epigenetic silencing complexes and (E) localize with these complexes at targeted loci
resulting in (C) epigenetic silencing of the lncRNA-targeted locus. (Figure taken from Weinberg
and Morris 2016)

pathway, proteins are polyubiquitylated that are recognized by the proteasome

complex which degrades the proteins.

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 Part III
Molecular Genetics II: Analysis of Genomes
Techniques of Molecular Genetics
14
Nidhi Sharma and Shrish Tiwari

14.1 Recombinant DNA Technology

In advanced molecular biology and genetics, recombinant DNA technology appears

as a boon that facilitates and opens up new challenges in the age of biotechnology.
This technology has overtaken the conventional genetics and given rise to the
opportunities to edit a genetic makeup of any organism. This has become possible
to identify a particulate target, cut, and insert into another organism. The very first
man-made recombinant product in the biotechnology industry was human insulin,
and it was only possible to develop by taking advantage of this technology. How this
recombinant technology story came in existence? It has begun with the discovery of
restriction enzyme. Werner Arber first time reported that bacteria has the machinery
which cuts the foreign viral DNA into fragments, and thus, he isolated this enzyme
known as “restriction enzyme.” With his discovery, geneticists came to know how to
“cut” and “paste” a DNA molecule and were encouraged to find novel restriction
enzymes. In a simple definition, recombinant technology is sum up into three main
tools as follows: (1) enzymes exploit for cutting, polymerization, and ligation,
(2) vectors as a vehicle for DNA transfer, and (3) host organism as a production
factory.
With the discovery of recombinant technology, it was further advanced by the
collaboration of Stanley Cohen and Herbert Boyer in 1972, and they successfully
established their first ever company, Gentech, in 1976 based on recombinant DNA
technology.

N. Sharma
La Sapienza University of Rome, Rome, Italy
S. Tiwari (*)
Aligarh Muslim University, Aligarh, Uttar Pradesh, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 635
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_14
636 N. Sharma and S. Tiwari

Fig. 14.1 Principle of recombinant DNA technology. Recombinant DNA technology includes few
stepwise processes where a DNA fragment is associated with vector-mediated (i) ligation,
(ii) recombination, (iii) replication, (iv) transformation, and (v) amplification to produce a recombi-
nant clone of desired DNA fragment (gene) in host cell

A recombinant DNA technology has five major steps: (1) cutting the DNA of
interest by the site-specific restriction enzyme, (2) amplification of DNA copies by
polymerase chain reaction (PCR), (3) insertion of amplified DNA into the appropri-
ate vectors, (4) incorporating the vectors in the desired host organism, and
(5) cultivating and harvesting the desired recombinant product (Fig. 14.1).
Currently, applications of recombinant DNA technology are not only limited in
recombinant protein production but also has been applied in gene therapy, clinical
diagnosis, and animal and plant transgenesis.
14 Techniques of Molecular Genetics 637

14.1.1 Vector-Vehicle of Carrying DNA

Vector is a DNA molecule other than genetic DNA that functions as a carrier to
transfer/insert a foreign gene, replicate, and express into another cell. Vectors are one
of the most essential and powerful tool for gen cloning. Vector also helps in the
detection whether insertion and expression of desired gene is done successfully in
the host organism. This is because vector encodes some biomarker-associated gene,
those only expressed in definitive growth condition. Cloning vector can be taken
from bacteria, virus, and cells (some higher organism like yeast) to be incorporated
to a foreign DNA particle for cloning purposes. As cloning vector adopts the
atmospheres in an organism, this vector too shows features that target a convenient
insertion and removal of gene or DNA. Cloning in vectors further can be followed by
subcloning in another vector that would show more specificity such as expression
vector, etc. Two most common used vector in recombinant DNA technology are
E. coli plasmid vector and bacteriophage λ vectors.
Mainly six major types of vectors are being used in recombinant technology so far
which are as follows:
Plasmid: Plasmid is an extrachromosomal bacterial DNA that contains the ability
of autonomous replication. Plasmid’s machinery allows it to invade the bacterial cell
and undergo self-replication inside the host cell. Plasmid does not kill host cell on
invasion, rather a plasmid containing antibiotic resistance gene helps the host to
survive in the presence of that antibiotic. Plasmid vector is structurally divided into
restriction enzyme site, origin of replication, and gene insertion site along with
reporter gene. Reporter gene distinguishes recombinant plasmid to nonrecombinant
plasmid (Fig. 14.2). Reporter gene can be either an antibiotic resistance gene or a
gene which produces colorimetric substance or a luciferase activity. All these
features will show up in cloning culture if the plasmid have received our gene of
interest. All plasmid does not have similar copy number, but usually, they have high
copy number, and pUC19 is among one of the plasmids that has highest copy
number of 500–700 per cell.
Phage: Foreign DNA can be delivered as an insertion into linear DNA derived
from the bacteriophage such as lambda phage. This insertion doesn’t diminish the
life cycle of phage. Genetically engineered lambda (λ) phage has the capacity to
carry an insert about 9–25 kb in size. At the structure point of view, this phage shows
two restriction sites at both ends of linear DNA – “stuffer fragment.” Stuffer
fragment is replaced by DNA insertion of interest through digestion with restriction
of endonuclease (Fig. 14.3). Larger fragments of DNA can easily be incorporated in
phage vectors than the plasmids. This is why phage is more often being used in
recombinant technology.
Cosmids: Cosmid vector is a pure hybrid construct that contains both the feature
of plasmid and phage to increase the capacity to carry a foreign DNA inside the
phage head. Cosmid is generally created by incorporating an antibiotic resistance site
from the plasmid and cos site from the phage. Antibiotic resistance site provides sites
for digestion, while cos helps in packaging of phage. Cosmids are deprived of
lambda genes responsible for progeny phage particle production after infection;
638 N. Sharma and S. Tiwari

Ampicillin-resistance
gene (Amp R)

EcoRl Digest plasmid EcoRl

with Pstl
1 Pstl

Pstl Pstl

2
Origin Mix with Pstl-
digested DNA

Plasmid Recombinant
vector plasmid vector

Fig. 14.2 Structural characteristics of a typical plasmid pUC19. A basic structure of plasmid and
its recombinant forms after insertion. The ampicillin resistance site in plasmid will allow the host
bacterium to grow on ampicillin-containing medium. Origin site is a point at which replication will
start as immediate as host bacteria will start to replicate in the culture

thus, cosmid has more capacity for acquiring more foreign DNA than a single
bacteriophage. As a result, a cosmid is able to receive 40–60 kb foreign DNA
fragments. Cloning through cosmid vector begins first with the insertion of foreign
DNA into cosmid and packaging of cosmid into phage head, as shown in Fig. 14.4.
Once packaging is done, cosmid-containing phages are allowed to infect E. coli
cells, cosmid injects its DNA fragment into the cultured E. coli where cosmid
replicates by using plasmid replication system, and positive-infected clones will be
identified by the presence of antibiotic resistance marker from the plasmid vector.
Cosmid vector has the advantage over others because it is the most desirable vector
in construction of the genomic library of higher organism that has the large genomic
DNA. Cosmid can accept up to 40 kb DNA, while phage is only able to receive DNA
of 20 kb, a literally half number of cosmid. However, cosmid also has a disadvantage
that some cosmid does not maintain stability on the propagation of E. coli in the
culture because E. coli has high copy number of plasmid replication system.
Bacterial Artificial Chromosomes: BAC is a slightly advanced vector from the
previous ones with new additional features. BAC has constructed with circular
bacterial F factor plasmid integrated with foreign DNA fragment of 180–200 kb.
BAC has predominantly been used for constructing genomic library of large DNA
such as plant genome. In the past decades, BAC is being more acknowledged for
engineered transgenic mice. These Tg mice had developed through the delivery of a
DNA fragment of interest via direct injection to the fertilized single-cell mouse
embryo. This gene delivery will result in a stable integration of transgene in mouse
embryo; however, this integration is a random process. A transgene construct
includes a gene fragment that upholds a eukaryotic promoter; associated regulator
elements, i.e., enhancer, suppressor, and locus control region, etc.; an open reading
14 Techniques of Molecular Genetics 639

Isolated DNA sample Bacteriophage λ DNA

EcoRl EcoRl
site site

50 kb
Enzymatic hydrolysis with
restriction endonuclease
EcoRl
λ End λ End

~ 20 kb
Stuffer fragment

Genomic DNA fragment

annealed with arms
50 kb

~ 20 kb Insert
Ligase Ligase

Infectious bacteriophage λ

Fig. 14.3 Structure of a typical bacteriophage such as lambda (λ). The image depicts the structure
and the recombination process of a λ bacteriophage (a typical bacteriophage mostly used for
cloning). A phage DNA with large linear DNA fragment (stuffer fragment) flanked with two
restriction will be removed by endonuclease. Always, target DNA is a replacement of a stuffer
fragment. Digestion with endonucleases produces arms with sticky ends possessing complementary
sequences that will help the target DNA to attach. Target DNA and bacteriophage arms put in a
mixture that also contains a ligase enzyme to ligate this arm with target DNA will also promote the
assembly of phage protein that begins the head formation

frame (ORF); and a eukaryotic polyadenylation (polyA) sequence. ORF contains

reporter genes that encode the essential marker gene such as lacZ, green fluorescent
protein, Cre recombinase, etc., to recognize the recombinants during the cloning.
BAC has been extensively studied in understanding virus genetics and its role in
human pathogenesis. For example, molecular function of most human viral genes is
not known. Recently, many of them including human cytomegalovirus
640 N. Sharma and S. Tiwari

amp r Restriction
site Partial digestion of high molecular
weight DNA to give 30–40 kb

or
i cos

Restriction
endonuclease
cleavage

amp r cos Ligation

amp r ori cos amp r ori cos

Packaging in vitro

Infect E. coli

Ampicillin resistant
colonies

Fig. 14.4 Cloning method using a cosmid vector. This vector contains a cos site, a restriction site
for inserting exogenous DNA, and a gene for ampicillin resistance. Exogenous DNA is cut with an
appropriate restriction enzyme, as is the vector. The vector and exogenous DNA are ligated
together, producing a recombinant molecule of 37–52 kb that can be packaged in λ by in vitro
packaging. The packaged vector infects E. coli, injecting its DNA into the host, where it circularizes
and multiplies. Escherichia coli cells that receive the cosmid are distinguished from cells that are not
infected by their ability to survive on media containing ampicillin

(herpesvirus), mouse CMV, pig CMV, pseudorabies virus, mouse gamma herpesvi-
rus, herpes simplex virus, and Epstein-Barr virus have been cloned and studied by
using BAC construct. In each case, BAC has replicated and cloned in
mammalian cell.
However, over the advantages, BAC has some limitations as well such as it
cannot produce recombinants at large scale due to its low copy number.
14 Techniques of Molecular Genetics 641

Centromere
Insert DNA
Telomere ARS (up to 1000 kb) Telomere

Fig. 14.5 YAC vector construct. A typical YAC consists of telomeres at both end as a part of
chromosome structure, autonomous replication sequence (ARS), and insertion part where foreign
DNA can be integrated

Yeast Artificial Chromosomes (YAC): This artificial chromosome engineered

in yeast cell has the capacity to clone genomic DNA of 1000 kb. YAC is an artificial
vector containing (i) telomeres at both end, (ii) origin of replication denoted as
autonomous replicating sequence (ARS), and (iii) centromere (center of the chro-
mosome) and a selectable marker along with DNA insert of 1000 kb (Fig. 14.5).
Above, we explained the BAC cloning system, and if we compare YAC and
BAC, it has considered that BAC is more advantageous over YAC because of clonal
instability, high chimerism, low quality of DNA, etc.
The first-time transgenesis was successfully achieved with YAC that cloned
approximately 500–600 lengthy genomic DNA and was cultivated in yeast as a
linear chromosome; however, this system has some limitations like it produces high
amount of chimeric (clone contains a genomic DNA from different region and
produces chimeric), instable clone, and it requires excess efforts to isolate relatively
large YAC, intact genomic DNA, and relatively high probability of YAC transgene
fragmentation.
Human Artificial Chromosome: HAC is an artificial chromosome that supports
the cloning of any gene into human cell. This vector is useful for expression study of
chromosomal genes and also is capable to clone large DNA fragment.

14.1.2 Cloning Strategies

Cloning is an engineered cellular machinery that has been used to create multiple
copies of certain genes which later undergo a study of their expression, functions,
etc. The first step in cloning is fragmentation of DNA and insertion into the vector to
be expressed and copied. In cloning, vector works as a carrier or vehicle to carry
DNA fragment as an insertion. Constructed plasmid with desired DNA insert is a
modified form which is reintroduced to the host cell for replication and multiplica-
tion. Plasmids divide and copy their DNA as bacteria dived and grow in the culture.
Bacterial DNA (host DNA) is being divided and copied along with the inserted DNA
in the plasmid. This inserted DNA (in cloning, it’s a human genome) in the plasmid
is usually referred to as “foreign DNA” to distinguish it from host DNA. A notable
characteristic of plasmid is ease with handling and integration of foreign DNA into
it. These vectors are characterized with specific DNA sequences to be recognized by
642 N. Sharma and S. Tiwari

Fig. 14.6 Palindrome structure of restriction enzyme. (a) A 6-nucleotide long sequence specific to
restriction enzyme recognition site is always palindrome in nature; sequence of nucleotide on 50 to
30 is the same as 50 to 30 on the complementary sequence, and (b) restriction enzyme cut the DNA
and (c) results into “sticky ends”

digestive enzyme like restriction endonuclease to cut the vector into short fragments.
This site-specific enzymes are usually produced by bacteria in the defense mecha-
nism against foreign DNA intervention.
Restriction enzyme cut both DNA strands in a way that each end has 2–4
nucleotide overhang after each cut, and such type of cut is known as “staggered
cut.” Restriction enzyme-specific sequence in dsDNA is a 4–8 nucleotide sequence
called “palindrome” sequence. Palindrome means a word can be read the same in
both forward and backward direction; likewise, nucleotide sequence in palindrome
reads in same order in both directions.
After stagger cut by restriction enzyme, overhangs on each strand are capable to
make complementary strand by hydrogen bonding in both forward and reverse
direction, and thus such ends are called “sticky end” (Fig. 14.6).
Sticky ends on single strand initiate making hydrogen bond to generate comple-
mentary strand. This process is annealing process and accompanied by the action of
additional enzyme such as ligase enzyme. DNA ligase helps in replication when
these two sticky ends come together, and ligase enzyme joins the DNA fragments
permanently (Fig. 14.6).
Cloned plasmid now contains two types of genomic information, one of itself and
other of foreign DNA, and therefore, as a result of cloning, this plasmid is known as
recombinant DNA molecule, and proteins produced through this cloning procedure
are known as recombinant protein (Fig. 14.7) However, in cloning, not all vectors
used for cloning will necessary express the respective protein, and thus, only
expression vectors are capable for protein expression and are genetically engineered
accordingly that in this organism this particular protein has to be expressed on some
stimulation. Expression of protein on the stimulation can be controlled by a scientist
if it is needed; therefore, this cloning strategy is safe.
14 Techniques of Molecular Genetics 643

Fig. 14.7 A schematic diagram of cloning strategies. A molecular cloning includes (i) DNA
cleavage by restriction enzyme, (ii) insertion of foreign DNA, (iii) ligation of DNA,
(iv) transformation in bacterial cell, and (v) selection of cloned DNA of interest

In the history of recombinant DNA technology, many plasmids have been

engineered to clone 1000–30,000 nucleotide pairs till date; however, larger DNA
weren’t possible to clone in conventional plasmid vector, and so researchers
introduced yeast artificial chromosome (YAC) in which large DNA can be inserted.
644 N. Sharma and S. Tiwari

Earlier, only plasmid is being used for gene delivery which was able to clone smaller
size of gene fragment; however, the capacity of plasmids has evolved, and its
derivative like YAC and BAC are able to clone 100,000 to one million nucleotide
pairs of foreign gene. F plasmid has been used in BAC which is far better than
conventional plasmid vector, whereas yeast chromosome has been converted into a
vector for mammalian gene cloning. In fact, BACs have low copy number, so it can
maintain the largely cloned sequence with stability in the E. coli. Also, it can avoid
the scramble issue; cloned gene sequence can recombine with other sequence carried
by other copies of plasmid. Therefore, having various adapting features like stability,
clone large fragment, and easy to handle, BACs have become the preferable choice
for constructing DNA libraries of complex organism, i.e., human and mouse
genome, as mentioned in previous section.
In recent years, researchers have developed a modified cloning system known as
“seamless cloning.” This strategy has become in existence to overcome those
limitations like (i) low efficiency and time-consuming restriction and ligation steps
and (ii) unwanted nucleotide insertion in desired sequence, which might result in
abruptly translated product of desired gene. Consequently, seamless cloning is an
enzyme-free, sequence-free, and vector-free cloning method. This method includes a
compatible set of tailed and non-tailed primers and linear vector with cohesive ends.
This innovative technique evades prolonged cloning steps such as cutting through
restriction enzyme and insertion into the vector, and rather, it allows an insert of
DNA fragment into linear vector. For example, in Gibson assembly, which was
created by Daniel G. Gibson in collaboration with the J. Craig Venter Institute,
plasmids and primers are designed with two identical sequences of about 40 base
pairs on each end. An exonuclease enzyme digests one strand of DNA back from
each 50 end, creating a single-stranded region that can anneal to its complementary
sequence on the vector or plasmid. DNA polymerase is used to close the gaps, and
DNA ligase links the joined segments together to create continuous sequence. The
entire process is carried out in a single isothermal reaction. Golden Gate Assembly
by New England Biolabs allows the insertion of multiple gene inserts into a vector
using a type IIS restriction enzyme (which recognizes sites outside of its recognition
sequence and cut them) and T4 DNA ligase enzyme. When the cleavage sites are
designed correctly, the plasmid is assembled without the original restriction site.

14.1.3 Polymeric Chain Reaction (PCR)

Kary Mullis in 1980 has invented a groundbreaking method to amplify the DNA. He
named this process a polymerase chain reaction (PCR). PCR technique is about the
ability of DNA polymerase enzyme to synthesize new strand on complementary site
of DNA template in vitro. Polymerization is carried out by heat-sensitive DNA
polymerase: Taq polymerase. Taq polymerase enzyme had been isolated from the
thermal bacterium “Thermus aquaticus” and is the only enzyme used for DNA
replication. Since this bacterium grow in extreme thermal condition, it has evolved
the protein which is temperature resistant.
14 Techniques of Molecular Genetics 645

PCR is a cyclic process that has a series of 20–40 thermal cycle of heating and
cooling that allows the enzymatic reaction to be carried out to amplify the target
DNA. The principle of PCR leads to the fact that target sequence doubles in each
thermal cycle, and this doubling is an exponential process, represented by 2N (here,
N is the number of cycles; if the cycle has 30 repeats, then it will generate
230 ¼ 1073741824 copies of DNA from a single template sequence).
The components of PCR are usually a mixture of small fraction of template DNA
(in few micrograms), forward and reverse primers that bind to the flank of the target
sequence, nucleotides (dNTPs or deoxynucleotide triphosphates), and small amount
of heat-resistant Taq polymerase. After performing the reaction, this mixture results
in a large amount of DNA as a product.
PCR reaction steps are as follow:
Initiation: This process allows the reaction to be first heated to 94–96  C
(or sometimes 98 degree if polymerase is highly thermostable) for 10–20 minutes.
Heating the reaction mixture activates the thermostable polymerase and denature the
other contaminants (if any). Initiation step on higher temperature will lyse the cell
and denature the other cellular components like unnecessary proteins and DNase
(enzyme which destroys the DNA). To avoid nonspecific amplification and primer
dimer, some reagent or antibody such as Hot Start DNA polymerase can be used.
Initiation step on high temperature is necessary to heat activate the polymerase
enzyme.
Denaturation: Reaction mixture heated on 94–98  C for 20–30 seconds
separates the double-stranded DNA into single-strand DNA as hydrogen bonds
break on high temperature. This process is sometimes known as melting process.
Annealing: Annealing process requires target-specific primer that is oligonucleo-
tide stretch, binds to template sequence, and guides the DNA polymerase to replicate
the DNA. After denature process, reaction has to be cooled down till 50–60  C to
allow the primer annealing. This process lasts for 20–40 seconds. It is important to
notice that optimal temperature for primer annealing depends on primer melting
temperature Tm: a temperature at which half of the duplex DNA is dissociated into
single strand. It has been observed that if temperature is too high, primer does not
anneal, and if temperature is too low, nonspecific priming and nonspecific DNA
amplification do occur. Therefore, an ideal annealing temperature can be considered
as 3–5  C below the Tm of the primers. This difference is high enough for specific
primer annealing and low enough for nonspecific priming. Primer concentration in
the mixture should always be higher than the DNA template, so once reaction will
start, the primer-template hybridization will replace the reannealing of the templates.
Once annealing between primer and template is done, polymerase starts
incorporating the oligonucleotide on the template strand. As soon as the primers
anneal to the template, DNA polymerase can start incorporating dNTPs onto the
template strand.
Elongation/Extension: Once primer anneals to the template, polymerase enzyme
incorporates dNTPs in 50 to 30 direction, and elongation of synthesis strand will
continue. The synthesis strand is complementary to the template. Optimum temper-
ature for extension step varies for different DNA polymerase used in the reaction.
646 N. Sharma and S. Tiwari

Fig. 14.8 A detailed mechanism of PCR. A PCR mechanism includes the following steps:
(1) Denaturation covers the breaking of hydrogen bonds of dsDNA on the temperature of
94–98  C for 20–30 seconds. (2) Annealing takes place between 50 and 65  C for 20–40 seconds
that allows primers to anneal to the template strands. (3) Elongation allows dNTPs to be added
continuously to the template strand, and exponentially amplified DNA will be the end product

The ideal temperature for most of DNA polymerase is 72–78  C. How much time a
reaction will take to finish the extension of the synthesis strand completely depends
on how longer the template strand is and on what speed DNA polymerase enzyme
adds dNTPs to the template strand. A common polymerization speed lies between
1 and 1.5 kb/min under optimal condition.
Final elongation: reaction mixture is kept on 72–78  C (which is an optimum
temperature for all types of DNA polymerase) for 5–15 min. This holding time will
ensure that any remaining single strand would get enough time to elongate in the end
of the PCR cycles (Fig. 14.8).
Final Hold: The final product can be stored in 4–15  C for an indefinite time for
short-term storage.

PCR – Primer Design

Potential primer design will ensure the fidelity of PCR product and avoid the
nonspecificity of primers. A few points that are described below should be taken
into account:

• Primer length should always be between 18 and 30 nucleotides. Lengthy primers

promote specific annealing and high melting temperature, while shorter primers
can bind easily but with less affinity.
• Optimal melting temperate (Tm) for primers are 50  C–65  C. Tm for forward and
reverse primers should have difference of 5  C so that both primers can anneal
simultaneously.
• During PCR, annealing temperature (Ta) should be set to 5  C of melting
temperature Tm. This difference lies between higher enough for specific binding
and lower enough for efficient annealing.
14 Techniques of Molecular Genetics 647

• The GC content of primer should be 40%–60% for efficient binding. The GC

content of target sequence affects the melting temperature as higher GC content
has higher melting temperature.
• If primer is ending with Gs and Cs on 30 terminal, it will promote target-specific
binding. However, Gs and Cs should not be more than three bases in the last five
bases.
• Secondary structure in target sequence will result in poor primer annealing and
low yields. Thus, it should be avoided if possible.
• Nucleotide repeats of four or more should be avoided (e.g., ATATATAT).
• Complementary sequence in either primers should be avoided; otherwise, that
may result in self-dimer or primer dimer. Moreover, excessive concentration of
primers than the template in a mixture will result in primer dimer and self-dimer,
and such dimers can compete template-primer annealing.
There are some primer designing tools available online, i.e., PrimerBLAST,
Primer3, and OligoCalc which can simplify the primer design process.

Technical Issues
PCR requires low amount of DNA sample, i.e., 5 μL to 100 μL, and such low volume
can face the issues of evaporation and insufficient pipetting. The second most
challenging issue is the amount of mixture solution. Large volume requires long
holding time for thermal equilibrium. Let’s say bigger amount of solution takes
longer time for an external temperature to be transmitted to the center of the solution.
Much solution will take longer time for thermal equilibrium at each cycle, and
therefore, longer holding time is required for each cycle. Thus, volume of solution
is linearly correlated to timing of entire thermal cycle. A standardized volume for
PCR mixture is 20 μL to 50 μL.
The sample mixture is pipetted into reaction PCR tubes which are thermostable.
Volume of reaction mixture is 20 μL to 50 μL and usually pipetted in thin wall
0.2 mL PCR reaction tube. PCR reaction tubes can be purchased as individual tubes
with or without caps or 8–12 tubes connected together called “strip tubes.” High-
throughput labs usually used 96- or 384-well plate for routine PCR. Other than
theses commercial tubes, PCR also has been performed in microscopic plate by
pipetting the sample spot on it, covered by coverslip and closed with mineral oil.

14.2 Construction of Library

14.2.1 Construction of Genomic Library

Molecular cloning is one of the promising tools these days which allow researchers
to study protein function and structure. Cloning also provides a platform to repro-
duce recombinant proteins. In this technique, genes for a particular protein of interest
will be cloned with the advantage of vector, PCR restriction enzymes, etc. Construc-
tion of genomic library is being associated with the range of multiple application in
the field of molecular cloning. The genomic library provides us the useful
648 N. Sharma and S. Tiwari

information about the source of DNA fragment which has been cloned and stored for
various use. The bright side of molecular cloning is that scientist can create and store
DNA fragments obtained from the different sources in a suitable host organism. This
cloned DNA is kept restored in a suitable microorganism which is protected by
indigenous cellular machinery to protect and replicate exogenous DNA. This type of
libraries can be a source for various genetic material such as cDNA, alleles, mutants,
mRNA, mitochondrial DNA, etc.
Genomic library construction takes a few steps as follows: Cells are collected or
grow to crush, and genetic DNA has isolated them from other proteins. In a cellular
extract when DNA is isolated from other cellular components, DNA/RNA will have
appeared in the aqueous phase since DNA is dissolved in water while other elements
will appear in the solvent phase (like phenol) and be taken out by a pipette. There are
several methods for precipitating, of which one is adding a solvent like alcohol to the
diluted DNA and precipitate it. However, DNA/RNA remains together, and addi-
tional RNase will degrade the RNA but not the DNA. Extracted DNA will be
followed by fragmentation through restriction enzyme on particular sites and
inserted to the vectors like plasmid, phage, and cosmid. These vectors containing
the DNA fragment will now be transferred to the bacterial host to replicate and
multiply to produce more copies. After a couple of cycle of bacterial growth, the
bacterial cell will produce phage particles or plasmid copies, contacting overlapped
genomic fragments. As a result, few clones contain the entire gene of interest, some
contains partial, and few contains no gene of interest. Therefore, the marker gene
will help to recognize the bacteria that contains a full genome of interest. The
genomic library must include the positive clones that have the entire genome
integrated into the vector, a library for human genome constructed by using cosmid,
and each cosmid is carrying the random gene of 30,000 to 40,000 bp long fragment.
Because of this feature (clone large fragments), cosmid provides 99% chances for
every gene to be presented in the human genome library. Establishment of
the genomic library will provide the sources for subsequent experiments or for the
primary purpose to which genomic library has been developed. To this end, the
genomic library should be stored carefully and safely for future purposes. For
example, a random genomic library produced with phage particle will contain a
suspension in the test tube and should be kept safe. At large scale, most of the
genomic libraries are stored at – 80  C. Other examples including bacterial cells
containing plasmids are stored and protected from the adverse effect of freezing by
adding glycerol as a cryoprotectant during freezing while phage particles are
protected by dimethyl sulfoxide (DMSO) which has the cryoprotective properties
(Fig. 14.9).
However, the main restriction of genomic library is that such kind of library is
accessible to create comparatively small genome like prokaryotic organism. A
genomic library of any eukaryotes seems difficult to construct and maintain since
it contains very large genome and small fraction of noncoding genome; therefore,
cDNA library omits this issue when it comes to eukaryotic genome.
14 Techniques of Molecular Genetics 649

Fig. 14.9 Schematic diagram of steps involved in construction of genomic library. DNA of interest
were first digested with restriction enzymes which give segmented DNA containing set of
overlapping fragments. This fragment and cut DNA molecules were joined to cloning vector.
Cloning vector were further cloned in the bacterial medium and screened for positive cloned
DNA of interest

14.2.2 Construction of cDNA library

After constructing a genomic library where the entire genome is settled down in the
form of a library which contains all the genomic information of an organism, we will
discuss the importance of cDNA library in this section. A cDNA library contains
many nucleotide sequences that are a complementary sequence to mRNA of the
same species. In simple words, cDNA library is constructed by using mRNA rather
than the whole genome, and mRNA further develops cDNA (complementary DNA)
which is kept stored as a form of a library for future purpose.
Most of eukaryote genome consists of repetitive sequences that are not tran-
scribed into mRNA, such as noncoding sequences, and cDNA library is deprived of
such noncoding sequences. Construction of cDNA library is applicable only for
650 N. Sharma and S. Tiwari

higher eukaryotic organism because prokaryote and lower eukaryotic organism

genome does not contain introns, and thus cDNA library is necessary for these
organisms. Hence, cDNA libraries have been produced only for higher eukaryotes,
and cDNA library is an alternate to create a genomic library for eukaryote organisms.
cDNA library has two additional advantages: (i) It is enriched with genes actively
transcribed into functional proteins, and (ii) cloning of eukaryote genes in bacteria
would not cause problem since it does not contain introns, and either bacterial
genome does not contain introns.
However, cDNA library also has some disadvantages over genomic library. The
major one is that it only stored sequences that are present in mature mRNA.
Sequences that are altered after transcription are not involved in the cDNA library.
Some sequences such as promoters and enhancers do not transcribe into RNA so that
cDNA of such sequences are not present in the library. Another important thing to be
noticed here is that cDNA library represents those mRNA sequence that is expressed
in the tissue, and RNA isolation is possible at the same time. Moreover, it has been
observed that the frequency of all genes in genome library is abundant and in equal
distribution, while the frequency of DNA in cDNA library corresponds to the
abundance of mRNA in a given tissue.

Method
cDNA library construction initiates with first step to isolate mRNA from the rest of
cellular RNA, i.e., tRNA, rRNA, rnRNA, etc. There are many methods available for
RNA isolation, but the most common is TRIZOL method till the time. As poly
adenine (poly A) tail at the 30 end is a prominent feature of most eukaryotic mRNA,
this long stretch provides a convenient hook for separating mRNA from the rest.
Oligo dT column, an oligonucleotide stretch with thymine (oligo dT chains), is a
very common technique which has been used for mRNA separation, based on the
concept that when mRNA pass through the column, adenine will pair with thymine
and be retained to the column while the rest of RNA will elute out from the column
(Fig. 14.10). Later in the steps, intact mRNA can be washed away through eluting
buffer which will break the hydrogen bonding between adenine and thymine, and
mRNA will be detached from the column.
Extracted mRNA will now be transcribed into cDNA upon the action of an
important enzyme – reverse transcriptase. Reverse transcriptase enzyme triggers
single-strand DNA synthesis from the template RNA (a reverse transcription).
This DNA synthesis process needs additional DNA nucleotide in the mixture to
bind on 30 OH group of primer (similar to normal transcription). Reverse transcrip-
tase is a retrovirus (i.e., HIV) enzyme, where the genetic material is RNA and is
transcribed into ssDNA. During this process, a short fragment of oligo dT will be
added to the mixture that works as a promoter. Primer will bind to poly (a) tail on 30
end of the mRNA and will provide free OH group for initiation of DNA synthesis.
Resulting RNA-DNA hybrid molecule is then interrupted by partial digestion by
RNase to separate the ssDNA from the RNA. Partial digestion will leave some gaps
on the hybrid strand on which the DNA polymerase can bind and initiate the
synthesis of complementary DNA strand. Undigested small RNA fragments will
14 Techniques of Molecular Genetics 651

Fig. 14.10 Schematic diagram of cDNA library construction. cDNA construction involves (a)
mRNA isolation through elution column of oligo dT and (b) cDNA construction from isolated
mRNA using reverse transcriptase enzyme. Reverse transcriptase synthesizes DNA strand on the
mRNA templates which were isolated by dT column. This mRNA-DNA hybrid lysed with RNase
to release the mRNA and to intact the DNA for DNA polymerase as a template that further
synthesizes the cDNA. DNA ligase used for ligation of cDNA. (Benjamin. A. Pierce., Genetics:
A conceptual approach)

be used as a primer, and DNA from the RNA-DNA hybrid will be used as a template.
While DNA is synthesizing, all the RNA fragments will be displaced eventually by
DNA polymerase, and nicks will be scaled by DNA damage machinery.

14.2.3 Construction of Chromosome-Specific Library

Large and complex genome in eukaryotes creates more difficulties in map saturation.
It is clear that disintegration of full genome into smaller parts will be easier to study
separately and could accelerate its analysis easily rather than the entire complicated
genome in once. Thus, chromosome-specific library would be ideal to construct
library from the subset of the genome instead of lengthy complexed genome.
Chromosome-specific library can be applied in (i) cytogenic genome mapping
652 N. Sharma and S. Tiwari

studies, (ii) region-specific marker isolation, and (iii) study of integration of genetic
and physical map. Chromosome-specific region has been separated by flow-sorting
separation (flow cytometry-based separation of chromosome regions) followed by
BAC, cosmid, bacteriophage, and YAC-based cloning. Cloning of fractionated
chromosome through these plasmids can be an advantage which represents an
individual chromosome type. Many human chromosomes have been mapped such
as chromosomes 19, 6, 21, and 22 and Y chromosome. Initially, the chromosome-
specific library construction was labor intensive and includes tedious methods which
require larger number of chromosomes. Pure chromosome was sorted by flow
cytometry, generation of somatic cell hybrid containing targeted chromosomes, or
a combination of both procedures. Thus, to eliminate such obstacles and improve the
quality of chromosome purity, many researchers have developed the new method
which has involved single flow-sorted chromosome which is also favorable and even
the resolution of chromosome population is poor. Single sorted chromosomes
technique has unique prevalence for the rapid generation of pure chromosome-
specific libraries for many genetic disorder or cancer-related chromosomes.
Chromosome-specific library (physical mapping) is eventually useful to map
transcribed sequence, i.e., mRNA and heterogenous nuclear RNA (hnRNA). This
advantage will allow us to spot the protein coding gene or transcribed sequence on
the chromosome. This mapping finally shows where those all estimated
50,000–100,000 human genes are located on the chromosome. However, it has
mentioned earlier that some unique sequence for promoters, enhancers, protein-
recognizing proteins, etc., does not present in genomic library; therefore, apart
from the protein coding and transcribed sequences instead, these unique sequences
also can be easily positioned on the human chromosome. Ultimately, chromosome
mapping or chromosome library will be advantageous for positioning sequences on
the chromosome and could significantly heighten the scope of human genome
functional map among others.

14.3 Screening of Gene Library

Once genomic library is prepared, it can be stored, can be used for purification of
proteins, and can be reanalyzed to check further whether a fragment of desired
sequence is present in our genomic library or not. Multiple strategies are developed
for screening purposes. Among all, probing is one of the most common method, and
the rest are involved in hybridization, colony hybridization, PCR, immunological
assay and protein functional analysis, etc. Here, we will understand the screening
strategies for any genomic or DNA library as follow:

14.3.1 Hybridization

Hybridization is a type of probing where the target sequence in DNA or cDNA

library can be determined with labeled DNA probe (Fig. 14.11). Probe must be a
14 Techniques of Molecular Genetics 653

Replica plate onto

nitrocellulose disk
placed on agar in dish

Transformant colonies
Nitrocellulose growing on agar surface
disk removed Retain
master
plate

Reference set of colonies

(Note: red colony has DNA
complementary to the probe)
Break open bacteria
with NaOH, neutralize,
treat with protease,
wash, bake at 80 C
to immobilize DNA on disk

Hybridize with
radioactive probe,
autoradiography

DNA print of colonies X-ray film

Fig. 14.11 Hybridization method to detect DNA probe from the library. Schematic diagram of
important steps involved in hybridization method to detect DNA probe hybridization on the nylon/
nitrocellulose membrane. The target complementary DNA for probe is identified on the master plate
of samples detected as red dot which further supposed to be isolated from the master plate. The
probe is radiolabeled so that autoradiography is being used on for detection

stretch single-stranded oligonucleotide or a double-stranded cDNA or a PCR prod-

uct of the same gene. The DNA probe can be either radioactively labeled or tagged
with fluorescent protein. Labeled probe is a key step for hybridization that will
produce signal or colored precipitation on the hybridization with target sequence.
Radioactively labeled probe gives radioactive signal by replacing phosphate back-
bone with radioactive phosphate 35 (p35). Whereas nonradioactive probe system of
which biotin has widely used up to now, for example, biotin is tagged to the probe
and, when it is hybridized with the target sequence followed by addition of substrate
antibody or solution, will result in colored precipitates that are produced after
efficient hybridization. Probe in some cases may be an RNA sequence.
The first step is labeling a nylon membrane with our DNA candidates from the
library and adding certain buffer or NaOH to denature the double-stranded DNA.
654 N. Sharma and S. Tiwari

Labeled membrane is exposed to the probes. Upon the hybridization between probe
and its complementary fragment, we get the signal of either fluorescence or colora-
tion. In case of radioactive probe signal, X-ray or autoradiography can be used to
measure the radioactivity coming from the hybridization which clearly indicates that
hybridization occurs successfully. This membrane further can be compared with
master plate to know where signal is indicating and where our desired fragment is
located on the master plate (Fig. 14.11).

14.3.2 Colony Hybridization

A successful cloning of desired gene in the E. coli bacteria further requires screening
to pick up cloned colony from the culture plate. Target DNA sequence is present in
the transformed colony that will be further detected by hybridization method with
radioactive DNA probes (sometimes labeled RNA probes can also be used). Colony
hybridization technique is also referred to as replica plating by some authors. The
technique is depicted in Fig. 14.12 and is briefly described. The transformed cells are
grown as colonies on a master plate. Samples of each colony are transferred to a solid
matrix such as nitrocellulose or nylon membrane. The transfer is carefully carried out
to retain the pattern of the colonies on the master plate. Thus, the nitrocellulose paper
contains a photocopy pattern of the master plate colonies. The colony cells are lysed
and deproteinized.
The DNA is denatured and irreversibly bound to matrix. Now, a radiolabeled
DNA probe is added which hybridizes with the complementary target DNA. The
non-hybridized probe molecules are washed away. The colony with hybridized
probe can be identified on autoradiograph. The cells of this colony (from the master
plate) can be isolated and cultured.
Many a times, multiple colonies are detected on hybridization by a DNA probe.
This is due to overlapping sequences. To identify which colony has the complete
sequence of the target gene, corresponding colonies can isolate from the master plate
and digested with the restriction enzyme, and data observed from the restriction
endonuclease analysis will be helpful to detect the exact transformed or cloned
colonies.
Moreover, sometimes overgrown colonies interfere with the signal, and therefore,
some published journal had been spotted that overgrown colonies can lead to
interference with background and are difficult to distinguish the labeled/hybridized
colonies from the negative ones. To prevent the variation in size of colonies, it is
advised that inoculation loop should be touched lightly to the agar plate instead
rubbing it forcefully.
The other thing to be noticed is accuracy in the placement of nitrocellulose
membrane on the agar plate. Nitrocellulose membrane usually adhere to the agar
plate because of a light layer of water that kept the membrane enough moist.
However, excess water makes movement of bacterial colonies along with displace-
ment of membrane, and thus, cultured colony that appears in fuzzy edges can be seen
in X-ray film.
14 Techniques of Molecular Genetics 655

Fig. 14.12 A flowchart

diagram of colony
hybridization. Transformants
containing DNA targets were
grown on nitrocellulose
membrane in the colonies.
Targeted DNA were identified
by radiolabeled DNA and
probed with labeled DNA,
mentioned in step 3. In step
4, identified hybrid probe
matches with master plate and
then subcultured to isolate
them from others for future
purpose
656 N. Sharma and S. Tiwari

14.3.3 PCR

Polymerase chain reaction (PCR) is as good as the hybridization technique for

screening DNA libraries. PCR offers an opportunity to identify complex and rare
sequences in a clone that has been produced by a complex bacteriophage, plasmid, or
cosmids. PCR facilitates the abundance of a unique and complexed sequence by
amplification, and thus it gives particular and sensitive technique in short time to
isolate a specific gene of interest which might not be possible by simple
hybridization. But adequate information on the flanking sequences of target DNA
must be available to prepare primers for this method. This method has been first
developed by Takumi and Lodish in 1994 for the screening of cDNA libraries.
PCR protocol first follows the culture of bacterial colonies in multiwell plates
same as they usually grow in L broth. Cultured bacterial colonies in the multiwell
plate contain phage particles or plasmid with a cloned target sequence. The phage
releases its DNA into the medium; that’s why the extraction of DNA is not needed
here. This phage culture is now ready to be used as PCR template for screening. The
rest of the following procedures are the same as usual, and we use them for a
conventional PCR. PCR primer is specific to the target sequence and will run for a
typical PCR reaction cycle protocol. After PCR run, the product should run on the
agarose gel, stained with ethidium bromide, and take the image. Later to this, the gel
is dried on 70  C to denature the DNA and further probed with complementary
oligonucleotide sequence for hybridization process. In this way, each well is
screened by PCR, and the positive wells are identified.

14.3.4 Immunological Assay

Immunological techniques can be used for the detection of a protein or a polypep-

tide, synthesized by a gene (through transcription followed by translation). The
procedures adopted for immunological assay and hybridization technique (described
already) are quite comparable. Screening procedure by immunological assay is
depicted in Fig. 14.13.
The protocol includes grown colonies on the master plate will be transferred on
the solid matrix such as nitrocellulose membrane. These colonies are then lysed, and
extracted protein will bind to the surface of the matrix. Fixed protein will be treated
with a primary antibody that is specific to the protein and will interact with the
protein (protein is the antigen here). The target DNA encodes a protein. The
molecular target of primary antibody is an epitope – a short amino acid sequence
that folds into a three-dimensional structure as a secondary protein structure on the
surface of antigen (protein). Incubation of antibody-antigen follows by removing the
unbound antibody by washings, and then a secondary antibody specific to primary
antibody is added to the membrane.
Again, the unbound antibodies are removed by washings. The second antibody
carries an enzyme label (e.g., horseradish peroxidase or alkaline phosphatase) bound
to it. The detection process is so devised that as a transparent substrate, it is acted
14 Techniques of Molecular Genetics 657

Fig. 14.13 Immunological assay for screening a gene library. Transformants from the gene library
were grown on the master plate and transferred to the solid matrix. Colonies were lysed on the solid
matrix using lysis enzyme. Exposed proteins are probed with primary antibody and secondary
antibodies. Signals on the solid matrix indicates the targeted protein or protein of interest which
further picks up from the master replica and subculture for further uses

upon by this enzyme, and a colored product is formed. The colonies which give
positive result (i.e., colored spots) are identified. The cells of a specific colony can be
subcultured from the master plate.
658 N. Sharma and S. Tiwari

14.3.5 Protein Function

Sometimes, gene library contains a few genes that directly synthesize protein straight
into the host cell and released in the form of the enzyme. It means that the target
sequence product is an active enzyme which is not produce by endogenous genes
(bacterial genes). In this case, screening is possible by measuring the enzyme’s
activity in the host cell. Enzyme activity is mainly measured by identification of
released product after enzyme-substrate reaction when the substrate is added onto
the membrane. The enzyme-substrate product will indicate the presence of the target
sequence/functional protein/enzyme in the host cells. For instance, α-amylase and
β-glucosidase are two natural enzymes that can be identified by this technique.

Chapter Summary
• Recombinant DNA technology (RDT) is a set of molecular techniques that enable
the possibilities to alter the genetic makeup of an organism by modifying or
altering the composition of genetic material like DNA or RNA. RDT provides
tools for locating, cutting, joining, analyzing, and changing DNA sequence and
for inserting the sequence into the cell.
• Restriction endonuclease enzyme plays a vital role that makes double-stranded
cuts in DNA at the specific sequence site. DNA fragments as a result of cuts by a
restriction enzyme can be separated on the gel through electrophoresis technique
and visualized by labeling the fragments with radioactive or chemical tag.
• Plasmids and bacteriophage are very essential and basic vectors (extrachromo-
somal DNA) in molecular techniques. Many other modified vectors have been
developed that contain the features of many in one and can replicate larger DNA
such as cosmid, phage, and plasmid-generated cosmid, bacterial artificial chro-
mosome (BAC), and yeast artificial chromosome (YAC). These modified vectors
have larger capacity to hold big DNA.
• Cloning is a strategy to generate identical copies of small DNA fragments
(molecular cloning) or entire organisms (reproductive cloning). In molecular
cloning, the desired DNA fragment is inserted into a bacterial plasmid using
restriction enzymes and transferred to the host cell to further multiply and
express.
• Polymerase chain reaction (PCR) is a method used for amplification of DNA
enzymatically without cloning. A solution for containing DNA is heated, so it
breaks down into single-stranded, primer (a complementary sequence of the DNA
template) bind to 50 and 30 end of the single-stranded DNA. High temperature
activates Taq polymerase to synthesize new strand from the primer. Each time
cycle is repeated, the amount of DNA doubles.
• Genes can be isolated and stored by creating a DNA library utilizing bacterial
colonies and viral plaque that has incorporated DNA fragment within. A genomic
library contains the entire genome of an organism, while a cDNA library contains
DNA fragment complementary to all different mRNA expressed in a cell.
14 Techniques of Molecular Genetics 659

• Screening of gene library requires a sensitive and powerful technique which can
be hybridization, colony hybridization, PCR, immunological assay, and protein
function.
• The immunological assay is one of the modified techniques of hybridization
where primary and secondary antibodies are hybridized with interest of DNA
and produce the signals to detect whether cloning is done or not. Protein function
refers to another method where enzymatic reaction signals as a function of protein
if the target DNA is specific to synthesize a protein which can bind with substrate
enzyme and produce signals.

14.4 Screening of the Genomic Library

Screening of the genomic library is a process that involves the identification of

clones carrying the gene of interest. Screening depends on the characteristic property
of a clone in a library. The genomic DNA library consists of a thousand clones in the
form of plaques or colonies on the plate.

14.4.1 Screening by Hybridization

• Screening with the help of the hybridization technique uses a gene probe (a short
nucleotide sequence specific for that particular gene) to identify the gene of
interest.
• The process begins with (Fig. 14.14) the production of a replica filter for each
plate that uses “colony lift” as a procedure. This procedure is quite similar to
southern blotting as discussed later in the book. A nitrocellulose or nylon
membrane is placed on the top of a Petri plate that contains the cells which
carry the desired gene of interest. This plate is kept for a short time (approxi-
mately 1 minute). When the membrane is kept on the top of the cell culture plate,
a part of each bacterial colony from the plate will bind to the membrane. The
membrane is removed and is soaked in a solution containing sodium hydroxide
(NaOH) that will help to release the bound DNA with the membrane and
additionally will denature it.
• In the neutralization step that uses buffer solution, the single-stranded DNA
molecules will be immobilized on the nitrocellulose membrane with either heat
or UV irradiation. The membrane is then hybridized with a suitable probe under
specific conditions.
• This is followed by autoradiography procedure which shows black spots repre-
sentative of the desired clones.
• The final step in the procedure is to align the X-ray film (autoradiograph result)
image with that of the plate and subculture the desired colony.

A diagram showing the process of screening by hybridization. The process starts

with the replication of a colony of cells from the library onto a filter. This filter is
660 N. Sharma and S. Tiwari

Fig. 14.14 A Schematic diagram representing the process of screening of clones by hybridization

nothing but a membrane made up of nitrocellulose or nylon fiber. A suitable probe is

then hybridized to the gene of interest for a short duration of time. This hybridization
makes a portion of the DNA colony to transfer from the library to the filter plate. The
filter plate is thus then washed with NaOH to release the binding of ssDNA. This is
then put under X-ray film to observe the cloned gene of interest. A black dot here
shows that the gene is being cloned. This gene is marked and compared with the
original Petri plate to screen the gene of interest.

14.4.2 Expression

Identification of clone within a cDNA library is an important task to achieve. This is

often done by immunological screening which uses antibody against purified virus
or a Coat protein. Antibody screening can be carried out on a cDNA-cloned gene on
a wide range of vectors that include plasmid and phage-based vectors. A wide range
of vectors λgt11 and λZAP are now available commercially that can be used for the
generation of expression library. These vectors include antibiotic screening. Anti-
body screening can also be done on a simple plasmid vector based on the blue-white
screening principle. cDNA inserts are expressed as fusion proteins with beta-
galactosidase after being induced by IPTG. The following methods of expression
are described in brief in this regard.
14 Techniques of Molecular Genetics 661

14.4.2.1 Library Plating

In this method, cDNA libraries are transformed into a suitable bacterial host, and
colonies are grown on plates at an optimum density to visualize single-cell colonies
at 37  C overnight.
A grid is drawn and numbered on a nitrocellulose circle that is a size of a Petri
plate which is then soaked in IPTG solution of 10 mM strength. This is followed by
air-drying and carefully placing it on a solid media plate. A similar grid is drawn on
the bottom of another solid media plate. Individual colonies are picked up from the
original cDNA library. They are then streaked at a numbered position of a nitrocel-
lulose grid; this streaking process is repeated on an agar plate using a sterile loop.
After enough colonies have been streaked out on to the plates are inverted and
incubated overnight at 37  C.

14.4.2.2 Western Blot Analysis

An antibody-positive colony is grown overnight in a liquid broth at 37  C in the
presence of 5 mM IPTG. The cells are harvested by microcentrifugation,
resuspended in SDS-PAGE-loading buffer, and boiled for 2 minutes. A
microcentrifuge is spined briefly to remove cell debris. The supernatant is also
removed into a fresh tube.
The sample is boiled for 2 minutes to separate it on 12.5% polyacrylamide gels.
This is followed by Western blotting onto a suitable membrane.

14.4.3 Hybrid Arrest and Release

14.4.3.1 HRT and HART (Hybrid Release Translation and Hybrid Arrest
Translation)
These are a set of two related techniques that find their usage in the identification of
translated protein products that are encoded by a cloned gene of interest in a cell-free
system. The frequently used cell-free systems are usually prepared from germinating
weed seeds or from rabbit reticulocyte cells since these cell-free systems are highly
active in protein synthesis. Cell extracts contain all the prerequisites needed for
protein synthesis including ribosomes, tRNA, and all other types of essential
machinery.
The process involves the addition of mRNA to a cell-free translation system with
a mixture of 20 amino acids that are common to all proteins. One of the most
commonly labeled amino acids is S35 methionine.
The mRNA molecules undergo a process of translation to produce a mixture of
radioactive proteins that can be separated by using gel electrophoresis and are then
visualized by autoradiography.
Each band on the autoradiogram represents a single protein coded by one of the
mRNA molecules present in the sample.
This technique works best as clone obtained from a cDNA library.
662 N. Sharma and S. Tiwari

Fig. 14.15 A diagram

representing the process of
hybrid release transition. The
processes include binding of
specific cDNA
complementary to a gene of
interest on a nitrocellulose
membrane. This is followed
by the addition of an mRNA
mixture that binds to cDNA.
The hybrid mRNA is then
recovered and is translated
into protein in the cell-free
system. The method thus
provides a pure sample of
protein products

HRT (Hybrid Release Translation)

This technique involves the following steps:
• cDNA is denatured (Fig. 14.15) and is then fixed on a nitrocellulose membrane
followed by an incubation with mRNA sample.
• This mRNA complementary to the cDNA hybridizes and remains attached to
the membrane.
• After removing the unbound molecules, the hybridized mRNA is recovered and
is translated in the cell-free system for protein synthesis.
• This method will provide a pure sample of a protein coded by the cDNA.
14 Techniques of Molecular Genetics 663

Fig. 14.16 A diagram

showing the process of hybrid
arrest translation. The steps
involved include mRNA
preparation which is
complementary to specific
cDNA. The hybridized
mRNA cannot be translated to
the protein product. Gel
electrophoresis is done that
shows protein bands. The gel
then shows results that could
represent protein coded by
cDNA and HART products

HART (Hybrid Arrest Translation)

• Hybrid arrest translation (Fig. 14.16) is a little different from the hybrid release
translation in the sense that in this technique, there is no need to denature the
cDNA, and the cDNA is directly added to the mRNA sample.
• This results in hybridization between the cDNA and the mRNA that are comple-
mentary to each other. The unbound mRNA is not discarded; rather, the whole
sample is translated in a cell-free system.
• As the hybridized mRNA is unable to translate, all the proteins except the ones
coded by the cloned gene are synthesized. The absent protein from the radiograph
is nothing but the protein product of the cloned gene of interest.
664 N. Sharma and S. Tiwari

14.4.4 Chromosomal Walking

It is developed by Welcome, Bender, Pierre Spierer, and David S. Hogness in the

early 1980s. It is a technique that is used for identifying DNA regions next to a
known segment of DNA by sequential hybridization of the clones (Fig. 14.17). It is
one of the first methods developed for assembly of clone contigs (set of overlapping
DNAs that together represent a consensus region of DNA).
Here in this example, the library contains 96 clones each containing different
inserts. The walks start with one of the clones that are used as a hybridization probe
against all other clones in the library. The clone in this example is the A1 probe, and
it hybridizes to itself and the other probes E7 and F6. The insert from the last two
clones must therefore have an overlapping sequence with the insert from clone A1.
The walk is continued, and the probing is repeated, but this time, the insert is from
F6, Thus, the hybridizing clones are A1, F6, and B12, showing that the insert from
the B12 insert has overlapped with that of the F6 fragment.
A schematic diagram representing chromosomal walking by PCR (Fig. 14.18).
The two oligonucleotides annealed within the end region of insert number 1. They
are used in PCR clones with all other clones in the library. Only clone 15 gives a
PCR product, and this result indicated that insert clone 1 and clone 15 show an
overlap. The walk could then be continued by sequencing the fragment from the

Fig. 14.17 A schematic diagram representing the process of chromosomal walking

Fig. 14.18 Chromosomal walking by PCR method

14 Techniques of Molecular Genetics 665

other clone 15, using them to design a second pair of oligonucleotides and using
them in a new set of PCR with other clones.

Limitations of Chromosomal Walking

• The drawback with chromosomal walking is that it can only sequence and map
small lengths of chromosomes.
• Chromosomal walking is a slow process, and it becomes very challenging to
assemble contigs of more than 15–20 clones by this method.
• To add the difficulty of walking through a repeated sequence that is scattered
through genes.
• Chromosomal walking can be stopped by unclonable sections of DNA.

Applications of Chromosomal Walking

• This disease can be used for the analysis of genetically transmitted disease. It can
also be used to look for mutations.
• It is used in the discovery of SNPs.
• One of the best applications of chromosomal walking was the identification of
cystic fibrosis gene.

Chapter Summary
• Genomic DNA library is a representative of the total genomic DNA of an
organism.
• Screening of genomic DNA library by hybridization is one of the most commonly
used technique to identify the gene of interest. This technique works on the same
principle that uses Southern blot. The autoradiograph is a representative of the
cloned gene of interest.
• Expression of libraries helps us to identify a clone within a gene library that is of
particular interest. Currently, a wide range of vectors is commercially available
for library screening. Immunological screening with the help of antibodies can
also provide great assistance to identify the gene of interest. This can be achieved
with the help of plasmid vectors that use blue-white screening strategies.
• Library screening and Western blot analysis are currently used techniques for
identifying a gene of interest from gene libraries.
• Hybrid arrest and release is a set of technique that is used to identify translated
protein products in cell-free systems. HRT (hybrid release translation) and HART
(hybrid arrest translation) use slightly two different approaches to identify a
specific protein of interest. These techniques work best with the cDNA library.
• Chromosomal walking is a technique that is used to map regions of DNA next to
an unknown sequence. It is the first method developed for assembly of clone
contigs.
666 N. Sharma and S. Tiwari

14.5 Site-Directed Mutagenesis by PCR

Site-directed mutagenesis is a technique that purposefully introduces a mutation at

the specific site of a DNA to monitor the effect of changed protein. A basic
understanding of the central dogma of biology says that DNA holds the genetic
information to synthesize any protein. Changes in the sequence of DNA can alter the
structure and function of the targeted protein. Site-directed mutagenesis allows us to
create a mutation in the segment of DNA. This technique was first reported to be
used by Charles Weissmann in 1974. With the advancement in science, this tech-
nique is developed, and it currently offers different methods of mutagenesis, namely,
cassette mutagenesis, primer extension method, and PCR method of site-directed
mutagenesis.
Cassette Mutagenesis: This is one of the early methods of site-directed muta-
genesis in which an artificially created DNA fragment containing the desired muta-
tion replaces the corresponding sequence in the wild-type gene (Fig. 14.19). Wells
et al. originally introduced this method to generate improved variants of enzyme
subtilisin. The efficiency of mutagenesis in this method was close to 100%. A major
drawback of this method was the requirement of unique restriction sites that flank the
region of interest.

Site-Directed Mutagenesis by Primer Extension

The single primer method of site-directed mutagenesis developed by Gillam et al.
and Zoller and Smith is the simplest method available for introducing mutation in the
DNA segment (Fig. 14.20). The first step of the process involves synthesizing a
DNA oligonucleotide that serves as a starting material for the polymerization
reaction. The oligonucleotide synthesized is typically 20 nucleotides long that
carries a base mismatch with a complementary sequence. The target DNA must be
single stranded, and cloning the gene in an M13-based vector makes the process
easy. A modification to this method was introduced by Dalbadie-McFarland et al.
in1982 where they clone DNA in a plasmid, obtained the duplex form, and
converted it to a partially single-stranded molecule that is suitable for the process.

Limitations of Primer Extension Method

• The double-stranded duplex molecules generated by this method have a high
chance of getting contaminated by any single-stranded nonmutant template DNA
that was not copied during this process.
• The presence of this single-stranded non-template DNA reduces the proportion of
mutant progeny. This can be corrected by sucrose gradient centrifugation or by
agarose gel electrophoresis but is a time-consuming process. A mixed population
of mutant and nonmutant progeny can be seen because the in vivo DNA synthesis
can lead to segregation of the heteroduplex molecule.
• Separating mutant progeny by cell’s machinery is a complex process and is
dependent on several factors which cannot be controlled.
• The requirement of a single-stranded template is an added disadvantage of this
method. This is because of two factors. First is the fact that single-stranded
14 Techniques of Molecular Genetics 667

Plasmid vector

YFG

Xbal BglIl

Cleave DNA with Xbal and BglIl

Remove wild-type Two synthetic

fragment oligonucletides

Anneal
Wild-type complementary
DNA fragment oligonucleotides

Xbal BgllI
Xbal BglIl DNA “Cassette”

DNa ligase

Transform competent Mutant DNA

bacterial cells

Prepare plasmid DNA

All resulting plasmids
contain mutant sequence

Fig. 14.19 The schematic diagram explaining the procedure of cassette mutagenesis. The plasmid
containing a copy of the desired gene (YFP; black segment) is digested with two well-known
restriction enzymes, for example, Xbal and Bglll. Both of them have only one unique restriction site
in the entire plasmid. The reaction mixture is separation on agarose gel by electrophoresis. The
larger fragment is purified from the gel. A pair of single-stranded oligonucleotide is synthesized by
automated DNA synthesis. These two strands are complementary to each other and differ from the
original sequence at only the single position containing the desired changes followed by
hybridization of the two strands because of their complementary nature. The vector and the two
strands are then ligated using the enzyme DNA ligase. The transformed cells contain mutation at the
desired location
668 N. Sharma and S. Tiwari

Fig. 14.20 The figure represents site-directed mutagenesis by primer extension method. Single-
stranded DNA is first prepared. It is then annealed to a synthetically made oligonucleotide. The
sequence is complementary to the wild-type sequence of the template except for the places that have
mismatched nucleotides that contain a mutant DNA sequence. The remaining strand is synthesized
by DNA polymerase using multigenic oligonucleotide as a primer followed by end join ligation
with the help of ligase enzyme. The resulting product contains one wild-type strand and another
mutant strand. This DNA is then introduced to E. coli cells. The DNA is sequenced and analyzed for
mutation

molecules are hard to prepare as compared to double-stranded molecules, and

secondly, gene insertion is less stable in single-stranded DNA and can be easily
degraded due to several factors.
14 Techniques of Molecular Genetics 669

14.5.1 PCR Method of Site-Directed Mutagenesis

PCR is considered one of the most revolutionary discoveries in science. A lot has
already been talked about PCR in the preceding sections. The main focus of this
topic will be to give readers an insight into site-directed mutagenesis by PCR-based
techniques. The key requirement of all PCR-based methods is to use high-fidelity
polymerase which is achieved by Taq polymerase with the error rate of 105 order
error/bp/duplication. This enzyme can be thus utilized to introduce nonselective
additional mutations in the gene of interest. Other enzymes such as Pfu with 106
error/bp/duplication and Phusion with an error rate of magnitude of 107 errors/bp/
duplication are preferred choices for site-directed mutagenesis over Taq polymerase.
PCR finds wide range of applications in the field of molecular biology, and site-
directed mutagenesis is not an exception to this. The central objective of PCR-based
site-directed mutagenesis technique is to separate/remove the template-strand DNA
from amplified strand to increase the efficiency of mutant clone after transformation.
If not removed, this can lead to a generation of false-positive results; therefore, it is
necessary to remove the template strand after PCR amplification.
The early works that used PCR-based site-directed mutagenesis are dated back to
the year 1986 when Scharf et al. showed the potential use of PCR in this technique.

PCR Conditions for PCR-Based Site-Directed Mutagenesis

PCR conditions for site-directed mutagenesis is quite unconventional, and the
number of cycles is reduced to 12–18 cycles only. The standardization of PCR
conditions is required for obtaining optimal amplification. This includes the use of
DMSO or glycerol as they aid in melting the strands whose Tm is close to 80  C.

Dpn1 Digestion
After the product amplification confirmation by agarose gel electrophoresis, the
template stranded is given Dpn1 treatment. This enzyme specifically recognizes
50 -GATC-30 as the sequence where adenine is methylated on both the strands, thus
specifically digesting parental plasmid, and will not digest unmethylated
PCR-amplified product.

14.5.2 Overlap Extension Method

Higuchi et al. have depicted a variety of the fundamental strategy which empowers a
transformation in a PCR-created DNA segment to be presented anyplace along its
length. His technique used two primary PCR reactions that produce two overlapping
DNA fragments both bearing the same mutation in the overlapping region
(Fig. 14.21). The overlapping in the sequence allows the fragments to hybridize.
This is followed by the extension of one of the two possible hybrids carried out by
DNA polymerase, and the other hybrid is degraded in the reaction mixture. One can
introduce addition/substitution and deletion by this method. The drawback of this
670 N. Sharma and S. Tiwari

Sense flanking primer Sense mutagenic primer

Antisense mutagenic primer

1st Round PCR 1st Round PCR Antisense
Mutation flanking
primer

Mix two PCR products

Denature and anneal

Over lap
2nd Round PCR

Add flanking primers

3rd Round PCR

Amplified mutant
DNA fragment
Ligate into cloning vector
Transform E. cloi

Fig. 14.21 The above schematic diagram shows site-directed mutagenesis by overlap extension
PCR. The initial two rounds of PCR created two covering segments of the original template, both
containing the mutation at the overlapping region. The two PCR products are annealed and then
subjected to the second round of PCR to create the entire segment with a mutation. The flanking
primers contain the restriction site for joining the segment back to the original vector

method was that it required four primers and three PCRs (two PCR cycles to amplify
the overlapping segments and the final PCR cycle to fuse these two segments).

14.5.3 Megaprimer Method

A relatively simpler method was developed by Sarkar and Sommer in 1990 which
uses three primers and two rounds of PCR. This modification used the product of the
first PCR as a megaprimer (Fig. 14.22) for the second PCR. This technique uses a
14 Techniques of Molecular Genetics 671

Mutation
Mutagenic primer
Wild-type
template DNA

1st Round PCR

Wild-type Mutation
template DNA

Hybridize 1st round PCR

product with wild-type DNA

2nd Round PCR “Megaprimer”

Add flanking primers

Mutation
Amplified mutant
DNA fragment
Ligate into cloning vector
Transform E. cloi

Fig. 14.22 The schematic diagram here represents site-directed mutagenesis by the megaprimer
PCR method. This method involves the use of two PCR cycles, and the first cycle makes the
fragment of the template DNA containing the desired mutation. The megaprimer thus formed is
hybridized to wild-type template DNA, and the second round of PCR is used to generate an entire
molecule with a mutation. Restriction sites present in the flanking primer is used to clone the
fragment back to the vector

single mutagenic primer to create changes in the target template. The first round of
amplification involves the wild-type template using a sense or an antisense muta-
genic primer and an appropriate flanking primer. The amplified product is utilized in
the second round of the PCR cycle with wild-type template and the other flanking
primer to create a fragment that is of the same length as the original target DNA
containing the mutation. The key to this technique uses the fact that an amplified
product of the first PCR cycle is used as the primer for the second cycle. The
overlapping of the template and the mutagenic strand is more extensive in the
megaprimer method.
672 N. Sharma and S. Tiwari

14.5.4 Inverse PCR-Based Technique

This technique requires the use of two primers to create the desired mutation
(Fig. 14.23). The unique feature of this method is that the entire vector gets amplified
while making the mutation. The two primers the one that contains the desired
mutation extend the circular DNA template in opposite direction. The amplification
results in a linear double-stranded DNA molecule containing the mutation at one
end. After amplification, the ends are ligated back, and the circular DNA molecule is
transformed in E. coli.
PCR-based site-directed mutagenesis offers an advantage of simplicity and speed
over other forms of site-directed mutagenesis; however, the possibility of
introducing unwanted mutations due to the error-prone nature of some thermostable

Double stranded
plasmid vector

DNA insert to be
mutagenized
Mutagenic primer
PCR

Amplified
product
Ligate ends
Mutation

Mutation
Transform E.coli

Fig. 14.23 The figure shows a process of site-directed mutagenesis using an inverse primer. As
depicted, this method uses two primers, one of them being mutagenic in nature and the other being
the normal simple primer. These primers extend the target DNA in opposite direction. After the
completion of the PCR cycle, the circular vector becomes linearized which contains the desired
mutation. The resulting linear DNA is religated and made circular. The DNA is then transformed
into E. coli cells
14 Techniques of Molecular Genetics 673

DNA polymers might offer some limitation to this method. With advancements in
science, a large number of thermostable polymerases with high fidelity are now
available that can improve this limitation, thus enhancing the potential use of
PCR-based site-directed mutagenesis.

Chapter Summary
• Site-directed mutagenesis is a technique that allows us to introduce mutations in
the DNA sequence for a better understanding of the protein structure-function
relationship.
• The early method involved in this technique could produce close to 100%
efficiency but have its own limitations.
• The use of unique restriction sites and lack of control in in vivo DNA synthesis
and the use of special single-stranded DNA molecules limited the use of non-
PCR-based site-directed mutagenesis.
• PCR-based site-directed mutagenesis used PCR reactions to introduce mutations
in the target DNA fragment.
• This includes techniques, namely, overlap extension method and the megaprimer
method inverse PCR method.
• The overlap extension method uses four primers, and three PCR cycles limited
the use of this method for site-directed mutagenesis.
• The megaprimer method was a bit advance than the overlap extension method
and used two PCR cycles and three primers.
• The inverse primer method used a set of two primers to create the desired
mutation.
• All these new advancements in PCR-based site-directed mutagenesis techniques
made it possible to introduce mutations in a specific part of DNA and allow us to
study how these mutations affect the structure and function of the resulted protein.
• PCR-based site-directed mutagenesis allows us to engineer proteins and enhance
their properties.

14.6 Visualization of Biomolecules

14.6.1 Visualizing DNA by Southern Blot

• Southern blot is a technique that is used to detect DNA molecule.

• E. M. Southern in 1975 is credited for the discovery of this useful technique.
• This technique aims at finding out a specific fragment of DNA in a sample.
• The basic principle of this technique is hybridization. This technique is used to
separate DNA fragments by size.

14.6.1.1 Procedure of Southern Blotting

Following electrophoresis (Fig. 14.24), the test DNA fragments are denatured in
strong alkali. As the electrophoretic gels are fragile and the DNA can diffuse within
674 N. Sharma and S. Tiwari

Fig. 14.24 A schematic diagram showing the basic structure of the Southern blotting technique.
The process involves the isolation of genomic DNA from the cell of bacterial, plant, or animal
origin. Once the genomic DNA is isolated, restriction endonuclease digests the sample of genomic
DNA. This is followed by agarose gel electrophoresis which separates DNA bands according to
size. These fragments are then transferred onto a nitrocellulose membrane or a nylon membrane.
The nitrocellulose membrane which now has DNA fragments is then probed with radioactive
phosphorus. After probing the DNA fragment with radioactive material, the nitrocellulose mem-
brane is exposed to X-rays. This is then viewed on an autoradiogram, and thus, the DNA of interest
is identified by the process of Southern blotting

the gel, it is usual to transfer denatured DNA fragments by blotting onto a durable
nitrocellulose membrane to which the single-stranded DNA binds readily.
Аfter trаnsfer, the DNА frаgments need tо be fixed tо the membrаne sо thаt they
саnnоt detасh. In саse оf nitrосellulоse рарer, nuсleiс асid immоbilizаtiоn оссurs
nоnсоvаlently аfter bаking fоr 2 hrs аt 80 С. In саse оf nylоn membrаne, either it is
bаking fоr 1 hоur аt 70 С оr UV irrаdiаtiоn аt 254 nm. Nuсleiс асid binds соvаlently
with nylоn membrаne аfter UV irrаdiаtiоn fоr 5 minutes. The individuаl DNА
frаgments beсоme immоbilized оn the membrаne аt роsitiоns whiсh аre а fаithful
reсоrd оf the size seраrаtiоn асhieved by gel eleсtrорhоresis. Fоllоwing the fixаtiоn
steр, the membrаne is рlасed in а sоlutiоn оf lаbeled (rаdiоасtive оr nоnrаdiоасtive)
RNА, single-strаnded DNА оr оligоdeоxy nucleotide whiсh is соmрlementаry in
sequenсe tо the blоt trаnsferred DNА bаnd оr bаnds tо be deteсted. Sinсe this lаbeled
nuсleiс асid is used tо deteсt аnd lосаte the соmрlementаry sequenсe, it is саlled the
рrоbe. The рrоbe is аllоwed tо hybridize tо its соmрlementаry single-strаnded tаrget
DNА sequenсes оn the membrаne. Соnditiоns аre сhоsen whiсh mаximize the rаte
14 Techniques of Molecular Genetics 675

оf hybridizаtiоn соmраtible with а lоw bасkgrоund оf nоnsрeсifiс binding оf the

membrаne. Аfter the hybridizаtiоn, reасtiоn hаs been саrried оut, and the membrаne
is then wаshed extensively tо remоve nоnsрeсifiсаlly bоund рrоbe. If the рrоbe is
rаdiоisоtорe lаbeled, then the membrаne is exроsed tо рhоtоgrарhiс film. If the
рrоbe is nоnisоtорiсаlly lаbeled with biоtin оr digоxigenin, the membrаne mаy be
with the сhemiluminesсent substrаte tо deteсt the lаbeled рrоbe аnd then exроsed tо
рhоtоgrарhiс film. The рrоbe will fоrm а bаnd оn the film аt а роsitiоn
соrresроnding tо the соmрlementаry sequenсe оn the membrаne.

14.6.1.2 Applications of Southern Blotting

• Identifying sрeсifiс DNА in а DNА sаmрle.
• It is used to construct RFLP maps. These maps are valuable in detecting and
studying various kinds of mutations including deletions, additions, substitutions,
and gene rearrangement.
• Diseases such as sickle cell anemia, cystic fibrosis, Duchenne muscular dystro-
phy, and others can be detected by Southern blot studies.
• Southern blotting finds its application in forensics particularly for criminal iden-
tification and DNA fingerprinting.
• This technique is of high value in identification of genetically engineered
organisms.
• Рrоgnоsis оf саnсer аnd рrenаtаl diаgnоsis оf genetiс diseаses.
• Determinаtiоn оf the mоleсulаr weight оf а restriсtiоn frаgment аnd tо meаsure
relаtive аmоunts in different sаmрles.
• It is used fоr the соnfirmаtiоn оf DNА сlоning results.

14.6.2 Visualizing RNA by Nothern Blot

This technique is used to study the level of gene expression. This study can be tissue
specific or condition specific. It is often seen that genes are transcribed in a tissue-
specific manner. A gene may have limited expression in normal circumstances, but it
may be highly expressed in diseased condition. The comparison of this healthy and
diseased could be made by Northern blot analysis. The level of gene expression can
be easily detected if one can find the amount of RNA transcribed from the gene of
interest.
The process of Nothern blotting (Fig. 14.25) involves the following steps:

• This technique measures the amount and size of RNA transcribed from genes and
estimates their abundance.
• Firstly, an RNA extract is electrophoresed in an agarose gel, using a denaturing
buffer such as formaldehyde that ensures that the RNA transcript does not form
any secondary structures.
• The agarose gel is blotted onto a reactive DBM (diazobenzyloxymethyl) paper
and hybridized with a labeled probe so as to detect RNA of specific interest.
676 N. Sharma and S. Tiwari

Fig. 14.25 A flow diagram representing the process of Nothern blotting. Nothern blotting is quite
similar to the above discussed Southern blotting. The only difference lies in the fact that in Nothern
blotting, the detected molecule is RNA and not DNA. A key point to mention during the process of
Northern blotting is the use of formaldehyde that removes any kind of secondary structures formed.
The secondary structures in RNA are quite common and may be formed because of inter- or
intramolecular hydrogen bonding. The process starts with collection of RNA sample which is
followed by electrophoresis that leads to separation of RNA fragments according to size. The RNA
fragments separated on agarose gel are transferred to a membrane, and then specific RNA is
visualized on an autoradiogram

• RNA bands can also be blotted onto nitrocellulose paper under appropriate
conditions and suitable nylon membranes.

14.6.2.1 Applications of Nothern Blot

• This technique is use to study the process of RNA degradation and alternative
splicing.
• Nothern blotting finds its use in the study of differential gene expression that are
tissue specific and condition specific that might offer an early detection of disease.
• Diagnosis of several diseases (Crohn’s diseases) including viral infection.
• This technique is widely used to study the overexpression of oncogenes and
downregulation of tumor suppressor genes in cancerous cells.
• Detect specific mRNA molecular weights and contents in a sample.
14 Techniques of Molecular Genetics 677

14.6.3 Visualizing RNA by RT-PCR

RT-PCR stands for Reverse Transcriptase Polymerase Chain Reaction. Another way
of detecting a specific mRNA is through PCR that enables to amplify the specific
message. This process requires copying of mRNA to cDNA with the aid of reverse
transcriptase. This is a highly specific method, and it becomes possible to detect
specific RNA species in a single cell (Fig. 14.26). This process also helps in
detecting low levels of mRNA and also makes it easy to analyze gene expression
that is difficult to obtain in huge quantity, for example, gene expression in cells from
tumors in order to pinpoint those genes that are expressed in such conditions. Tissue-
specific expression or expression of cell under stress conditions can be monitored by
this technique. RT-PCR also requires less quantity of mRNA and hence requires
fewer cells to achieve same goal as compared to conventional Northern blot.
RT-PCR is a boon during the current times of SARS-COV2 (coronavirus pan-
demic). It has gained a lot of repute in recent times and has helped researchers and
medical doctors to know the exact viral load in the patient’s body. This helps in
proper diagnosis and relevant treatment of the diseased person.

14.6.4 Visualizing Proteins by Western Blotting

Western blotting (also called immunoblotting) is a technique used for analysis of

individual proteins in a protein mixture (e.g., a cell lysate). In Western blotting
(immunoblotting), the protein mixture is applied to a gel electrophoresis in a carrier
matrix (SDS-PAGE, native PAGE, isoelectric focusing, 2D gel electrophoresis, etc.)
to sort the proteins by size, charge, or other differences in individual protein bands
(Fig. 14.27). The separated protein bands are then transferred to a carrier membrane
(e.g., nitrocellulose, nylon, or PVDF) (blotting). The proteins adhere to the mem-
brane in the same pattern as they have been separated due to interactions of charges.
The proteins on this immunoblot are then accessible for antibody binding for
detection.
Antibodies are used to detect target proteins on the Western blot (immunoblot).
The antibodies are conjugated with fluorescent or radioactive labels or enzymes that
give a subsequent reaction with an applied reagent, leading to a coloring or emission
of light, enabling detection.
The term Western Blotting is also based on a play of words. The Southern blot,
which is a method to detect specific DNA sequences, is named after Ed Southern,
who first described this procedure. The Western blot (immunoblot), as well as the
Northern blot (for RNA detection), play on the meaning of this name.

Applications of Western Blotting

• The confirmatory HIV test.
• Used as the definitive test for Bovine spongiform encephalopathy (mad cow
disease).
• Some forms of Lyme disease testing employ Western blotting.
678 N. Sharma and S. Tiwari

Fig. 14.26 A schematic diagram representing the process of reverse transcription. The primer
base-pairs with the mRNA, and it extends along the length of mRNA molecule with the help of
enzyme reverse transcriptase. The RNA-DNA hybrid thus formed is cleaved with the help of
RNAse H, an enzyme that specifically digests the RNA segment. The DNA thus left is actually
called cDNA (c, complementary). Another primer anneals to this cDNA and adds nucleotides that
are complementary to this cDNA. This completes the first round of PCR cycle. The cycle is repeated
multiple times by using primers 1 and 2. This generates the PCR product. This technique has
recently become an advantage in dealing with coronavirus pandemic
14 Techniques of Molecular Genetics 679

Fig. 14.27 A schematic showing the process of Western (immunoblotting) blotting

Table 14.1 Difference between southern blot, northern blot, and western blot
Characteristics Southern blot Northern blot Western blot
Molecule to be DNA RNA Protein
detected
Extraction Alcohol precipitation Cellulose Differential
chromatography configuration
Separation (gel Agarose gel Agarose gel SDS-PAGE
used) electrophoresis electrophoresis
Denaturation Alkali (NaOH) Not required Not required
Blotting method Capillary blotting Capillary blotting Electroblotting
Membrane used NC or nylon Nylon or DBM NC or PVDF
Blocking Pretreatment Not required BSA/milk powder
Probe used Radiolabeled ssDNA Radiolabeled ssDNA One or two antibody
Hybridization DNA-DNA DNA-RNA Ag-Ab complex
Detection Autoradiography Autoradiography Colorimetric
Application DNA fingerprinting Disease diagnosis HIV and hepatitis B

• Western blot can also be used as a confirmatory test for hepatitis B infection.
• In veterinary medicine, Western blot is sometimes used to confirm FIV+ status
in cats.
• This technique is also employed in the gene expression studies.
• It is used in the definitive test for BSE.Chapter Summary
• Working with DNA, RNA, and proteins is very important part in the field of
molecular biology.
680 N. Sharma and S. Tiwari

• Blot: A membrane on which biological molecules such as protein and nucleic

acids are adsorbed or immobilized.
• The process of transferring these molecules (proteins and nucleic acids) from a
gel to the membrane followed by their detection on the membrane is known as
blotting.
• Generally, the process of transferring molecules from agarose gel to nitrocellulose
membrane is often done by capillary blotting which uses capillary action to
transfer DNA, RNA, or protein onto a nitrocellulose membrane; however, in
some cases, electroblotting is also observed that uses current electricity for the
same purpose.
• Dot blot: A technique often used to detect proteins. DNA/RNA can also be
detected. This is performed to screen the binding capabilities of antibodies.
Simplification of Western blot with the exception that proteins to be detected
are not first separated by electrophoresis. Fast method and is cheaper than
Western blotting techniques.
• A method of Eastern blotting is also present. This is nothing but a modification of
Western blotting that could detect posttranslational modifications.

14.7 Analyses of Gene

14.7.1 Physical Mapping by Restriction Endonuclease

Physical mapping is a technique used in molecular biology that is used to determine

the physical structure of the genome (Fig. 14.28). This includes the exact position of
restriction sites, the positions of specific clones, and the complete sequence of gene.
Restriction endonucleases are the enzymes that recognize specific sequence of

Fig. 14.28 A diagram showing practical application of physical mapping

14 Techniques of Molecular Genetics 681

Fig. 14.29 A schematic diagram representing the procedure for construction of a physical map

nucleotide and cut at a specific position. Restriction endonuclease can either produce
blunt ends or they can produce sticky ends. Physical maps are used to arrange
fragments of a cloned DNA.

Mapping of a Sequence with the Help of Restriction Endonuclease

This is one of the most common used techniques to create a physical map. In order to
know the exact location of restriction sites, a sample of DNA is cleaved with a
restriction enzyme, and another sample is cut with a different restriction enzyme
(Fig. 14.29). A third sample is cut with both the restriction enzymes that are
previously used to cleave sample of DNA. After restriction endonuclease digestion,
the DNA segments produced are separated by gel electrophoresis, and their relative
sizes are compared. Overlapping in the size of the fragments produced can be used to
position restriction sites on the original DNA molecule. These maps are often created
for genomic analysis. The basic difference between a genetic map and a physical
map is that genetic maps are based on recombination rates and are measured in
percent recombination, whereas physical maps are based on physical distances and
measured in base pairs.

14.8 Sequencing of Genes

Biomolecules are basic structural and functional unit of a living cell. Nucleotides
(DNA and RNA) form the genetic basis of inheritance. We now know that proteins
are in fact the translated message of the RNA which in turn is transcribed from DNA.
A gene by definition is a set of three consecutive nucleotides that codes for a
functional polypeptide chain or an RNA molecule. The functional properties of a
gene can only be decoded once we know the gene sequence. DNA sequence is
682 N. Sharma and S. Tiwari

Table 14.2 A brief history of DNA sequencing

Year Milestone achievement
1963 The alanine tRNA of yeast was sequenced by Robert Holley
1972 MS2 bacteriophage was sequenced using RNA sequencing by Walter Fiers
1973 With wandering spot analysis, Walter Gilbert and Allan Maxam sequenced lac operator
1975 The plus-minus sequencing of FX174 bacteriophage was developed by Sanger and
Coulson
1977 The chemical cleavage method of DNA sequencing was discovered by Maxam and
Gilbert
1977 The chain termination method of sequencing was discovered by Frederick Sanger
1980 The Nobel Prize for DNA sequencing was awarded to Sanger and Gilbert
1986 The semiautomated DNA sequencing machine was invented by Leroy E. Hood
1997 Fully automatic sequencing machine with capillary electrophoresis
2005 Next-generation sequencers like Genome Sequencer GS20, 454 Life Sciences, Roche
2006 Genome Analyzer, Solexa/Illumina
2007 SOLID Applied Biosystem

perhaps the most crucial technique that is currently available to molecular biologist
that allows to determine the precise order of nucleotide in a piece of DNA. DNA
sequencing nowadays has become a vital activity of most labs. These methods are
about 50 years old. Gene sequencing is defined as a method of determining the order
and arrangement of nucleotides in DNA fragments. Any segment of RNA or DNA
can be used to derive the sequence of the gene. The DNA sequence provides
valuable information about the presence of regulatory regions, coding regions,
homologous sequence, and sequence variation in two forms of genes or alleles. In
the mid-1970s, rapid and efficient DNA sequencing was made possible, but earlier,
these techniques were restricted to individual genes, but with advancement in
technologies, it has been made possible to sequence the entire genome since
1990s. A number of different methods are devised to sequence DNA and are broadly
classified into the chain termination method and the next-generation sequencing
method The chain termination sequencing method will be discussed in detail, and for
next-generation sequencing, please refer to high-throughput sequencing.

Sanger Dideoxy Sequencing Method of Gene Sequencing

This method of DNA sequencing was developed by Frederick Sanger and his
colleagues in the mid of 1970s. This technique found wide range of application in
sequencing the DNA segment. This is also known as chain termination method or
dideoxy method of DNA sequencing. The basic principle behind this technique is
that single-stranded DNA molecules that differ in size by a single nucleotide can be
separated from one another by polyacrylamide gel electrophoresis.
Chain termination sequencing (Fig. 14.30) is performed by DNA polymerase. It
makes a copy of DNA molecule that is being sequenced. This method begins with a
short nucleotide being annealed to the template DNA oligonucleotide acts as a
primer for the synthesis of new DNA strand that is complementary to the template.
14 Techniques of Molecular Genetics 683

Fig. 14.30 A schematic diagram representing the process of chain termination

The basic science behind the chain termination reaction is that DNA polymerase
cannot discriminate between the deoxy and dideoxynucleotide. Once incorporated, a
dideoxynucleotide blocks further elongation because it lacks the 30 OH group needed
to form a linkage with the next nucleotide. Since the ratio of deoxyribonucleotide to
dideoxynucleotide is relatively higher, strand synthesis does not always terminate
close to primer, and a DNA may be extended to several hundred nucleotides before
the incorporation of dideoxynucleotide. As the consequence to this formation of new
molecule takes place, these new molecules are of different length, each of them
ending in a dideoxynucleotide.
A schematic diagram representing the process of chain termination. Figure 14.30a
represents a primer that is annealed to a template DNA that extends from 50 to 30
direction. Figure 14.30b represents a deoxynucleotide which lacks a 30 OH crucial
for elongation of DNA. This molecule halts the process of DNA synthesis. The DNA
polymerase is unable to distinguish between deoxynucleotide and
684 N. Sharma and S. Tiwari

Fig. 14.31 A diagram representing the detailed sequencing of a DNA fragment with the help of a
detector. Usually, this detection is achieved by a fluorescently labeled molecule. Here is this
diagram: (a) A is labeled as orange color, T is blue, C is green, and G is red. So where there is a
green signal, one can say the nucleotide is adenine. (b) The diagram (Fig. 14.32) represents a
sequence of nucleotide in the form of a printout. This sequence could also be stored for future
reference

dideoxynucleotide. The dideoxynucleoside is purposely added to terminate chain

elongation. Figure 14.30c represents the incorporation of dideoxynucleotide that
results in the formation of DNA fragments of different chain lengths.
To correctly sequence the DNA fragments, it is important to identify the
dideoxynucleotide at the end of each chain-terminated molecule. This step is done
by electrophoresis in polyacrylamide gel. This is done to separate the molecules
according to their length.
After separation, molecules are run through a fluorescence detector that helps in
discriminating labels attached to deoxynucleotide (Fig. 14.31). The detector there-
fore determines that each molecule ends in A, C, G, or T. The sequence can be
printed by the operator or could be saved for future reference.
A unique feature of chain termination reaction is that not all DNA polymerases
can be used in this procedure. This is due to the fact that a DNA polymerase have
diverse enzyme activities. It can degrade as well as synthesize DNA. DNA degrada-
tion can occur in 50 –30 direction and can occur in 30 –50 direction. If it occurs in 30 –5
direction, this could adversely affect chain-terminating sequence. This is due to the
fact that 30 –50 activity prevents chain termination from occurring as this activity
removes the dideoxynucleotide immediately after it has been added at the 30 end of
strand of this being synthesized.
14 Techniques of Molecular Genetics 685

Fig. 14.32 Determining the DNA sequence through Sanger method. This figure represents the
method to determine the exact sequence of DNA through Sanger sequencing method. It is clearly
visible that gel runs from top to bottom where top represents larger- or bigger-sized fragments, and
the bottom represents the smaller size fragments. However, the DNA sequence is determined from
bottom to top and read in 50 –30 direction. One key point to mention here is that the sequence that is
determined here is of the non-template strand. It is complementary to the template strand

In the actual method of chain-terminating sequence, the Klenow polymerase was

used as a sequencing enzyme. This enzyme was a modified version of DNA
polymerase I in the sense that its 50 –30 exonuclease activity was removed, and a
mutation was done so as to hamper its 30 –50 exonuclease activity.
However, Kelow fragment is not suitable for DNA sequencing due to its low
processivity, i.e., it can only synthesize a short DNA strand before dissociation from
the template due to natural causes. This limits the length of sequence to about 250 bp
in a single experiment.
These drawbacks in Klenow fragments lead to the use of Taq DNA polymerase
with high processivity and no exonuclease activity, making it suitable for chain
termination sequence.
Chain termination sequence method that uses Taq polymerase is called thermal
cycle sequence as it is quite similar to PCR with the slight modification that uses just
single primer, and the reaction mixture includes four dideoxynucleotides. The use of
single primer makes the amplification linear and not exponential as in the case of
real PCR.
686 N. Sharma and S. Tiwari

Limitations of Chain Termination Sequencing

Sanger dideoxy chain termination has a limited use and can lead to problems that can
negatively affect result analysis of the sequenced data. The following are the most
common drawbacks of Sanger sequencing:

1. Sanger method of sequencing is mainly used to sequence short pieces of DNA

about 300–1000 bp.
2. The quality of sanger sequencing is often compromised for the first 20–40 bases
because it is where primer binds.
3. Sequence quality tends to reduce after 700–900 bases.
4. Some unwanted DNA segments might also be sequenced. This problem may arise
due to the fact that if a DNA fragment being sequenced has been cloned, one
might also find some vector sequence in the final result.
5. Sanger sequence is relatively expensive in terms of the fact that it takes about 500
$ per 1000 bases. The Human Genome Project used Sanger sequencing to
sequence human genome.

14.8.1 High-Throughput Sequencing (HST)

High-throughput sequencing is a set of techniques that uses modern machines to

sequence DNA. This has greatly influenced our understanding of biology, human
diversity, and disease. Advancement in DNA sequencing technologies have opened
up new opportunities and introduced us to the era of personal genomes and genomic
medicine. These technologies are quickly reliable and often automated.
Human Genome Project presented its first draft in 2001 which was followed by
genomic sequencing of several other organisms. Sanger sequencing allowed all these
projects to accomplish but was not desirable due to limited throughput and large cost
investments. It is estimated that the first Human Genome Project costed around 0.5 to
1 billion dollars. Due to this high-cost investment, the National Human Genome
Research Institute (NGHRI) created a DNA technology which cut down the original
cost to 70 million dollars, aiming to achieve a 1000 dollars genome sequencing in a
decade. HST thus emerged to fulfil this ambitious dream.
To begin, some improvements were made in conventional Sanger sequencing
techniques which lead to a cost reduction by 100 folds; however, to reach a 1000-
dollar genome threshold, an additional jump of five orders was much needed. The
cost of genome sequencing (without interpretation) is less than 2000 dollars, all
thanks to HST technologies. The following paragraph will be discussing some of the
most popular HST technologies just to give an overview about recent trends in
genome sequencing.

14.8.1.1 Illumina
This biotech giant released Genome Analyzer II in 2006, and advancements in
Illumina’s technologies over the past years have set a pace for huge profits in
terms of output and reductions in cost. As an outcome, Illumina technologies rule
14 Techniques of Molecular Genetics 687

the HST business. This technology uses a fluorescently labeled molecule 30 -O-
azidomethyl-dNTPs to halt the polymerization reaction, thus enabling the removal
of bases that didn’t incorporate and allow fluorescent imaging to determine the
added nucleotide. An added advantage common to all Illumina models is that overall
error rates are below 1% and the most common type of error encountered is
substitution type.
Illumina currently provides a variety of sequencing machines optimized for
various uses. The most common sequencers are MiSeq, NextSeq500, and the
HiSeq series. The MiSeq and HiSeq are the more accepted platforms. The MiSeq
is a fast personal benchtop-sized sequencer and could sequence small genomes in
just 4 hrs. HiSeq, on the other hand, is designed for high-throughput applications
which can generate about 1 Tb outputs in 6 days (pretty fast!). Illumina also
launched NextSeq and HiSeq X Ten in 2014. NextSeq 500 was also designed as a
benchtop sequencer for individual labs. NextSeq is capable of producing 120 Gb of
data in less than 30 hrs. NextSeq also uses a unique two-channel sequencing strategy
in which cytosine is labeled red, thymine is labeled green, and adenine is labeled
yellow (labeled with a mixture of red and green), and guanine remains unlabeled.
The four-channel sequencing strategy is used in HiSeq and MiSeq platform.
Two-channel sequencing strategy is favored because it reduces data processing
times and increases the throughput.

14.8.1.2 Life Technologies/ThermoFisher/Ion Torrent

Ion Torrent’s semiconductor sequencing technology was commercialized by Life
Technologies in 2010 in the form of benchtop Ion PGM sequencer. This sequencer
senses pH changes induced by hydrogen ions during DNA extension. These changes
are monitored by a sensor placed at the bottom of a microwell and changed into a
voltage signal. The voltage signal is directly proportional to the number of bases that
are incorporated during sequencing. In addition to this, Ion Torrent avoids optical
scanning to distinguish nucleotides during cycles of sequencing. This speeds up the
sequencing process and is cost-effective.
Ion Torrent launched another machine in 2012 named the Ion Proton that has an
advantage to increase the output over the PGM by a magnitude of the order of
1 Gb vs. 10 Gb. Proton is currently limited to 200 bp reads as opposed to 400 bp read
by PGM. PGM found its use in targeted resequencing and small genome analysis,
whereas Proton has the ability to sequence exomes and perform whole transcriptome
analysis.
The speed of these sequencers can range from 2 to 8 hrs that depends on factors
like the machine and chip used for sequencing. This property makes these
sequencers quite useful in terms of clinical applications. The most common types
of errors that could occur during this type of sequencing include additions and
deletions. A homopolymer tail of more than 6 bp could increase the error rates.

14.8.1.3 Pacific Biosciences

They are credited for pioneering single-molecule real-time sequencing (SMRT).
They used strand displacing polymers that allowed the original DNA molecule to
688 N. Sharma and S. Tiwari

be sequenced multiple times, which leads to an increase in accuracy. This method

involves direct sequencing of native and potentially modified DNA. DNA synthesis
takes place in zeptoliter-sized chamber termed as zero-mode waveguides (ZMWs) in
which a single polymerase is fixed at the bottom of the chamber. The science behind
these chambers adds value to this technique by reducing background noise such as
phosphate-labeled versions of all four nucleotide as a result of this polymerization
can occur continuously, and DNA sequence can be read in real time from fluorescent
signals recorded in the video.
RS II is the only sequencing machine made commercially available by Pacific
Biosciences. This was launched in 2010. This machine can have enhanced perfor-
mance when the chemistry involved in the systems gets altered. SMRT cell can
produce up to 1 Gb reads of data in just 4 hrs. The error rates in these machines can
be as large as 11%, which is a disadvantage to single-molecule sequencing
platforms. This machine finds useful application in projects involving de novo
assembly of small bacterial and viral genomes. Reconstruction of structural variation
(SV) in genomes and isoform usage in transcriptome is an important area where
SMRT sequencing is advantageous over short-read technologies.
Lower throughput and higher per-base sequencing cost can limit the scope of
most genome-wide studies. SMRT sequencing has a distinguishing feature of
polymerization monitored in real time. This technology can also map
6-methyladenine and 5-methylcytosine genome-wide in bacteria.

14.8.1.4 Oxford Nanopore Technologies

Nanopore-based sequencing is a recent single-molecule strategy that has made
remarkable progress in recent years. It majorly relies on the movement of DNA
nucleotides through a small channel. The sequencing is done by measuring charac-
teristic changes in the current. The first available device for nanopore sequencing is
MinION, which was a USB-powered portable sequencer. The error rates reported in
this sequencing were 4.9%, 7.8%, and 5.1% for insertion, deletion, and substitution,
respectively. MinION reads have been successfully used to figure out the positional
and structural aspects of bacterial resistance island. With lower throughputs and
higher error rates, this sequencing tool is not a preferred choice in current times.

Use of HTS Application

1. Genome sequencing and variation: The first de novo sequencing was done for
Acinetobacter baumannii. HTS has also been able to characterize SV in the
human genome. HTS are frequently used to give an insight about human diversity
and disease.
2. Mapping regulatory information of genome: HTS can map regulatory elements of
DNA at high resolution. This mapping of sequences back to the genome may
reveal location of chromatin modification.
3. Mapping the 3-D organization of the genome: Global organization and compart-
mentalization of chromosomes are advanced by HST technologies. Hi-C was one
of the first technique to allow impartial genome-wide probing of chromatin
organization and revealed open and closed genome states.
14 Techniques of Molecular Genetics 689

4. Characterizing the transcriptome: HTS technologies could characterize various

classes of RNA. This also includes characterization of RNA structure,
RNA-protein interactions, and genomic localization. HST has led to the develop-
ment of microRNA target discovery. HTS application have also made it possible
to determine transcript structure both in vitro and in vivo.
5. Microbiome sequencing: HST technologies have enabled classifying
metagenomic samples. This can provide a knowledge of the microbial diversity
from wide variety of sources. In fact, this has also given rise to the concept of
personal microbiome.
6. Genome sequencing of rare disease: The ability to sequence large genome,
exomes, and transcriptomes has added to our current knowledge about human
health and disease. This is particularly relevant for rare mendelian disorders and
cancer. To quote an example, exome sequencing of a child with severe inflam-
matory bowel disease uncovered a mutation in an important regulator of inflam-
mation, X-linked inhibitor of apoptosis (XIAP). Based on the severity of the
child’s symptoms as well as the molecular diagnosis, a bone marrow transplant
was given to the patient, which subsequently reduced his symptoms.
7. Cancer genome sequencing: The Cancer Genome Atlas and International Cancer
Genome Consortium performed sequencing on thousands of cancer and normal
cell pairs. These studies thus provide a better understanding of cancers at molec-
ular level. Limitations of HTS: Although HTS has a lot to offer, its limitation
includes low hit rate due to incompatible libraries and potential false-positive
results.

14.8.2 Whole Genome Sequencing

The genome of each individual organism contains its entire genetic information.
Whole genome sequencing is a powerful technology that helps researchers to obtain
the entire genetic information of the genome and reveals the complexity and
diversity of the genome. Whole genome sequencing can detect variants including
single nucleotide variants (single nucleotide polymorphism, SNPs), and insertions,
deletions, and copy number changes are large-scale structure variance. Whole
genome sequencing can be divided into de novo sequencing and resequencing
based on whether there is a reference genome. A reference genome can make
genome assembly easy and rapid.
Methods of Whole Genome Sequencing: In the early 1980s, Sanger successfully
completed the genome sequencing of the lambda phage by using shotgun method,
and the method was successfully applied to large viral DNA, organelle DNA, and
bacterial genome. Shotgun sequencing is the classic strategy for genome sequencing.
Shotgun strategy provides a technical guarantee for large-scale sequencing. This
technology first randomly interrupts a complete target sequence into small fragment
sequence, separating and then splicing them into a consistent sequence by using the
overlapping relationship of this small fragments. For large genomes, it mainly
690 N. Sharma and S. Tiwari

includes two methods: the hierarchical shotgun sequencing with the clone-by-clone
method and the other is the whole genome shotgun sequencing.
Clone-by-Clone Method: This method was once adapted by the Human Genome
Project consortium, and this method can generate high-density maps, making the
genome assembly easier. It generally includes four steps:

• Preparation of BAC clone library.

• Preparation of clone fragment.
• BAC clone sequencing.
• Sequencing assembly.

However, this method is time-consuming and costly, so it is seldom used at

present.

Whole Genome Sequencing

This generally involves four steps: random fragmentation of genomic DNA, size
selection using electrophoresis, library construction, and sequencing sequence
genome assembly. Two different sizes of DNA fragments including longer insert
of 2–2.5 kb and short insert of 0.5 to 1.2 kb are selected from the agarose gel. The
longer inserts are cloned in phage or cosmid vectors. The short inserts are cloned in
plasmid vectors.

Advantages of Whole Genome Sequencing

• Do not require genome map.
• Less time-consuming.
• Cost-effective.

Disadvantages Include
• Genome assembly of eukaryotic genome is difficult to abundant repetitive
sequences.
• Genome sequence using this method is not a reliable source and might produce
false results.

Advanced Sequencing Technologies Promote Whole Genome Sequencing

In this method, genomic DNA is first randomly fragmented using sonication or
nebulization. They are then ligated to the platform specific to sets of double-stranded
adapters to generate shotgun library. Subsequently, these library fragments are
sequenced by next-generation sequencing instruments. All NGS instruments utilize
microfluidic device to contain the amplified fragment of the shotgun library followed
by an imaging step that collects data from fragments being actively sequenced. The
data analyses workflow for whole genome sequencing includes raw read quality
control, data preprocessing, alignment, variant calling, genome assembly, genome
annotation, and other analyses. NGS instruments require the PCR amplification step
before sequencing, while minION and PacBio RSII rely on single molecule for
sequencing. This sequencing technology omits the amplification step and can
14 Techniques of Molecular Genetics 691

perform direct sequencing. Although NGS has enabled population scale analyses of
small variants, it is difficult to identify larger structural variations. De novo assembly
using next-generation sequencing is often at lower quality compared to early
methods. The single-molecule sequence technology can get over these difficulties
which span nearly the entire chromosome and not sensitive to GC content. These
have been used to produce highly accurate de novo and reference assemblies for
microorganisms, plants, animals, and humans, enabling new insights to revolution
and sequence diversity.

Box 14.1: Scientific Concept: Efficient Method for Site-Directed

Mutagenesis in Large Plasmids Without Subcloning – Louay K. Hallak
et al.
Conventional site-directed mutagenesis (SDM) techniques allow easy manip-
ulation of DNA sequence in small plasmid; however, introduction of mutation
in larger plasmids is often considered a daunting task. This is so because
introduction of mutation in larger plasmids involves the necessity of
subcloning. Subcloning is a time-consuming and labor-intensive technique.
In addition to this, subcloning method is not reliable for large plasmids. A
relatively new DNA mutagenesis technique has been developed in this regard
and named URMAC (Unrestricted Mutagenesis and Cloning). The basis of
this technique is to use quick biochemical reactions and replace it with
conventional subcloning technique. UMRAC allows the use of a wide range
of plasmid size and allows simultaneous introduction of multiple mutations.
UMRAC uses two PCR reactions each followed by ligation step that allows it
to circularize the product with an optional third enrichment PCR step which is
followed by traditional cloning step that requires two restriction sites.

URMAC Method
The basic principle involved in this method is to convert a linear PCR product
generated from a plasmid template to a circular DNA that can be opened at second
site. This involves the amplifications with primers that contain required mutations
circularized by religation again and amplified with original primers to replicate the
DNA that contains the required mutations. URMAC uses a set of two primers,
namely, the starter primers and the opener mutagenic primers. These primers are
phosphorylated at the 50 end so that they can participate in the ligation step.
Starter primers are used twice, firstly in the initiation step that amplifies the
modification target sequence and secondly in the enrichment step.
The open primers are used to introduce the mutation of interest.
The URMAC process requires six sequential steps (Fig. 14.33) that include a
PCR reaction followed by ligation followed by another PCR reaction. This is
followed by another ligation which is often succeeded by another PCR enrichment
step. The final step in this procedure involves digestion with restriction enzymes
(Fig. 14.34).
692 N. Sharma and S. Tiwari

Fig. 14.33 A schematic diagram representing the process of URMAC method. This diagram
represents an imaginary insertion within the modification target (black lines) in the original DNA
plasmid. The first PCR generates the starter DNA copy of the modification target that is positioned
between specific restriction sites X and Y in this figure. This is achieved by the use of thermostable
DNA polymerase using starter primers S1 and S2 represented by black arrows. T4 DNA ligase then
14 Techniques of Molecular Genetics 693

URMAC offers the following advantages over other conventional SDM

techniques:

1. PCR reactions have a higher success rate in small fragments rather than full-
length plasmids.
2. URMAC tends to avoid the errors of introducing polymerase errors as reported in
the QuickChange Method and inverse PCR method because the fragment being
amplified is much smaller.
3. URMAC do not require sequence verification as any region of plasmid that is not
the direct target of DNA mutagenesis remained unaffected by DNA polymerase.
4. URMAC is very fast as compared to traditional SDM subcloning techniques with
an average of a single day to complete URMAC and an additional 3 days to clone
the final product into the original plasmid. It’s quite fast when compared to
3–4 weeks required for subcloning.
5. This technique can reduce the challenge of high GC-containing plasmids by
avoiding PCR amplification of those parts of plasmids. URMAC is cost-effective
when compared to subcloning as it requires less labor and materials.
6. It also offers versatility for handling any combinations of deletions, additions, and
substitutions.

Box 14.2: Scientific Concept: The pPSU Plasmids for Generating DNA
Molecular Weight Markers
Nucleic acid visualization by gel electrophoresis is one of the most common
techniques in molecular biology. The visualization aims to know the exact size
of the DNA bands, and molecular weight markers or ladders are commonly
used for this purpose. With the advancement in the basic understanding of
plasmid creation, a group of researchers constructed a pair of cost-effective
plasmids. The pPSU1 and pPSU2 pair of molecular weight marker plasmids
can produce both 100 bp and 1 Kb DNA ladders when digested with two
common restrictions, enzymes Pst1 and EcoRV, respectively. The 100 bp
ladder fragments have been optimized in such a way that they can be migrated
appropriately on both agarose and native polyacrylamide. The pPSU molecu-
lar weight marker plasmids were constructed in such a way to provide low

(continued)

⁄


Fig. 14.33 (continued) circularizes the starter DNA in the next step that forms closed starter DNA.
The closed starter DNA acts as a template for the next PCR cycle that uses opener primers OP1 and
OP2 yielding a mutated intermediate DNA. OP1 incorporates an insertion mutation that have the
sequence of interest attached to its 50 terminal end. The intermediate DNA is circularized by the aid
of T4 DNA ligase. The SP1 and SP2 primers are used in the enrichment step amplifying the linear
modified DNA. The linear modified DNA and the original plasmid are digested with restriction
endonuclease that cleaves at unique sites X and Y. The appropriate fragments are joined to produce
the modified original DNA
694 N. Sharma and S. Tiwari

Fig. 14.34 A schematic diagram representing the validation of URMAC method by insertion,
substitution, and deletion of some restriction site in pUC 18 plasmid. An illustration (a) showing
modification target relative to the positions of restriction sites that occur between the starter primers
S1 and S2. As a result of first PCR reaction, the starter DNA migrated as expected, 532 bp on a 1%
agarose gel. A DNA ladder of 100 bp size is shown for comparison. (b) is showing a diagram that
introduces different kinds of mutations using a closed starter DNA from a PCR product. a is used as
a template and is used to create opener/mutagenic primers. The top picture at the right corner shows
intermediate DNA which contains mutations. The figure at the bottom shows modified DNA after
enrichment with SP1 and SP2 primers. (c) Validation of URMAC mutagenesis for the three
different types of mutations by restriction analysis
14 Techniques of Molecular Genetics 695

Box 14.2 (continued)

molecular weight markers when digested with Pst1 restriction enzyme. Once
digested with Pst1, both plasmids produce a ladder of 50, 100, 200, 300,
400, 500, 600, 700, 800, 900, 1000, 1500, 2000, and 4100 bp reference
fragments.
An additional site of 500 bp Pst1 fragment is deliberately added to provide
a visual landmark for the fragment on the electrophoresis gel. EcoRV diges-
tion of the same pair of plasmids produced a 1 Kb ladder which gives 500, 750,
1000, 1500, 2000, 3000, 4000, and 5000 bp reference fragment by making the
10 kb pPSU plasmid linear. The pPSU1 and pPSU2 plasmids can be digested
individually with appropriate restriction enzyme, or they can be combined and
digested in a single reaction. These two plasmids can be linearized with Nco1
and BglII to yield bands that can be up to 10 kb in size. Ppsu1 alone can
produce 0.5, 1.0, 1.5, 2.0, 3.0, 5.0, 7.0, and 10 kb fragments by combining
EcoRV, EcoRI, and Nco1 digest. The plasmid hence digested can empower us
with large number of bands to be visualized simultaneously. In addition to this,
these plasmids also confer ampicillin resistance and can be grown on standard
E. coli strains and media which adds to its benefits of being a cost-effective
and multipurpose use plasmid.

Fig. 14.35 A figure representing the plasmid maps of pPSU1 and pPSU2. A nesting approach is
used to create a 64 kb of 100 bp and 1 kb ladder fragments generated by the use of Pst1 enzyme of
each plasmid shown in red and 1 Kb fragments produced by EcoRV digestions. pPSU1 contains the
500, 700, 800, 900, 1000, 2000, and 4100 bp PstI fragments, and 500, 1000, 1500, 2000, and
5000 bp EcoRV fragments. pPSU2 contains 50–600, 1500, and 4100 bp PstI fragments, and
750, 3000, and 4000 bp EcoRV fragments
696 N. Sharma and S. Tiwari

Fig. 14.36 The figure here represents an actual ladder on a polyacrylamide gel (10%) after
electrophoresis. Lane 1: reference Thermo Scientific GeneRuler 100 bp ladder (ref1). Lane 2:
PstI digestion of the intermediate pPSU1 plasmid contains 800, 900, and 1000 bp lambda DNA
fragments which migrate anomalously slowly. Lane 3: pPSU1m contains replacements for the
800 and 900 bp fragments from lambda DNA and the 1000 bp fragment from the human Bmi1
RING domain gene. Lane 4: PstI digestion of the final pPSU1 plasmid containing a replacement
800 bp fragment from the human G9a histone methyltransferase gene. Lane 5: reference New
England Biolabs 2-Log DNA ladder (ref2)

Figures 14.36, 14.35, 14.36, 14.37, and 14.38

The above three figures from 14.36 to 14.38 are the representatives of different
ladders that are produced by the restriction endonuclease digestion under different
conditions. The results of the ladder generated with different set of restriction
enzyme are compared with the standard (Thermo Scientific GeneRuler) in this
case. This helps easy identification of the DNA segment and thus helps in determin-
ing the actual size of the DNA segment.
14 Techniques of Molecular Genetics 697

linear linear,
100 bp 1 kb 1+2 EcoRl
ladder ladder & &
Pstl EcoRV EcoRV EcoRV
pPSU 1+2 1 2 1+2 1+2 1 ref2
10 000 10 000
7 750 7 000
5 000 5 000
4 000 4 000
3 000 3 000

2 000 2 000
1 500 1 500

1 000 1 000
800
750

500 500

1 2 3 4 5 6 7

Fig. 14.37 This is an actual representation of the ladder in Lane 1: when the pPSU plasmids
(pPSU1 and pPSU2) is cleaved by Pst1 restriction enzyme. Lane 2: pPSU 1 digested with EcoRV.
Lane 3: pPSU 2 digested with EcoRV. Lane 4: shows when both the plasmids are joined and
digested simultaneously. Lane 5: linear pPSU plasmids (pPSU1 and pPSU2) digested with EcoRV.
Lane 6: linear set of plasmids digested with EcoRV and EcoRI. Lane 7: represents the reference
ladder that is used to know the exact size of DNA

14.9 Summary

• Sequencing is a process that determines the correct order of nucleotides in a piece

of DNA.
• The sequencing technology was introduced by Fedrick Sanger in mid-1970s
(1977) with the aim to sequence genomes of small organisms.
• His technique used chain termination strategy also known as dideoxy method
which uses dideoxynucleotide molecule which stops the elongation reaction. This
generates a variety of different sized fragments that are detected radiolabelly to
sequence a DNA fragment.
• However, the chain termination method of sequencing had its own limitations. It
was a very expensive technology to use. The sequencing process was often very
time-consuming, and sometimes, these sequencing produced false-positive
results.
• All these limitations lead to the advancements of next-generation sequencing
technologies.
698 N. Sharma and S. Tiwari

Pstl digest Pstl

pPSU 1 2 1+2 ref1 1+2 ref2

4 100
2 000 2 000
1 500 1 500
1 200
1 000 1 000
900 900
800 800
700 700
600 600
500 500*
400 400
300 300
200 200

100 100

1 2 3 4 5 6

Fig. 14.38 The ladder pattern is shown in this diagram. Lane 1: pPSU1 digested with Pst1. Lane 2:
pPSU 2 digested with Pst1. Lane 3: pPSU plasmids (pPSU 1 and pPSU 2) digested with Pst1. Lane
4–6: Thermo Scientific GeneRuler lane

• The next-generation sequencing technologies used a set of advanced machines

that produced better sequencing results at a faster rate and were cost-effective.
• Some recent advancements in next-generation sequencing technologies resulted
in formation of high-throughput sequencing.
• The use of high-throughput sequencing made human health and disease simpler
to understand, thus creating a branch of personalized medicine and
pharmacogenomics.
• Whole genome sequencing provides a thorough insight of the genome-wide
studies that could lead to a new era of medical and health research.
• DNA sequencing developed around the 50s is now an advanced area of study and
research and can potentially reveal the secrets of biology.
Genomics
15
Sai Krishna AVS, Sonali Patle, Parampreet Kaur, Shama Omkumar,
and Aarti Sharma

15.1 Overview of Genomic Analysis

The field of genomics is interdisciplinary, and it is defined as the study of genes

present in an organism along with the various techniques involved. It studies the
functions, structures, mapping, and various techniques that are used to edit genomes.
Genome, on the other hand, is defined as the information repository of an organism.
It is the entire set of genetic information that is important for the proper functioning
of the living being over its period of life. Genomes include the genes that are coding
in nature as well as the noncoding DNA, mitochondrial DNA, as well as the DNA of
the chloroplast.
The word genome was first formulated in 1920 to explain “the haploid chromo-
some set, which, together with the pertinent protoplasm, specifies the material
foundations of the species” (Fig. 15.1). The discovery of Mendelian genetics was
done in the year 1900, and the identification of chromosomes as the carrier of genetic
information was done in the year 1902. The fact that DNA carries genetic informa-
tion did not come into light till the late 1920s.
When referred to the Google ngram analysis, it states the case-dependent episodes
of the words “gene,” “genome,” and “chromosome” in the aggregation of books
written in English from 1920 to 2008.
Genome analysis necessitates the prediction of genes, and a vast range of both
animals and plants are sequenced, including microorganisms (Table 15.1), but the

S. KrishnaAVS (*) · P. Kaur · S. Omkumar

Ramaiah University of Applied Sciences, Bangalore, India
S. Patle
Ajay Kumar Garg Engineering College, Ghaziabad, India
A. Sharma
NIMHANS, Bangalore, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 699
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_15
700 S. K. AVS et al.

Fig. 15.1 The interchange of the word “genome” compared to similar expressions.

Table 15.1 Estimated sizes of certain genomes and the number of genes in them (SCFBio, IIT
Delhi)
Species Genome size (mb) Number of genes
Mycoplasma genitalium 0.58 500
Streptococcus pneumoniae 2.2 2500
Escherichia coli 4.6 4400
Saccharomyces cerevisiae 12 5800
Caenorhabditis elegans 97 19,000
Arabidopsis thaliana 125 25,500
Drosophila melanogaster 180 13,700
Oryza sativa 466 45–55,000
Mus musculus 2500 29,000
Homo sapiens 3300 27,000

pace at which genome annotation is done is not at par with the pace of genome
sequencing. There is a high demand for making computational tools to predict genes.
The main question that fascinates scientists regarding the genome is to identify what
part of the genome codes for proteins and what part of it is junk and the classification
of junk DNA.
To successfully sequence a genome, firstly, an organism is selected and the
sequencing of DNA is done, followed by the assembly of the sequence (also
known as sequence compilation) to represent the actual chromosome, and finally,
annotation and analysis of the sequence are done.

DNA Sequencing
The first step in exploring the genome is to determine the DNA sequence. The term
refers to the biochemical methods utilized to determine the accurate representation of
adenine, guanine, cytosine, and thymine, respectively.
There are three main types of methodologies that are used for sequencing DNA:
15 Genomics 701

1. Chain termination method.

2. Chemical degradation method.
3. Pyrosequencing method.

Chain Termination Method

This method was developed by Frederick Sanger and his coworkers in the year 1977.
It is also called the dideoxy method, where the complementary DNA is produced by
the DNA polymerase along with dNTPs and ddNTPs. The termination of the
reaction occurs at random sites due to the addition of ddNTP with the oligonucleo-
tide chain.

Chemical Degradation Method

The establishment of the procedure was done by two scientists named Allan Maxam
and Walter Gilbert in the year 1977. The sequence of a dsDNA molecule is
determined following chemical treatment. The chemicals make cuts at specific
nucleotide sequences.

Pyrosequencing
In the year 1996, Mostafa Ronaghi and Pal Nyren invented the procedure of
pyrosequencing. The procedure necessitates the addition of a deoxynucleotide to
the end of the growing strand, and this is detected due to the emitted light.

DNA Sequencing Methods

High-Throughput Sequencing Methods
A wide range of methods called next-generation or second-generation sequencing
methods were invented in the later part of 1990s and implemented in the 2000s. All
of the methods were named second generation or next generation to create a clear
differentiation from the previous techniques used for sequencing including Sanger
sequencing. This includes next-generation short-read and the second-generation
long-read sequencing methods.
Long-Read Sequencing

1. Single-molecule real-time sequencing—This is a synthesis-dependent approach

where DNA is produced in zero-mode waveguides by using an unmodified
polymerase and freely floating fluorescently labeled nucleotides.
2. Nanopore sequencing—This approach works by monitoring changes to an elec-
tric current when the nucleic acid is passed through the nanopore. The resulting
signal is then decoded to provide the specific sequence.

Short-Read Sequencing

1. Polony sequencing—In 2005, this technique was first used to sequence the
genome of Escherichia coli. This involves an automated microscope, in vitro
coupled tagged library, and ligation-based sequencing chemistry.
702 S. K. AVS et al.

2. 454 pyrosequencing—It was invented by “454 Life Sciences,” after which

“Roche Diagnostics” took its possession. This technique necessitates utilization
of emulsion PCR and pyrosequencing.
3. Illumina (Solexa) sequencing—This method was developed by Shankar
Balasubramanian and David Klenerma, and it involves the use of polymerases
that are engineered and reversible dye terminators technology.
4. SOLiD sequencing—This procedure was invented by Applied Biosystems. It was
an approach that used sequencing by ligation.
5. Ion Torrent sequencing—The technique is developed using the basic chemical
reactions in sequencing, although it has a unique detection system which utilizes
semiconductors.
6. DNA nanoball sequencing—This technique amplifies the little pieces of genetic
material to a DNA nanoball utilizing rolling circle replication mechanism, and it
is followed by sequencing by ligation to determine the nucleotide sequence.

15.1.1 Sequence Compilation

The second step in genome analysis is the assembly or compilation of the fragment
of DNA to recreate the original sequence. The process of DNA sequencing cannot
read the whole-genome fragment at once. Instead, it reads shorter fragments around
50–30,000 bp long, and the size of the fragment depends on which technology of
sequencing is being used.
To explain what sequence compilation means, take the example of a paper being
shredded and rejoined again to form the original one by just looking at the shredded
pieces.

History
The very first assemblers or compilers were developed in the late 1980s and 1990s.
They were developed to join the large number of fragments generated by using
automated sequencers.
Scientist faced tons of problems to sequence the first eukaryotic genome—
Drosophila melanogaster in 2000 and a year later techniques such as Celera
assembler and Arachne. These assemblers can handle genomes from 130 million
to 3 billion base pairs in size.
The basic definition of sequence compilation is to align and merge fragments, and
there are two approaches to assemble the genome.

i. Mapping and assembly—If a previously sequence genome is available as a

reference, then the newly sequenced genome is first plotted by referencing
genome by alignment, followed by assembling in a proper order.
Example: Bowtie—It is a read aligner which maps and assembles the genome.
ii. De novo assembly—When there is no reference genome available, the de novo
approach of assembling is used as explained in Fig. 15.2. In this technique, paired
reads are used instead of single reads as they help in generating scaffolds.
15 Genomics 703

Fig. 15.2 The illustration of the pipeline of de novo assembly (Liao et al. 2019). “The subgraph (a)
shows all reads. The subgraph (b) shows the principle of building de Bruijn graphs. The subgraph
(c) shows the principle of building OLC/String graph. The subgraph (d) shows the principle of
scaffolding and gap filling. The subgraph (e) shows the consensus operation. The subgraph (f)
shows the final genome sequence”

Sequence assembly is a systematic approach where the “assembly of the

sequence reads into contigs, assembly of the contigs into scaffolds (supercontigs),
and assembly of the scaffolds into chromosomes.”
704 S. K. AVS et al.

The process of genome assembly is algorithm driven and automated, and the
following three approaches can be used:

1. Greedy—It is a program that assembles sequences that are most similar to each
other, and it does so by first comparing the sequences in a pairwise manner with
overlaps followed by merging the best overlaps. The gaps generated due to this
are filled by paired-end sequencing (e.g., phrap and CAP).
2. Overlap-layout-consensus (OLC) Hamiltonian path—This approach is based on
pairwise comparisons, and after the comparison, a graph is generated using reads
and overlaps.
The graph represents each sequence as a node, and the nodes which overlaps
generate an edge. The algorithm then determines the Hamiltonian transversal
pathway in the graph, and this contains the nodes and the overlaying nodes which
are then combined together to form the sequence of the genome (e.g., Arachne).
3. De Bruijn graph and Eulerian path—It is mostly used in assembling short reads
but also has some experience with long reads (e.g., Euler-SR, Oases, Velvet,
ALLPATH)

Evaluation of Assembly
Evaluation is an important step as this will inform us whether the sequence assembly
has met the standards (e.g., QUAST, evaluation tool).
The following criteria can be used to evaluate assemblies:

1. N50: The minimum length of contig required to cover 50% of the total length.
2. L50: Number of contigs that are longer than N50.
3. NG50: The minimum length of contig required to cover 50% of the reference
genome.
4. LG50: Number of contigs that are longer than NG50.
5. NA50: Minimum length of aligned blocks that are required to cover 50% of the
total length.
6. LA50: Number of contigs that are longer than NA5.
7. Genome fraction (%): Percentage of bases that align to the reference genome.

15.1.2 Sequence Annotation

Annotation is defined as the procedure which marks the genes in order to identify
their functions, locations, and the coding regions, and the steps are shown in
Fig. 15.3.
The process of sequence annotation comprises of three steps:

1. Recognition of the part inside the genome which is noncoding for a protein.
2. Recognition of genome elements.
3. Addition of elements and the biological information.
15 Genomics 705

Fig. 15.3 A genome annotation flowchart. The flowchart represents the steps in genome annota-
tion, locating the positions of all the genes, feedback from gene identification, general database
search, specialized database search, statistical gene prediction, and prediction of structural features

These steps are performed by automatic annotation tools via computer analysis.
One such very simple method which is most widely used is BLAST. A BLAST
search for homology, and that data helps in annotating the genomes and genes. Any
discrepancies can be handled by manual annotators. A few other annotations can be
done via a database that utilizes the context information of the genome along with
similarity scores and experimental information through their various subsystems
approach.
Annotation can be broadly classified into two types:

1. Structural.
2. Functional.

The structural annotation part deals with recognition of elements in the genome
such as:
706 S. K. AVS et al.

• The localization of ORFs.

• Gene structure.
• Coding locations.
• Regulatory motifs regions.

The second part involving functions deals with addition of genomic elements and
the biological information such as:

• Information regarding biochemical function.

• Information regarding biological function.
• The required interactions and regulation.
• Expression.

A wide range of ongoing projects are going on related to annotating the genome,
for example, ENCODE, Entrez gene, Ensembl, GENCODE, and GeneRIF.
Project GeneQuiz was the first automatic system for genome analysis. It
performed similarity searches followed by automatic evaluation of results and
generation of functional annotation.

15.2 Functional Genomics

This branch aims in determining the functions and interactions of genes. It uses the
huge amount of data that is generated by a variety of transcriptome and genomic
projects.
The main focus of functional genomics is transcription, translation, and gene
expression along with their protein-protein interactions.
The function genomic analysis measures the various changes in DNA, i.e., the
genome as well as the epigenome, RNA, and different interaction between protein,
DNA, and RNA which in turn influence the sample’s phenotype.
The branches of functional genomics are:

1. Genotyping
2. Transcription profiling.
3. Epigenetic profiling.
4. Nucleic acid-protein interactions.
5. Meta-analysis.

Techniques
A variety of techniques are used at DNA, RNA, and protein level, and these
techniques are as shown in Fig. 15.4.
These techniques at different levels are defined as follows:

– At the DNA level, genomics and epigenomics.

– At the RNA level, transcriptomics.
15 Genomics 707

Fig. 15.4 Functional genomics is the study of how the genome, transcripts (genes), proteins, and
metabolites work together to produce a particular phenotype (EMBL-EBI)

Fig. 15.5 Genetic interaction mapping. GI mapping entails perturbing genes in pairs (e.g.,
knockout, knockdown, or overexpression) to see how one gene influences the phenotype of the
other. Cell viability is commonly used as a phenotypic readout, with GIs that increase cellular
fitness being labeled “positive” and GIs that decrease cellular fitness being labeled “negative”
(Krogan Lab, UCSF)

– At the protein level, proteomics.

– At the metabolite level, metabolomics.

Genetic interaction mapping—A systematic approach for deleting genes or

inhibiting their expression is one way to identify genes with related functions even
if there is no interaction between them physically.
The technique involves pairwise perturbation (e.g., knockout, knockdown, or
overexpression) of genes in order to determine how the genes modulate each other’s
phenotypes as explained in Fig. 15.5.
DNA/protein interactions—Techniques such as ChIP sequencing and
CUT&RUN sequencing are developed in order to identify the interaction between
DNA and protein, and this is due to the fact that proteins help in regulating
expression of genes (Fig. 15.6).
708 S. K. AVS et al.

Fig. 15.6 ChIP sequencing workflow. ChIP-Seq can be used to map global binding sites for a
protein by identifying the binding sites of DNA-associated proteins. Cross-linking of DNA-protein
complexes is usually the first step in ChIP-Seq. The fragmented samples are then treated with an
exonuclease to remove any unbound oligonucleotides. The DNA-protein complex is
immunoprecipitated using protein-specific antibodies. The DNA is extracted and sequenced,
yielding high-resolution protein-binding site sequences
15 Genomics 709

Fig. 15.7 An overview of DNA microarray technology. RNA is isolated from the control and the
target samples, and labeled cDNA is then hybridized (Muhammad Afzal, Researchgate)

DNA accessibility assays—ATAC-seq, DNase-Seq, and FAIRE-Seq are few

such assays that help in identifying the accessible regions on the DNA such as
open chromatin region, etc.
Microarray—It helps in detection of a thousand gene expressions at a single time.
They are microscopic slides with thousands of spots indicating a defined position,
and each spot contains a known amount of DNA or gene (Fig. 15.7).
SAGE—It stands for Serial Analysis of Gene Expression. This approach is
established on RNA sequencing as explained in Fig. 15.8. The transcriptomic
technique generates a small overview of mRNA present in the sample in the form
of small tags, and these tags correspond to fragments of those transcriptomes.
From the desired samples, RNA is extracted and purified. Reverse transcriptase
uses biotinylated poly-T tails as primers to transform mRNA to double-stranded
cDNA. Anchoring enzymes recognize and cut at their respective restriction sites,
which are found naturally every few hundred base pairs, to fragment biotinylated
cDNA. The fragmented cDNA’s biotinylated ends are bound to streptavidin beads
and separated into two parts. At the cleaved end of each cDNA fragment, appropriate
adaptors with an endonuclease site for tagging enzymes are ligated. Tagging
enzymes are added to release fragmented cDNA (up to 26 bp) from the beads by
cleaving a small distance from their endonuclease sites found at each adaptor. The
blunt ends of each of the two fragmented cDNA segments are ligated together to
form a ditag, which is then amplified by PCR. Ditags are cleaved at their adaptors,
710 S. K. AVS et al.

Fig. 15.8 SAGE. Gene expression analyses in sequence (SAGE)

15 Genomics 711

Fig. 15.9 Affinity purification and mass spectrometry (Thermo Fisher Scientific)

concatenated, and cloned into plasmids following PCR. Plasmids are sequenced
after cloning, mapped back to the gene, and then analyzed for gene expression.
RNA sequencing—This is the most efficient way to study gene expression and
transcription; it is widely used as compared to microarray and RNA sequencing. In
this technique, the quantity and sequence of RNA can be analyzed using next-
generation sequencing.
Yeast two hybrid systems—In order to determine protein-protein interaction, this
tests one “test” protein against a variety of potential proteins. This approach is
mostly based on the GAL-4 transcription factor. If two proteins are being tested,
one of their genes will bind with the DNA binding domain of GAL-4 (GAL4-DBD),
while the other will bind with the activation domain of GAL-4 (GAL4-AD).
Affinity purification and mass spectrometry—This technique is used to determine
the protein that interacts with one another, present in complexes. The affinity
enrichment approach uses specific binding property between molecules to success-
fully isolate the protein of interest as explained in Fig. 15.9.
Using the proximity biotinylation approach, affinity purification mass spectrom-
etry can be used to look at particular protein-protein interactions within protein
complexes or to look at protein complexes more broadly at the interactome level.
Interactions can be studied under different conditions by combining affinity purifi-
cation with quantitative MS, resulting in a much more dynamic view of protein-
protein interactions. This workflow also allows for PTM analysis, allowing
researchers to investigate the role of posttranslational modifications (PTMs) in
facilitating protein-protein interactions.
RNAi—This method is being used to silence or knock down genes. It uses siRNA
or shRNA.
CRISPR Screens—As explained in Fig. 15.10, it deletes genes in a multiplexed
manner and quantifies the amount of guide RNA in order to determine essential
genes. The enzymes “Cas9 and dCas9” are required in deletion and inhibition of
gene and its expression.
The Cas9 nuclease modifies a specific sequence by repairing the susceptible DNA
through the pathway of NHEJ or homologous recombination accompanied by a
customized template of DNA. Then the dCas9 binds the DNA and hinders the RNA
712 S. K. AVS et al.

Fig. 15.10 CRISPR-based tools are usually compliant to high-throughput screening

polymerase, thereby obstructing the gene expression. The fusion of dCas9 and a
transcription activator domain starts the transcription. The mutation of nucleotides
happens by new base-editing technologies without the insertion of DNA nicks by
using the integration of Cas9 nickase (nCas9) and in some cases dCas9 into adenine
or cytosine deaminase. The utilization of retron systems to give rise to multi-copy
single strands of DNA (msDNA) like a template for editing is another way. Lastly, a
contrived guide RNA and a merged nCas9 and reverse transcriptase introduce
changes encrypted in the guide sequence.

Bioinformatics Approaches for Functional Genomics

As the data produced is very large in quantity, the bioinformatics method is very
useful in the analysis of genomic data.
Data clustering, unsupervised machine learning, as well as artificial intelligence
techniques are used. A variety of computational analysis methods are developed in
the last decade to understand deep mutational scanning experiments, and phydms is
one such software, and the mechanism is explained in Fig. 15.11.
Phydms runs the results of the experiment with the phylogenetic tree in order to
provide accurate information.
Deep mutational scanning synthesizes every amino acid change in a protein, their
activity is then assayed, and the comparison to the wild-type variant determines the
mutation.
15 Genomics 713

Fig. 15.11 Phydms workflow (A-D). Phydms is a program that allows you to compare the results
of deep mutational scanning experiments to natural selection on genes. Phydms allows rigorous
comparison of how well different experiments on the same gene capture real natural selection when
given a phylogenetic tree topology inferred with another program

The technique is also useful in determining the structure of protein and protein-
protein interactions. Phydms use experimentally informed codon models, and it is
written in Python by Bloom laboratory.

15.3 Features of Prokaryotic Genome

15.3.1 Introduction

The prokaryotic genome is fundamentally very different from eukaryotic genome.

There is a lack of physical separation in prokaryotic eel between DNA and cyto-
plasm. The size of prokaryote genome is much smaller; however, it is observed that
an overlay in size extends between one of the largest prokaryotic genomes and one of
the smallest eukaryotic genomes. E. coli K12 is known to have a genomic size of
4639 kb, i.e., 2/fifth the extent of the genome of yeast with only 4405 genes. A small
714 S. K. AVS et al.

DNA molecule, i.e., circular in shape is known to carry the whole prokaryotic
genome in addition to which there may be genes that occur in plasmids. These
genes are highly efficient and are known to express genes responsible for resistance
toward antibiotics. They are also able to make use of toluene, i.e., a complex
compound as a carbon source. In prokaryotes, there is diversity in the organization
of genome with few that possess unipartite genomic components such as E. coli.
Other organisms have a genome which is multipartite in nature. An example of this is
the organism Borrelia burgdorferi B31 that is found to contain a chromosome, i.e.,
straight and linear of the length 911 kb while consisting of 853 genes. This genome
may also be accompanied by 17–18 circular and linear molecules that altogether
generates around 533 kb and 430 genes.
John Cairns, in 1963, has proved that the genomic constituents of E. coli consist
of a chromosome, i.e., single and circular. Later, in 1979, the pioneering plasmid in
Streptomyces, which is linear, was identified, and in 1989, scientific evidences
pointed out that the chromosome of Borrelia burgdorferi is straight and linear.
This formed an evidence that bacterial DNA molecules need not be circular.
Furthermore, the “megaplasmid” was identified in Sinorhizobium meliloti in the
early 1980s, disputing the hypothesis wherein almost the complete genome of
bacteria is present on the chromosome. Eventually, a scientific development regard-
ing this emerged that the organism Rhodobacter sphaeroides was composed of a
“second chromosome.” This development led to many more fundamental cell
function theories revolving around the fact that a critical cell function can be
expressed by several replicons present in the bacterial genome. Nearly 9–10% of
the genome in bacteria do not possess chromosome, i.e., single and circular, such as
E. coli. Rather, it has been found to contain multiple critical and large replicons that
may be straight or circular. This type of genome composition regarding a chromo-
some with one or more than one big extra replicon constitutes to be a multipartite
genome (also known as divided genome).

15.3.2 Concept and Classification of Replicons

The genome of prokaryotes is composed of a single replicon which consists of its

own replicator and initiator. Several terms have been used to establish the classifica-
tion of DNA molecules which might be present in the genome that is multipartite in
nature.
Replicon and Secondary Replicon: Irrespective of its exact nature, any DNA
molecule is in general referred to as replicon, and a single replicon is again branched
into further subtypes on the basis of exact features. A secondary replicon is one that
is not the primary chromosome belonging to the cell. Replicon can be
subcategorized as such: chromosomes, second chromosomes, chromid,
megaplasmid, and plasmid (Fig. 15.12).
Chromosome: This is the primary replicon and is also the largest. It consists of the
critical genes. It is found that the chromosome forms the core for nearly all genetic
material in prokaryotes. However, there are a few anomalies such as the
15 Genomics 715

Replicon

Is it the largest replicon with

most of the core genes?

Yes No

Chromosome Secondary replicon

Does it have any

essential core genes?

Yes No

Did it result from a split of Is it larger than

the chromosome into two? 350 kilobases in size?

Yes No Yes No

2nd Chromosome Chromid Megaplasmid Plasmid

Fig. 15.12 Flowchart representing the classification of bacterial replicons. (Adapted from “The
Divided Bacterial Genome: Structure, Function, and Evolution” authored by George C. diCenzo,
Turlough M. Finan Department of Biology, McMaster University, Hamilton, Ontario, Canada)

chromosome present in Burkholderia xenovorans LB400 and Sinorhizobium

meliloti 1021 are responsible for around 50% of their respective genomic
constituents.
Plasmid and Megaplasmid: They are defined as type of replicons that lack the
core genes. The majority of secondary replicons in bacterial genomes are redundant
for cell viability as they do not carry core genes and are nonessential. There is
significant amount of difference from chromosome in terms of GC percentage and
composition of dinucleotides. Megaplasmid is nothing but a plasmid of larger size.
716 S. K. AVS et al.

Chromid: It is an arrangement of plasmid with chromosome. In terms of replicon,

it forms a common link for chromosome and plasmid. Chromids contain regulatory
controls that help in integrating their replications into the cell cycle. They also carry a
core gene that is important for viability of the cell wherein its loss could result in
possible death of the cell. Chromids can be further classified as primary chromids
and secondary chromids. Primary chromids are critical for viability of the cell, while
chromids that are secondary in nature might be unnecessary but are required by the
organism for survival in its natural habitat. A replicon is considered as secondary
replicon when it is important for its native habitat. The tRNAarg and engA genes on
the pSymB replicon of S. meliloti represent few of the critical genes present on the
chromid.
Second Chromosome: This constitutes a term used in describing a rare occurrence
wherein a replicon which is secondary is composed when the ancestral chromosomal
is divided into further two replicons.

15.3.3 Genomic Signatures of Bacterial Replicons

Multiple prokaryotic genomes differ in their features such as usage of codons (ratio
at which similar codons occur in the genome), relative abundance of dinucleotides
(the frequency at which a certain base-pair of nucleotides can occur within a
genome), and content of GC nucleotides (the percentage of guanine and cytosine
in a genome).

15.3.3.1 Usage of Codon

The usage of codon depends on the level of expression in a gene wherein genes that
are expressed in high levels constitute a codon usage which is similar to the
comparative tRNA presence. This is not the case with low expression of genes.

15.3.3.2 GC Content
GC content differs quite relatively in prokaryotes and can lie in the range of 15–75%.
The GC content of an organism can also be influenced by factors such as environ-
mental adaptation and recombination. Furthermore, GC content can differ quite
notably within a genome and can thereby be used to recognize and identify genes
recently obtained by horizontal gene transfer. Adequate studies and research have
depicted that the process of replication starts by a bound, fixed origin of replication
(ori) which further sets forth in a bidirectional manner replicating the two arms
known as replichores till the replication complex extends unto the replication
terminus (ter) site, i.e., positioned directly opposite to the ori (Fig. 15.13).
Replication, one of the most basic and critical phenomena of bacterial cell cycle,
employs selection and mutational pressure that spans across the genome. This aids in
deciding the polarity of the genome by addition of nucleotides that are biased in an
asymmetrical manner within the lagging and leading strand. This constitutes a
certain skew in the composition that is identified with ease while marking a graph
of standardized abundance value containing guanine content (G) with respect to
15 Genomics 717

Fig. 15.13 DNA polymerases at work replicating a chromosome as the replication fork extends
from ori to ter. The blue strand is directed clockwise, and the green strand is directed counterclock-
wise. (Phillip Compeau, Programming for lovers, Chapter 1: finding replication origins in bacterial
genomes)

cytosine content (C). This is called as a GC skew graph, and it aids in segregating a
genome into further two subregions, i.e.:

(a) One having an abundance of guanine above cytosine that corresponds to the
base composition of the leading strand.
(b) One having an abundance of cytosine above guanine that corresponds to the
base composition of the lagging strand.

The shift points that are marked in the graphs of the GC skew are found to
correspond to the loci region of ter and ori. Several studies have indicated that the
GC skew can be identified only in bacterial organisms that have a circular chromo-
some and not in organisms with a straight or linear chromosome.

15.3.3.3 Relative Quantity of Dinucleotide

Studies regarding the topic of quantity of dinucleotide of the organizational genome
reveal that they are distinguished for every single genome of bacterial origin and are
representative of phylogeny of bacteria. It has also been illustrated by several studies
that relative quantity of dinucleotide has the ability to distinguish plasmids and
chromids from chromosomes.
718 S. K. AVS et al.

15.3.4 Physical Structure of the Prokaryotic Genome

• Size: The size of majority of prokaryotic genome is less than 5 Mb which is

present in a DNA molecule which is adapted to the nucleoid with an exception
being B. megaterium which has a genome of 30 Mb. The most commonly studied
prokaryote is the E. coli which has a size of just 1.0
2.0 μm with the
chromosome having a circumference of 1.6 mm to sequence the entire genome.
Packaging of the genome is done with help of DNA binding proteins. Studies of
E. coli chromosome revealed that it is negatively supercoiled. Supercoiling
happens in positive manner or negative manner, i.e., if surplus turns are made
to enter in the double helix of the DNA, it is known as positive supercoiling. If the
turns are excised or removed, it is known as negative supercoiling (Fig. 15.14).
Because the circular chromosome of E. coli has no free ends, the strain or
torsional stress cannot be released by rotation. Instead, the molecule responds
to the strain by writhing itself in a more compact structure. The enzymes involved
in supercoiling are DNA gyrase and DNA topoisomerase I. There is restricted
freedom to rotate in E. coli DNA molecule if a break is introduced at any point in
the chromosome. However, there is loss of supercoiling in a limited region of
DNA when a break is made.
• The genome of prokaryotes has to fit into a comparatively small space. For
example, the circular chromosome of E. coli is known to have a circumference
of 1.6 mm while the cell size of E. coli is around 1.0
2.0 um. The organization
of DNA in the nucleoid is essentially studied from E. coli. Supercoiling has been
identified and studied to be an ideal technique of packaging the circular DNA
molecule into a relatively smaller space. Supercoiling of the circular DNA
molecule structure winds over and around itself to lead to the formation of a
more closed and compact structure, and this can be aided by two important
enzymes. Those enzymes are DNA gyrase and DNA topoisomerase I.
• Further studies regarding nucleoids and cells that are carefully isolated and living
have revealed the DNA of E. coli to be restricted in their ability to rotate once a
break is introduced. This has been found to be caused due to the attachment of
proteins to the DNA molecule, thereby restraining its capacity in relaxing. Upon a
single rotation at the DSB site, the supercoiling is lost from a section of the DNA.
The present profile of the DNA molecule of E. coli has been found to be attached
to a protein core from which over 39–50 loops that are supercoiled in nature are
known to emerge radially outward to the cell. A separate DNA loop is found to
comprise around 90–100 kb of DNA, i.e., supercoiled. This constitutes the
quantity of DNA that is separated as a consequence of the double-stranded single
• The enzymes DNA gyrase and DNA topoisomerase I are critical in sustaining the
supercoiling of the DNA molecule. A set of around four proteins are also found to
accompany the enzymes with a special ability to aid in the packaging of the DNA.
Hu, a packaging protein, is morphologically very distinguished from histones of
eukaryotes. However, they perform their function in an identical manner by
making a tetramer leading to unwinding of over 60 bp of DNA (Fig. 15.15).
15 Genomics 719

Fig. 15.14 This figure depicts the unwinding of a double-stranded circular molecule of DNA
leading to the formation of supercoiling that is negative. (Chapter 2, Genome Anatomies, Genomes,
TA Brown 2002)

15.3.5 Repeats in Prokaryotic Genome

A repeat in a prokaryotic genome is mathematically represented by a subsequence of

a given genome which bears strong resemblance to another subsequence in the same
genome. In order to correctly define a genomic repeat, one has to examine biological
and mathematical factors that influence the occurrence of the repeat. Repeat that
occur in an unexpected and random assembly out of the genetic context cumulates
interest as such repeats have clearly not occurred as a consequence of certain
phenomena and generally may discover a critical significance of biological origin.
720 S. K. AVS et al.

Fig. 15.15 A diagram depicting the formation of structure of a nucleoid of E. coli. Around 39–50
loops of DNA that are supercoiled emerge radially outward with the protein core in the center. A
loop is depicted in its unwound circular form representing a break that has formed in that section of
DNA, thereby causing the loss of supercoiling. (Chapter 2, Genome Anatomies, Genomes, TA
Brown 2002)

It has been found that the repeats are of biological significance as they are formed as
a direct consequence of significant overlapping and sequence recombination. An
association between genetic elements can lead to the overlapping of the genes.
Co-expression of genes may also occur when identical copies of a repeat are
present in their regulatory genes. In such cases, repeats are known to efficiently give
rise to associations within genes in genomic complexes. Repeats can also constitute
operons or entire genes, wherein functional redundancy occurs as a subsequence of
intersection by overlapping of functional genes in the two copies.

15.3.5.1 Recombination and Repeats

Homologous recombination involves the promotion of strand exchange by RecA
between homeologous and homologous sequences which are primary critical steps.
RecA is considered to be the critical component of the process. However, it is absent
in certain small genomes of obligatory endomutualists, like Buchnera, which lack
repeat elements and thereby cannot repair themselves by the common repair
mechanisms.

15.3.5.2 Origin of Repeats

Evidences have pointed out that repeats can occur in several different possible
methods wherein generic repeats are known to make an appearance by the common
recombination processes; however, more special families of families are known to
originate through specialized recombination processes like site-specific integration
for prophages, transposition for insertion sequences (ISs), or poorly understood
mechanisms for elements like retrons or clustered regularly interspaced short palin-
dromic repeats (CRISPR). After their genesis, repeats can associate in general
recombination processes independently of their origin (Fig. 15.16).
15 Genomics 721

Fig. 15.16 Different methods by which a repeat is created. Δ stands for the size of the spacer
between the two repeats expected under the different mechanisms. (FEMS Microbiology Reviews,
Volume 33, Issue 3, May 2009, Pages 539–571, Genesis, effects and fates of repeats in prokaryotic
genomes- Todd J. Treangen, Anne-Laure Abraham, Marie Touchon, Eduardo P.C. Rocha)

15.3.5.3 Repeats of Generic Nature

Horizontal gene transfer and homologous recombination between shorter degenerate
repeats are known to form generic repeats. Repeats are created as a consequence of
horizontal gene transfer whenever the incoming DNA is comprised of elements of
elements that are identical to preexisting information.
Integration of information, i.e., laterally transformed, can occur through site-
specific recombination. Homologous recombination that occurs in between degen-
erate and short repeats can lead to conversion of the gene and thereby efficiently
enlarge the area of likeness in between the elements, thus generating larger repeats.
This conversion of the gene can allow the integration of similarity between
sequences intact.
Illegitimate recombination can occur between small degenerate closely spaced
repeats that may cause “Dislocated Mutagenesis” as a consequence of which the
assembly of a constricted repeat is formed by a degenerate one. Owing to the
properties of slipped strand mispairing, the repeats have to be in close proximity
in the genome with a length of only few nucleotides.
722 S. K. AVS et al.

15.3.5.4 Selfish Elements

The production of SE in a genome is known to be a major source of repeats by which
TEs are undoubtedly the best observed and ubiquitous in presence. Based upon their
structure and transposition mechanism, they can be classified into two major groups:

(a) Class I elements that constitute retrotransposons that are known to transpose by
an RNA intermediate.
(b) Class II elements that constitute DNA transposons that are known to transpose
by a DNA intermediate.
(c) Furthermore, retrotransposons have been subclassified into three main
categories.

15.3.5.4.1 Group II introns

The only transposable elements to be distributed to all three domains of life belong to
Group II introns. They have the ability to self-splice introns, can grow by reverse
transcription, and are efficient in maintaining both retro mobility reactions and
splicing of introns by itself.

15.3.5.4.2 Retrons
Genetic elements that can generate several DNA, i.e., single stranded and linked
covalently to RNA via transcriptase enzyme, are known as retrons.

15.3.5.4.3 Diversity-generating retroelements

A recently discovered family of genetic elements, diversity-generating retroelements
can distribute specific benefits in certain conditions by injecting large quantities of
diversities in the sequence of the target gene.

15.3.5.5 Interspersed Repeats

Interspersed repeats are isolation of highly recognized families of DNA repeat
elements from a vast classification of eukaryotic and prokaryotic organisms. How-
ever, the exact function and origin are unknown for these elements. They are of
ubiquitous nature in archaeal and bacterial plasmids and chromosomes. These
repeats are known to perform important functions in genetic evolution, organization
of the genome, and genome plasticity. Interspersed repeat sequences in prokaryotes
are highly intergenic (Table 15.2).

15.4 Eukaryotic Genome

15.4.1 Introduction

The typical genome that has been studied quite often with regard to the eukaryotic
genome is the human genome. The human system forms a good model for studies
regarding the eukaryotic genome. The eukaryotic nuclear genome can be divided
15 Genomics 723

Table 15.2 Families of interspersed repeats (Adapted from: Todd J. Treangen, Anne-Laure
Abraham, Marie Touchon, Eduardo P.C. Rocha, Genesis, effects and fates of repeats in prokaryotic
genomes FEMS Microbiology Reviews, Volume 33, Issue 3, May 2009, Pages 539–571)
Repeats Acronyms Features
Repetitive extragenic palindromic REP or 21–65 bp imperfect palindrome, extragenic
or palindromic units PU sequence, potential stem-loop structure
probably transcribed
Bacterial interspersed mosaic BIME 40–500 bp mosaic combination of REP
elements separated by other sequence motifs
Clustered regularly interspaced CRISPR Noncontiguous direct repeats (DR, 24–47 bp)
short palindromic repeats separated by stretches of similarly sized
unique spacers (26–72 bp)
Potential stem-loop structure, i.e., probably
transcribed
Miniature inverted repeat MITE 100–400 bp nonautonomous element
transposable elements (mobilizable in trans by full-length
transposase)
Probably derived from IS by internal deletion
Intergenic repeat unit or IRU or 69–127 bp large palindromic sequence
Enterobacterial repetitive ERIC
intergenic consensus
Insertion sequence IS 0.7–3.5 kbp autonomous element
Transposons Tn Autonomous element that codes for
transposase and a number of gene products
(e.g., antibiotic resistance, virulence factor)

into two or more linear DNA molecules, each of which is stored in a chromosome
which is distinguished.
All eukaryotes are known to also contain shorter mitochondrial genomes that are
generally circular in nature. Plants are observed to have an additional genomic
structure, i.e., present in their chloroplast known as the chloroplast DNA. This is
unique to the eukaryotic genome of plants alone and cannot be identified in any
human or animal eukaryotic model.
Despite the similarity in the fundamental morphology of all eukaryotic genomes,
they differ in their genomic size and can thus be distinguished from one another. The
smallest eukaryotic genome has been found to be shorter than 10 Mb in length, and
the largest eukaryotic genome is larger than 100,000 Mb. By observing the table
below, the range of eukaryotic genome size is known to correspond to a specific
level to the organism’s complexity.
Fungi, a simple eukaryotic system, can be observed to have the smallest genome
with respect to its highly generic and simplified nature, while higher eukaryotes like
vertebrates and flowering plants have larger genomic sizes. One of the well-known
exceptions to such an understanding is the nuclear genome of S. cerevisiae that at
12 Mb is of 0.004
the size of a human genome. It would be estimated to contain
0.004
35,000 genes, i.e., 140 genes. However, S. cerevisiae is comprised of 5800
genes.
724 S. K. AVS et al.

Table 15.3 Sizes of Species Genome size (in Mb)

eukaryotic genome
Fungi
(Chapter 2, Genome
Anatomies, Genomes, TA Saccharomyces cerevisiae 12.1
Brown, 2002) Aspergillus nidulans 25.4
Protozoans
Tetrahymena pyriformis 190
Invertebrates
Caenorhabditis elegans 97
Drosophila melanogaster 180
Bombyx mori 490
Locusta migratoria 500
Vertebrates
Homo sapiens 3200
Mus musculus (mouse) 3300
Plants
Arabidopsis thaliana (vetch) 125
Oryza sativa (rice) 430
Zea mays (maize) 2500
Triticum aestivum (wheat) 16,000

The complete absence of corresponding link between the organism’s genome

complexity and its genomic size led to the theory of the C-value paradox.
S. cerevisiae is a common illustration that depicts this point (Table 15.3).

15.4.2 Packaging of DNA into Chromosome

Scientific evidences have suggested and pointed out that the chromosomes are
generally smaller and shorter than the DNA molecules that they are comprised
of. Therefore, a specific organized packaging system is required to package a
DNA molecule into its chromosome. The most critical findings regarding the
packaging of DNA were formed in the early 1970s by a combination of analysis
by biochemical methods and electron microscopy techniques. The presence of
DNA-binding proteins known as histones is very crucial in packaging the DNA
molecules in a chromosome. In the 1970s, several groups of scientists carried forth
nuclease protection on chromatin (DNA-histone complexes) which were carefully
removed from the nuclei via methods fabricated to preserve the chromatin structure
as much as possible. In a nuclease protection assay, the complex is subjected to
treatment with an enzyme that cleaves the DNA at certain sites of the DNA that is not
bound to the protein. The sizes of the obtained DNA fragments depict the location of
the protein complexes on the original DNA molecule. It has been found that the bulk
of DNA fragments varies in lengths of nearly 200 bp and multiples of 200 bp, i.e.,
suggestive of a common spacing of histone proteins along the DNA (Fig. 15.17).
15 Genomics 725

Fig. 15.17 Nuclease protection analysis of chromatin from human nuclei. (Chapter 2, Genome
Anatomies, Genomes, TA Brown 2002)

Electron micrographs depict the biochemical results, and a subsequent inference

was made with regard to the purified chromatin. The spacing of histone binding
along the length of the DNA at regular intervals of distance was drawn as conclu-
sion. The histone protein was found to bind to the DNA molecule in a manner similar
to the presence of “beads on a string.” More biochemical assays have revealed that a
single bead, known as nucleosome, consists of eight histone protein molecules, i.e.,
two each of histones H2A, H2B, H3, and H4.
However, it was observed that the “beads on a string” appearance is known to
depict an unpacked form of chromatin which forms less frequently in nuclei that are
living. Techniques to break the cell have been conducted in a very gentle manner and
have revealed a much compact version of the complex known as the 30-nm fiber
(approximately 30 nm in width). Many developments have been proposed regarding
the 30-nm fiber among which the solenoid structure is one of the most popular.
The formation of nucleosomes as a form of DNA packaging can shorten the fiber
length around sevenfold. This means that a stretch of DNA, i.e., a meter in length,
will lead to formation of a “string of beads” chromatin fiber of just 14 centimeters
length (approx. 6 inches) long (Fig. 15.18). The chromatin is coiled further to lead to
generation of a much thicker and shorter fiber, i.e., termed as the 30-nm fiber (the
size is nearly 30 nanometers in diameter).
Histone H1 has been observed to perform a critical function of stabilizing the
chromatin of complex structures, and 30-nm fibers are generated with ease due to the
presence of H1 protein. Modification of histones by enzymes is performed to
acetylate, methylate, or phosphorylate the protein molecule to enable transcription
and replication (two of the major fundamental mechanisms) to occur without any
726 S. K. AVS et al.

Fig. 15.18 Chromosomes are made up of DNA tightly wound around histones. (Adapted from
Pierce, Benjamin. Genetics: A Conceptual Approach, 2nd ed.)

hindrance. Chromatin remodeling complexes can replace histones to reveal the DNA
sequences and allow the binding of polymerase enzymes to the DNA sequence.

15.5 Human Genome Project

The Human Genome Project, a global research study, aims to interpret the human
genome sequence, define all of the genes found within it, and develop the research
insights to explore all genetic data that has been generated. This groundbreaking
project is focused on the matter of fact that isolating and analyzing the genetic
component found within the DNA will allow scientists with new powerful keys to
address the progression of the disease and develop modern prevention and treatment
strategies. Except for physical injuries, almost all medical conditions are linked with
15 Genomics 727

Table 15.4 Disease- Year Disease

related genes identified
1986 Chronic granulomatous disease
using positional cloning
Duchenne’s muscular dystrophy
(Collins and Fink 1995)
1989 Cystic fibrosis
1991 Fragile X syndrome
Familial polyposis coli
1992 Myotonic dystrophy
Norris’s syndrome
1993 X-linked A gammaglobulinemia
Neurofibromatosis type 2
Huntington’s disease
Wilson’s disease
1994 McLeod’s syndrome
Polycystic kidney disease
Diastrophic dysplasia
1995 Spinal muscular atrophy
Ataxia telangiectasia
Alzheimer’s disease
Hereditary multiple exostoses

a change in the structure as well as the function of DNA due to mutations. These
disorders comprise heritable “Mendelian” diseases associated with single mutations,
complex and common disorders caused by genetic alterations in multiple genes, and
disorders caused by DNA mutations acquired during childhood, such as several
cancers. Although many of these activities and research have been carried out by
scientists for decades, the Human Genome Project stands out because of its efforts.
The human genome is made up of three billion nucleotides, enough for it to cover
1000-thousand-page phone books if every other nucleotide is defined by a single
letter. To process large quantities of data rapidly, affordably, and precisely,
researchers must develop new methods, given the size of the human genome.
These methods will be used to classify DNA for disease studies in families, to
construct genomic maps, and to identify the gene sequences and other large signifi-
cant fragments of DNA.
The Human Genome Project’s primary objective is to create three important
research facilities that will enable researchers to recognize genes that are relevant
in both typical biology and rare and common diseases. Advanced techniques like
positional cloning enable researchers to scan the genome for disease-linked genes
without first determining its function of the corresponding protein. Since 1986, when
investigators used positional cloning to discover the gene for the chronic granulo-
matous disease, this approach has resulted in the sequestration of nearly 40 genes
that are disease related and will enable the identification of several additional genes
forthcoming (Table 15.4).
All the three methods that are being created by this Genome Project help to
narrow down the gene that is being searched. The genetic map, for example, is made
up of thousands of distinctive features—small, distinct pieces of DNA—that are
pretty much equally distributed along the length of chromosomes. Researchers may
728 S. K. AVS et al.

use this technique to pinpoint the position of a gene on a chromosome. Investigators

use another method, the physical map, to locate a particular gene once this area has
been established. Physical maps are considered as imbricating DNA sequences that
can cover throughout the chromosome. These collections have been cloned and
preserved in anticipation of potential testing. Instead of digging through the
chromosomes all over again, researchers will indeed be allowed to retrieve the
exact segment of DNA required once the physical map is complete. The develop-
ment of a detailed sequence map of the DNA, which will include the perfect
sequence of all the nucleotides that form up the human genome, will be the final tool.

15.5.1 Origin of the Project

The Human Genome Project (HGP) has radically altered the field of biology and is
accelerating the medical revolution. Renato Dulbecco proposed the HGP in a review
issued in 1984, arguing that the sequence of the human genome would aid in the
knowledge of cancer. In May 1985, Robert Sinsheimer convened a conference solely
based on the HGP, with 12 experts debating the project’s merits. The session
finished that the plan was technically feasible but would be incredibly difficult to
implement. However, there was some debate on whether it was a good idea, with six
people in attendance in favor and six against. The skeptics contended that big science
could be bad as it can divert the assets away from “true” small science, that the
genome is all junk and its sequencing is worthless, that they are not ready to take
over such a massive task and should await once the technology is up to the task, and
also that sequencing and mapping the genome is a straightforward and necessary
task. Approximately 80% of biologists, as well as the National Institutes of Health,
were opposed to the HGP during its early years of advocacy (mid to late 1980s). The
US Department of Energy (DOE) is driven for the project at first, claiming that
understanding the sequence of the genome would facilitate us to better comprehend
the impact of radiation upon the genome caused by atomic explosions as well as
other forms of transmitted energy. This DOE activism was crucial in igniting the
controversy and, eventually, the HGP’s approval. Surprisingly, the US Congress was
more supportive than most biologists. The attraction of foreign competition in
medicine and biology, the prospective for economic gains and technological spin-
offs, and the capability for more innovative ways to treat the disorders were all
acknowledged by many in Congress. In 1988, a National Academy of Science
committee approved the HGP, and the flow of thought shifted: The initiative was
launched in 1990, and the completed series was written ahead of time and on a
budget in 2004.
As genomics technology advanced, this three-billion-dollar, 15-year initiative
progressed significantly. The HGP’s initial goal was to create a human genetic
map, followed by a physical map of the human genome, and eventually a sequence
map. The HGP was a driving force behind the advancement of high-efficiency DNA
planning, sequencing, and mapping technologies throughout its life. There was hope
when the HGP was established in the 1990s that the then-current technologies used
15 Genomics 729

for sequencing could get a replacement. The whole process, now known as “first-
generation sequencing,” used agarose gel electrophoresis to build up the ladders as
well as fluorescent or radioactive-based tagging techniques to execute base calling. It
seemed a too time-consuming and low degree of efficiency for effective sequencing.
In the end, a 96-capillary version of first-generation sequencing technology was used
to decode the original human genome reference sequence. Alternative methods, such
as multiplexing and hybridization sequencing, were tried but failed to scale
up. Meantime, the HGP saw gradual enhancements in the momentum, cost, produc-
tion, as well as precision of first-generation fluorescent-based preprogrammed
strategies for sequencing. The objective of the acquisition of generating a full-
blown physical map was disposed of in the subsequent stages of the project in
favor of establishing the complete sequence earlier than expected because
researchers were clamoring for sequence results. Craig Venter’s ambitious idea to
build a corporation (Celera) to use the technique of the whole-genome shotgun
approach to decode the entire sequence rather than the fragmentary approach that
uses the clone-by-clone method, used by the International Consortium, utilizing the
vectors of bacterial artificial chromosome (BAC) intensified this push. Government
funding organizations backed Venter’s proposal, which called for the development
of a clone-based prototype sequence to every chromosome, with the final version
coming later. These concurrent efforts sped up the process of creating a genome
sequence that would be invaluable to biologists.
The Human Genome Project generated a carefully selected and precise sequence
as reference for every other different chromosome, with only a few discrepancies and
major heterochromatic regions excluded. The sequence that has been used as a
reference has proved important for both the advancement as well as eventual
extensive use of second-generation sequencing technology, started in the
mid-2000s, in the supplement to provide a basis for the succeeding research in the
variation of the human genome. Second-generation cyclic array platforms of
sequencing generate many hundreds of millions of short reads (initially 30–70
bases, even several hundreds of bases) in such a single cycle, which are usually
mapped against the genome of reference maintaining extreme redundancy through-
out the coverage. The HGP paved the way for several sequencing techniques (ChIP-
Seq, RNA-Seq, and bisulfite sequencing) that have greatly sophisticated research
surveys of gene transcription and control and genomics.

15.5.2 Mapping of Human Genome

The process of determining human genome starts with mapping or characterizing the
chromosomes that lead to the development of a physical map followed by its
sequencing of the order of DNA bases on chromosomes that will ultimately generate
a genetic map. The order of genes or other markers on each chromosome, as well as
the spacing between them, is described by a genome map. Human genome maps
have been generated on a variety of dimensions and resolution levels. Genetic
linkage maps, which represent the comparative positions of a chromosome with
730 S. K. AVS et al.

DNA markers (functional genes as well as other recognizable DNA sequences)

based on their pattern of inheritance, have the lowest resolution. The biochemical
properties of DNA molecules are defined by physical maps.

15.5.2.1 Mapping Strategies

15.5.2.1.1 Genetic Linkage Maps

The relative positions of DNA markers that are specific throughout the entire
chromosome are depicted on a genetic linkage map. Any inherited molecular or
physical trait that varies between different persons and can be accessed in the
research lab is a probable genetic marker. You can use fragments of DNA that
have been expressed (genes) or any segment of DNA as markers with no defined
coding mechanism but a predictable pattern of inheritance. Since DNA sequence
variations are abundant and to be successful in mapping, the DNA markers should be
polymorphic, i.e., different patterns should prevail between different individuals and
that they can be detected within various members of the family in an investigation.
Polymorphisms are variants in the DNA sequence that appear every 300 to 500 base
pairs on average. Exon sequence variations can cause visible changes including
variations in blood type, the color of eyes, and susceptibility toward a disease. The
majority of differences exist among the noncoding DNA sequences and have very
little to no impact on the nature or functionality of an entity, but they can be detected
at the nucleotide level and can be utilized as markers. Two examples for these kinds
of markers comprise (1) restriction fragment length polymorphisms (RFLPs), which
represent variations within different DNA sequence sites that are able to be cleaved
by restriction enzymes, and (2) variable number of tandem repeat sequences, which
are very short repetitive fragments that differ in the number of repeated units and,
thus, in length (an easily measurable characteristic feature). The genetic linkage map
of humans is derived from the frequency at which the inheritance of two markers is
together.
The markers within the same chromosome that are close to each other are more
likely to be passed down from parent to infant. DNA strands split and rejoin in
various positions on the same chromosome or the other copy of the same chromo-
some during normal sperm and egg cell development (i.e., the homologous chromo-
some). Recombination during meiosis is a mechanism that allows two separate
markers that have been previously on the very same chromosome to be separated
(Fig. 15.19). Centimorgans (cM), called for geneticist Thomas Hunt Morgan, are
used to calculate the distance between markers within the genetic map. If recombi-
nation separates two different markers 1% of the time, they are said to be 1 cM apart.
A genetic distance of 1 cM is approximately equivalent to a physical distance
covering up to 1 Mb. Most human genetic map regions have a resolution of around
10 megabytes. A genetic map with high resolution (2 to 5 cM) is one of the genome
project’s short-term goals; modern consensus maps of certain chromosomes have
ranged from 7 to 10 cM between genetic markers. The use of rDNA technology,
such as chromosomal fragmentation induced by radiation and fusion of cells to build
15 Genomics 731

Fig. 15.19 Constructing a genetic linkage map. The vertical lines in this diagram represent pairs of
chromosomes 4 for each member of a family. A short-established DNA sequence used as a genetic
marker (M) and Huntington’s disease (HD) are two characteristics that can be identified in any child
who inherits them from the father. The implication that only one infant inherited a single trait
(M) from that chromosome suggests that the father’s hereditary material recombined during the
sperm development process. This event’s frequency aids in determining the distance between two
DNA sequences on a genetic map

a group of cells that is particular and variable elements of a chromosome, has

improved genetic mapping precision.

15.5.2.2 Restriction Enzymes: Microscopic Scalpels

Restriction enzymes are bacteria-derived enzymes that identify very small sequences
of DNA and cleave them at particular locations. (One of these enzymes’ common
biological functions is to defend bacteria by targeting genetic material of the virus
and other foreign DNA.) Few enzymes cleave the DNA occasionally, resulting in the
formation of few huge fragments (several thousand to a million bp). The majority of
the enzymes cleave DNA at a higher rate, resulting in a huge number of small
fragments (few hundreds to more than a thousand base pairs).
On average, restriction enzymes yield:

• Pieces of 256 bases (4-base recognition sites).

• Pieces of 4000 bases (6-base recognition sites).
• Pieces of 64,000 bases (8-base recognition sites).

15.5.2.3 Physical Maps

The degree of resolution varies between different types of physical maps. The
chromosomal (also known as cytogenetic) map is the lowest-resolution physical
map, and it is based on the distinct banding patterns observed by light microscopy
of stained chromosomes. On a chromosomal map, a cDNA map depicts the locations
732 S. K. AVS et al.

of expressed DNA regions (exons). The cosmid contig map shows the order of
overlapping DNA fragments across the genome in greater detail. The order and
distance between enzyme cleavage sites are described by a macrorestriction map.

15.5.2.4 Low-Resolution Physical Mapping

Chromosomal Map—Genes or other recognizable DNA fragments are attributed to
their corresponding chromosomes in a chromosomal map, with distances measured
in base pairs. An approach which is known as in situ hybridization includes labeling
the markers with a specific identifiable label that may be used to physically associate
these markers with specific bands (identified by cytogenetic staining). Soon after the
probe that is labeled binds with one of the DNA strands that is complementary in an
integral chromosome, its position can be determined. Even the finest chromosomal
maps could only identify a DNA fragment to a region of about 10 Mb, the size of a
standard chromosome band, until recently. FISH techniques have improved,
allowing DNA sequences as near as 2 to 5 Mb to still be aligned. In situ hybridization
techniques have been tweaked while using chromosomes during the interphase
phase of cell division, because they’re less compact, resulting in a resolution of
about 100 kb. Additional advancements in banding may enable chromosomal bands
to be linked to unique fragments of DNA that have been amplified, which could help
assess recognizable physical characteristics linked to chromosomal abnormalities.
Complimentary DNA Map—The locations of exons concerning unique regions
of chromosomes are shown on a cDNA map (those transcribed into mRNA are
known as expressed DNA regions). When cDNA is being generated in the lab while
utilizing the mRNA strand as a guiding template, base-pairing rules are followed
(a nucleotide G on the mRNA will pair only with a C on the newly synthesized DNA
strand). The cDNA would then be traced back to regions within the genome. Since
cDNAs reflect genomic regions that have been expressed, they are believed to
classify the sections of their genome with the most medical and biological relevance.
The chromosomal position of genes whose roles are unknown can be calculated
using a cDNA map. When the estimated position of a disease gene has been
identified using genetic linkage techniques, the map may also indicate a range of
candidate genes to test for disease gene hunters.

15.5.2.5 High-Resolution Physical Mapping

Top-down (producing a macrorestriction map) and bottom-up (producing a
microrestriction map) are the only two approaches that are currently being used for
high-resolution physical mapping (generating a contig map). Maps illustrate
organized DNA fragment sets formed by restriction enzymes cleaving genomic
DNA with any of the strategies. Cloning or polymerase chain reaction (PCR)
methods are used to amplify the fragments.
Macrorestriction Maps: Top-Down Mapping—A single chromosome is split into
large pieces (with rare-cutter restriction enzymes) and then sorted and divided up;
the small fragments will then be mapped further. The corresponding macrorestriction
maps show the sequence in which rare-cutter enzymes cleave and the distance
between them (Fig. 15.20). This method produces maps with far more consistency
15 Genomics 733

Fig. 15.20 Physical mapping strategies (P. R. Billings et al.). (a) Top-down physical map is
generating fewer gaps within the map, but the position of particular genes may not be allowed by the
resolution of the map. (b) Bottom-up strategies produce highly detailed maps with smaller areas, but
they also have a lot of gaps. Both methods are being used in tandem

with smaller gaps separating the fragments compared to contig maps; however, the
resolution of the map being lower might not be suitable for identifying a unique set
of genes. Additionally, this method seldom produces long stretches of mapped
locations. Currently, this method can locate DNA fragments in regions ranging
from 100,000 to 1 million bytes.
Contig Maps: Bottom-Up Mapping—Breaking the chromosomes into smaller
parts, in which every fragment is ordered and cloned, is the bottom-up method.
Adjacent blocks of DNA are created by the arranged fragments (contigs). The
resultant clone library presently ranges in size from 10,000 bp to 1 Mb. The fact
that these stable clones are available to other researchers is a benefit of this approach.
The FISH technique may verify contig construction by localizing cosmids with a
particular segment within chromosomal bands.
Large DNA fragments can now be cloned synthetically using vectors that can
hold the fragments of human DNA as big as 1 MB, thanks to technological advances.
These vectors are kept as artificial chromosomes in yeast cells (YACs). (See DNA
amplification for more information.) Before the creation of YACs, the largest
cloning vectors (cosmids) only had inserts of 20 to 40 kb. The YAC method
significantly decreases the number of clones that must be ordered; several YACs
span whole human genes. Subcloning, a method in which actual inserts are cloned in
the form of fragments into relatively small vectors, will create a much more
elaborated map consisting of a huge YAC insert. High-capacity bacterial vectors
(those that can handle large inserts) are indeed being established since some YAC
regions are unstable.
734 S. K. AVS et al.

15.5.3 Sequence of Human Genome

The use of paired-end sequences (also known as mate pairs) originating from
subclone repositories with distinctive insert sizes and cloning characteristics was a
crucial element of the sequencing approach used in this mega base-size and larger
genome. The effectiveness of using end sequences from long segments (18 to
20 kbp) of DNA cloned into bacteriophage lambda in the assembly of microbial
genomes led to the suggestion of using end sequences from 150-kbp bacterial
artificial chromosomes to simultaneously map and sequence the human genome
(BACs).

15.5.3.1 Sources of DNA and Sequencing Methods

This part examines the reasoning and moral principles overseeing giver choice to
guarantee ethnic and sex variety alongside the procedures for DNA extraction and
library development. The plasmid library development is the principal basic advance
in shotgun sequencing. On the off chance that the DNA libraries are not uniform in
size, are nonchimeric, and don’t arbitrarily address the genome, at that point, the
ensuing advances can’t precisely remake the genome grouping.

1. Library construction and sequencing—The organization of excellent plasmid

libraries in a variety of supplement estimates is vital to the entire genome shotgun
sequencing process, as it allows for the acquisition of sets of grouping peruses
(mates): one read from each plasmid embeds two closures. Great libraries have an
equal representation of all genome parts, few clones without embeds, and no
contamination from sources like the mitochondrial genome or Escherichia coli
genomic DNA. Every giver’s DNA was used to build plasmid libraries in at least
one of three sizes: 2 kbp, 10 kbp, and 50 kbp. The major focus is on creating a
simple method that could be executed in a consistent and repeatable manner and
monitored effectively when developing the DNA-sequencing process
(Fig. 15.21).
2. Trace processing—An automated trace-processing pipeline was built to process
each sequence file. After consistency and vector trimming, the average trimmed
sequence length was 543 bp, and sequencing accuracy was exponentially
distributed, with a mean of 99.5% and fewer than 1 in 1000 reads being less
than 98% accurate.
3. Quality assessment and control—Each sequence read must be put in the genome
in a specific way, and even a small error rate will reduce the assembly’s effec-
tiveness. Furthermore, for the algorithms discussed below, preserving the validity
of mate-pair information is important. As sequencing reactions progressed
through the process, procedural controls were developed, including strict rules
built into the laboratory information management system, to ensure the validity of
sequence mate pairs.
15 Genomics 735

Potential Entry Points Potential Exit Points

Process Management
Human Samples
[Medical Affairs]
Workflow Process
-sample screening
QA Process
Tissue Samples DNA/RNA Extraction QC: size and clarity DNA/RNA
[DNA Resources] [DNA Resources] [DNA Resources]

QC: insert size,

DNA/RNA (External) QC: size & concentration libaray complexity Libraries
Library Construction
[DNA Resources] [DNA Resources] [DNA Resources]

Libraries QC: titer & functional test Pre-Sequencing Fluorescently Labeled

[DNA Resources] [Pre-Sequencing Lab] DNA
[Pre-Sequencing Lab]
QC: monitor statistical
Fluorescently Labeled Sequencing summary data Trace Files [NT]
DNA [Sequencing Lab] [Sequencing Lab]
[Pre-Sequencing Lab]

-validate trace files vector & contaminant

Trace Files [UNIX] -load QCDS quality info Post-Sequencing screening Trimmed Fragments
[Sequencing Lab] [Content Systems] [Content Systems]

QC: byte count, External Data

External Fragments remove duplicates Processing Pre-Assembly
[Content Systems- EDA] [Content Systems] [Content Systems]

QC: “gatekeeper”
External &Trimmed syntax, duplicates&
Fragments Proto I/O File Generation quality values Proto I/O Files
[Content Systems] [Content Systems] [Content Systems]

Chromosome
Proto I/O Files “gatekeeper” run again Assembly Team QA review Assemblies
[Content Systems] [Informatics Research] [IR / CT]

Fig. 15.21 Flow diagram for sequencing pipeline (J. Craig Venter et al.). With an emphasis on
consistency inside and across divisions, samples are collected, selected, and processed by standard
operating procedures. Each method has its collection of inputs and outputs, as well as the ability to
share samples and data with both internal and external organizations when adhering to quality
standards

15.5.3.2 Genome Assembly Strategy and Characterization

The two main approaches that are being used to assemble the genome are discussed
in this section. To create an independent, nonbiased view of the genome, one
approach involves computationally combining all sequence reads with shredded
data from GenBank. Based on mapping details, the second method involves cluster-
ing all of the fragments to a region or chromosome. After that, the clustered data was
shredded and placed through a computational assembly process. Both methods
yielded a virtually identical reconstruction of the assembled DNA sequence, com-
plete with proper order and orientation. Shotgun sequence assembly is a classic
inverse problem: Given a random sample of reads from a target sequence, recon-
struct the order and location of those reads in the target sequence. Celera assemblies
are made up of a series of contigs that are grouped and oriented into scaffolds, which
736 S. K. AVS et al.

Fig. 15.22 Anatomy of whole-genome assembly. To generate a contig and a consensus series,
overlapping shredded BAC contig fragments (red lines) and internally derived reads from five
separate individuals (black lines) are combined (green line). Using mate-pair details, contigs are
connected into scaffolds (red). Scaffolds are then mapped to the genome (gray line) using physical
map knowledge from STS (blue star)

are then mapped to chromosomal locations with the aid of known markers. The
contigs are made up of overlapping sequence reads that form a consensus recon-
struction of a contiguous region of the genome. Mate pairs are an integral part of the
assembly process. They’re used to create scaffolds in which the size of gaps between
contigs is known to a reasonable degree of accuracy. This is achieved by noting that
a pair of reads, one of which is in one contig and the other in another, means a
distance and orientation between the two contigs (Fig. 15.22).
Assembly strategies—It was decided to take two separate approaches to assem-
bly. The first was a whole-genome assembly process that used Celera data
and Publicly Funded Human Genome Project (PFP) data as synthetic shotgun
data, and the second was a compartmentalized assembly process that partitioned
Celera and PFP data into sets localized to large chromosomal segments and then
conducted ab initio shotgun assembly on each set (Fig. 15.23).

1. Whole-genome assembly (WGA)—The WGA assembler is made up of five

stages: Screener, Overlapper, Unitigger, Scaffolder, and Repeat Resolver. All
microsatellite repeats with less than a 6-bp element are found and labeled, and all
recognized interspersed repeat elements, such as Alu, Line, and ribosomal DNA,
are screened out. Marked regions are checked for overlaps, while screened
regions are not but maybe included in an overlap with unscreened matching
segments.
15 Genomics 737

5.11X Celera Read s Public Bactig s

39X mate pair s (from 33,421 BACs )

Shredde r Matcher

2.96x Faux
Reads Celera-unique Bactigs & Celera pair s
read s (binned by BAC )

WGA Combinin g
WGA Assemble r

Unique BA C
Scaffold s Scaffold s

Tile r

Components 1

Components 2

WGA+Shredder

Components n

WGA Assembly CSA Assembly

Fig. 15.23 Architecture of Celera’s two-pronged assembly strategy. Each oval represents a
computation process that performs the function indicated by its mark, with labels on arcs between
ovals representing the types of objects generated and/or consumed by the process. The discussion in
the text that describes the words and phrases used is summarized in this diagram

Every read is compared to every other read in the Overlapper to find full end-to-
end overlaps of at least 40 bp and no more than 6% differences in the match. Early in
the process, the assembler must avoid selecting repeat-induced overlaps. Unitigger
helps in achieving this aim. Unitigs are the contigs produced from these
subassemblies (for uniquely assembled contigs). These unitigs are uncontested
interval subgraphs of the graph of all overlaps in formal terms.
The Unitigger generated a series of correctly assembled subcontigs that covered
an estimated 73.6% of the human genome. The Scaffolder then proceeded to connect
these into scaffolds using mate-pair details. When two or more mate pairs mean that
a pair of U-unitigs are at a certain distance and orientation concerning each other, the
likelihood of this being incorrect is approximately 1 in 1010, assuming that mate
pairs are false less than 2% of the time. A consensus sequence of each contig is
produced at the end of the assembly process, as well as at several intermediate
738 S. K. AVS et al.

points. The theory of maximum parsimony guides an algorithm, which uses quality-
value-weighted measures to evaluate each foundation.

15.5.4 Features of Human Genome

The human haploid genome comprises approximately 30,000 genes and is and
around three billion bp in length. Since each base pair can be encoded with two
bits, this amounts to approximately 750 M of data. A single somatic cell comprises
two times as many base pairs or around six billion. Males have comparatively lesser
base pairs than a female since the Y chromosome has around 57 million while X had
around 156 million. Since human genomes differ by only about 1% in sequence, the
differences of any given genome of a human from a standard reference perhaps
reduced approximately to 4 MB without losing any data. The genome’s entropy rate
varies greatly among noncoding and coding sequences. It is similar to the limit of
2 bits per base pair for coding sequences (approximately 45 Mbp) but less for
noncoding pieces. The entropy rate for each chromosome varies from 1.5 to 1.9
bits per base pair. Y chromosome is an exception, which has 0.9 bits of entropy rate
per base pair.

15.5.4.1 Noncoding DNA vs. Coding DNA

Coding and noncoding DNA sequences are widely used to separate the human
genome’s material. Coding DNA refers to DNA that has the potential to synthesize
mRNA by transcription and ultimately leading to the production of proteins; these
sequences account for just 2% of the entire genome. The sequences that don’t encode
for any proteins are considered as noncoding DNA (roughly 98%). RNA molecules
with essential roles can be found in certain noncoding DNA (rRNA and tRNA). The
ENCODE (Encyclopedia of DNA Elements) initiative, which attempts to explore the
entire genome of the human by utilizing a variety of analytical methods whose
findings are representative of biological activity, is one example of contemporary
genome research focusing on the role and evolutionary origins of noncoding DNA.
The analyzed genome framework has evolved into a much more focused analytical
category than the conventional concept of DNA-coding genes since noncoding DNA
exceeds the number of coding DNA.

15.5.4.2 Coding Sequences (Protein-Coding Genes)

Protein-coding sequences are the most researched and well-understood part of the
human genome. Though many biological processes (such as rearrangement of DNA
and alternate splicing of pre-mRNA) can result in the development of far more
unique proteins than that of the genes encoding for the proteins, these sequences
eventually contribute to generating all the proteins. The exome contains the
genome’s entire functional protein-coding ability and is made up of nucleotide
sequences that are encoded by coding sequences and can then be translated to
functional proteins. The exome was considered as the first big achievement of the
15 Genomics 739

Fig. 15.24 Human genes classified according to the function of transcribed proteins, expressed as
many encoding genes and as a percentage of all genes (PANTHER Pie Chart)

Genome Project for its biological importance as well as the reality that it makes up
only around 2% of the entire genome (Fig. 15.24).
1. Number of protein-coding genes—In databases like UniProt, around 20,000
human proteins have been annotated. Historically, estimates for the number of
protein-coding genes have ranged from 200,000 in the late 1960s to 2000,000 in
the early 1970s, and many scientists noted in the early 1970s that only about
40,000 for the total number of functional loci are present which includes func-
tional noncoding as well as protein-coding genes. This is because mutational load
arising through deleterious mutations sets a maximum limit. The number of genes
encoding the protein is comparable to that of several fewer complex species such
as roundworms and fruit flies. This distinction may be due to humans’ widespread
utilization of alternative splicing of pre-mRNA, which helps them to create a wide
range of functional proteins by selectively adding exons.
2. Chromosome’s protein-coding capacity—Genes encoding the proteins are
unequally scattered throughout the chromosomes, varying from several hundred
to over 2000, with chromosomes 1, 11, and 19 having the highest gene abun-
dance. Gene-abundant and gene-poor regions can be located on each chromo-
some, which can be connected to bands on the chromosome and GC material. The
importance of these established gene density arrangements is still unknown.
3. The magnitude of protein-coding gene—Within the human genome, the scale
of protein-coding genes varies greatly. For instance, the HIST1HIA gene, which
codes for the histone H1a, is small and straightforward. A 781-nucleotide mRNA,
lacking introns, encodes a protein with 215 amino acids arising from 648 bp ORF.
According to the 2001 human reference genome, dystrophin (DMD) was consid-
ered as the biggest protein-coding gene, spanning 2.2 million nucleotides, while a
fox-1 homolog 1, which is an RNA binding protein, RBFOX1, which spans 2.47
million nucleotides, was discovered in a much more recently published statistical
analysis of modified human genome results.
740 S. K. AVS et al.

The longest coding sequence (114,414 nucleotides), consisting of the highest

number of exons (363), and the lengthiest exon (17,106 nucleotides) are all found in
Titin (TTN). The mean size of a protein-coding gene is 66,577 nucleotides, the mean
exon size is 309, the mean of the number of exons is 11, and the mean of the encoded
protein is 425 amino acids in length, according to estimates in accordance with a
carefully selected range of protein-coding genes across the entire genome.
Table 15.5 provides few examples of human protein-coding genes.

15.5.4.3 Noncoding DNA (ncDNA)

The nucleotide sequences in a genome that are not included within protein-coding
exons and thus never appear in the amino acid composition of gene products are
referred to as noncoding DNA. According to this definition, ncDNA makes up as
much as 98% of entire human genomes. Genes that encode for noncoding RNA
(e.g., transfer RNA and ribosomal RNA), untranslated regions of mRNA,
pseudogenes, introns, repetitive DNA sequences, regulatory DNA sequences, and
sequences linked to transposons are all examples of noncoding DNA. Noncoding
DNA refers to a variety of sequences that are found within genomes.
The human genome contains less than 1.5% protein-coding sequences (specifi-
cally, coding exons). Furthermore, introns make up about 26% of the human
genome. The human genome includes noncoding DNA regions in addition to
genes comprising both introns and exons and regions that regulate gene expression
(8–20%). The precise amount of noncoding DNA that contributes to cell physiology
has been a point of contention. According to a recent report by the ENCODE project,
80% of the human genome is either transcribed, binds to regulatory proteins, or is
involved in some other biochemical activity.
Most of these sequences control chromosome structure by restricting heterochro-
matin formation regions and controlling chromosome characteristics like
centromeres and telomeres. Few other noncoding regions function as origins of
replication. Finally, some regions of the genome are transcribed into functional
noncoding RNA, which controls protein-coding gene expression, translation of
mRNA and its stability, and chromatin remodeling [including DNA binding protein
modifications, methylation of DNA, DNA recombination]. It’s also probable that
many of the transcribed noncoding regions have no function and are the outcome of
the nonspecific activity of RNA polymerase.

15.5.4.3.1 Pseudogenes
Pseudogenes are nonfunctional transcripts of protein-coding genes that have
accumulated due to inactivating mutations. They are also produced by gene duplica-
tion. The total number of pseudogenes with the human genome is on the order of
13,000, and the number of functional protein-coding genes in certain chromosomes
is approximately the same. During molecular evolution, gene replication is a popular
process for generating new genetic material. The gene family of the olfactory
receptor is one of the most well-studied models of a pseudogene. In humans, as
many as 60% of the genes in this given family are dysfunctional pseudogenes. In
contrast, just 20% of the genes within the olfactory receptor gene family of the
 15
Genomics

Table 15.5 Common examples of human protein-coding genes (Ensembl genome browser, July 2012)
Protein Chromosome Gene Length Exons Exon length Intron length Alt splicing
Breast cancer type 2 susceptibility protein 13 BRCA2 83,736 27 11,386 72,350 Yes
Cystic fibrosis transmembrane conductance regulator 7 CFTR 202,881 27 4440 198,441 Yes
Cytochrome b MT MTCYB 1140 1 1140 0 No
Dystrophin X DMD 2,220,381 79 10,500 2,209,881 Yes
Glyceraldehyde-3-phosphate dehydrogenase 12 GAPDH 4444 9 1425 3019 Yes
Hemoglobin beta subunit 11 HBB 1605 3 626 979 No
Histone H1A 6 HIST1H1A 781 1 781 0 No
Titin 2 TTN 281,434 364 104,301 177,133 Yes
741
742 S. K. AVS et al.

mouse are pseudogenes. According to research, the most closely related primates all
have proportionally fewer pseudogenes, implying that this is a species-specific trait.
This evolutionary observation explains why humans have a less acute sense of smell
than other mammals.

15.5.4.3.2 Untranslated Regions and Introns of mRNA

The preliminary transcripts of protein-coding genes normally include substantial
sequences which don’t code for any functional product in the form of 50 - and
30 -untranslated regions, introns in addition to the few ncRNA transcripts encoded
by distinct genes. Introns are 10 to 100 times longer than the exons of most protein-
coding genes in the human genome.

15.5.4.3.3 Regulatory DNA Sequences

Many different regulatory sequences exist in the human genome, all of which are
essential for regulating gene expression. According to conservative figures, these
sequences make up 8% of the genome, but extrapolations from the ENCODE project
say that gene regulatory sequence makes up 20–40% of the genome. Noncoding
DNA is a form of genetic “switch” that does not code for functional proteins but does
control the temporal and spatial expression of genes (enhancers). Since the late
1960s, regulatory sequences have been identified. Recombinant DNA technology
was used to identify the first regulatory sequences within the genome. The identity of
these sequences might later be deduced through evolutionary conservation, thanks to
the advent of genomic sequencing. For example, the evolutionary split between
primates and mice occurred 70–90 million years ago. As a result, computerized
evaluations of gene sequences will reveal noncoding sequences that are being
conserved, which will indicate their role in functions such as gene regulation.

15.5.4.3.4 Repetitive DNA Sequences

Approximately half of the human genome is composed of repetitive DNA
sequences. Low complexity repeat sequences with many adjacent copies, tandem
repeats, make up around 8% of the human genome (e.g., “CAGCAGCAG...”).
Tandem sequences can vary in length from a few nucleotides to tens of thousands
of nucleotides. These sequences are used for forensic DNA and genealogical DNA
analysis because they are extremely inconstant among nearly connected persons.
Microsatellite sequences (e.g., the dinucleotide repeat (AC)n) are recurring
sequences with less than 10 nucleotides. Trinucleotide repeats are especially impor-
tant among microsatellites because they can occur in the exonic regions of protein-
coded genes, causing genetic disorders. Huntington’s disease, for example, is caused
by the expansion of a trinucleotide repeat (CAG)n within the gene on human
chromosome 4. A hexanucleotide repetition of the sequence (TTAGGG)n, which
ends the telomeres, is also a microsatellite. Tandem repeats of comparatively longer
sequences (10 to 60 nucleotides in length) are termed as Minisatellites.
15 Genomics 743

15.5.4.3.5 Transposons (Mobile Genetic Elements) and Their Remnants

Transposable genetic materials, or DNA sequences that can duplicate and introduce
clones of themselves in the other sites within the same host genome, are prevalent in
the human genome. Alu, the most common transposon line of descent, has around
50,000 operating clones and can inject itself into both intergenic and intragenic
locations. LINE-1 is another lineage with about a hundred active clones within a
genome (the number differs among various people). They make up more than half of
total human DNA, along with the nonfunctioning remnants of obsolete transposons.
Transposons, also known as “jumping genes,” have been playing a significant role in
shaping the human genome. A few of these sequences are retroviruses that have been
generated endogenously, which are copies of viral DNA that have been incorporated
permanently within the genome and are crossed down the generations. LTR
retrotransposons (8.3% of the total genome), SINEs (13.1% of the total genome)
including Alu elements, LINEs (20.4% of the total genome), SVAs, and Class II
DNA transposons are the various types of mobile elements present in the human
genome (2.9% of the total genome) (Fig. 15.25).
A diagram depicting the general structure of various types of mobile components.
The proportion of the human genome that is predicted to be present is shown on the
right. ITR stands for the inverted terminal repeat, LTR stands for the long terminal
repeat, and UTR stands for the untranslated region. Gag, which codes for viral
structural proteins; pol, which codes for viral enzymes; and env, which codes for
the viral envelope protein are all HERV genes. The sense and antisense promoter
regions are indicated by black arrows. DNA is represented by black and gray double
helices. AAAA stands for polyA tail.

15.5.5 Human HapMap Genome

The International HapMap Project’s mission is to identify the general characteristics

of variations of a DNA sequence within the human genome and enable this

Fig. 15.25 Transposable elements in the human genome

744 S. K. AVS et al.

knowledge publicly accessible. By identifying the genetic traits of one million or

even more expression levels, their patterns, as well as the direction of the association
among them in genetic data from communities with ancestors from areas of Asia,
Africa, and Europe, global collaboration is constructing a map of such variations
across the genomes. The HapMap will aid in the identification of genetic variations
that influence common diseases, as well as the development of screening methods
and the selection of targeted therapies. The project aims to classify variation in the
sequence, their prevalence, and associations within them in DNA specimens from
inhabitants with lineage from Asia, Africa, and Europe to establish common
schematics of DNA sequence variation in the human genome. As a result, the project
will develop tools that will make it simple to apply the indirect association method to
any operational candidate gene, any area proposed by familial linkage assessment,
and, eventually, the entire genome for disease risk factor scans.
Variation in the human DNA sequence—Approximately 0.1% of nucleotide
positions vary between each replica of the human genome (on average, one per
thousand bases). An SNP, the most common type of variant, is a variation in the base
present at a specific location in the DNA sequence between chromosomes
(Fig. 15.26). Few chromosomes in a community, for example, can have a C allele
at a particular location, while other people have a T allele. It is assumed that
approximately ten million locations (on average, one alternative per 300 bases)
differ in the human population in such a way that both the alleles are ascertained
at an incidence of 1%. These ten million standard SNPs account for 90% of the
divergence in the population. The remaining 10% is due to a wide variety of
variations, each of which is extremely rare in the population. A genomic DNA
sample is tested (“genotyped”) to determine the existence of specific SNP alleles in
an organism.
DNA samples and populations—The project will analyze 270 DNA samples,
including 90 samples from a US Utah population, gathered by the “Centre d’Etude
du Polymorphisme Humain” (CEPH) and new samples gathered from 90 Yoruba
individuals, 30 trios of Nigerian, 45 distinct Japanese in Yokohama, and 90 Yoruba
people. A survey of 45 irrelevant persons must be enough to identify 99% of
haplotypes in a population with a prevalence of 5% or higher. Random individual
samples, trios, or larger pedigrees may be used in Linkage Disequilibrium
(LD) studies; every structure has benefits (easier sampling) and drawbacks (larger
pedigrees) (with a rising number of related individuals, productivity is dwindling).
Irrelevant persons and trios have a lot of credibility when it comes to predicting
localized LD patterns, according to established smart computing algorithms. The
trios may have valuable insights into the project’s genetic analysis platforms’
precision.
To accurately explain genetic discrepancy throughout the whole genome, a high
density of SNPs is needed. The mean abundance of markers in the shared database,
dbSNP, when the task began was about one per kb (2.8 million SNPs), although
many domains had a much lower density due to their variable distribution. There are
three methods for determining the consistency of a genotype. To begin, each center
received the same arbitrarily chosen set of 1500 SNPs for analysis and genotyping
15 Genomics 745

Fig. 15.26 SNPs, haplotypes, and tag SNPs (the International HapMap Project). (a) SNPs are
single nucleotide polymorphisms. A small piece of DNA from four separate people’s variety of the
exact chromosomal area is seen. The majority of the DNA sequence in these chromosomes is
similar, but three bases are seen where there is the difference. Each SNP has two potential alleles;
the alleles C and T are contained in the first SNP. (b) A haplotype is composed of a specific set of
alleles found at neighboring SNPs. The identified genetic makeups for 20 SNPs spanning 6000
bases of DNA are depicted here. Just the varying bases, like the 3 SNPs in section a, are displayed.
Demographic data revealed that many of the chromosomes in this zone have haplotypes 1–4. (c) It is
adequate to genotype only the three tag SNPs out from the 20 SNPs to distinguish such four
haplotypes. For example, if these three tag SNPs on a specific chromosome have the sequence A–
T–C, this configuration represents the pattern defined for haplotype1. Multiple chromosomes in the
populations bear the standard haplotypes

with the 90 CEPH DNA samples used for the study. Genotyping centers provided
data that was beyond 99.2% complete and 99.5% accurate on average (comparative
with the concordance of at least two other forums). Second, samples for convergent
validity checks are used in each genotyping test, with replicates of five different
samples with a blank in every 96-well plate. Also, data from trios can be used to
verify that SNP alleles are inherited in a consistent Mendelian manner. The informa-
tion from the independent samples serves as a review to ensure that perhaps the
SNPs seem to be in Hardy-Weinberg equilibrium across all communities (a test of
genetic mating patterns). Although there are a tiny proportion of SNPs that failed
such assessments due to biological factors, they seem to be more prone to failure
even when a genotyping system creates recurrent errors, like under-calling
heterozygotes. Third, a sample of SNP genotypes from every center will indeed be
selected at random and re-genotyped by several other centers. These comprehensive
third-party quality assessments will ensure that the data that has been generated
during the project is complete and reliable.
746 S. K. AVS et al.

While using the HapMap to analyze vast genomic areas, several comparative
studies can occur when evaluating tens to hundreds of thousands of SNPs as well as
haplotypes for disease correlations. It would be difficult to distinguish true from
false-positive outcomes as a result of this. To validate the findings and classify the
functionally significant SNPs, functional analyses, analytical techniques, and vali-
dation tests of variants would be needed. The HapMap has a lot of potential as a
modern method for discovery—to help us better understand the genetic factors that
influence health and disease. Fundamental science researchers, population
geneticists, physicians, epidemiologists, sociologists, theologians, and the public,
in general, will work together to reap the maximum benefits.

15.5.6 Human Genome Vs. Chimpanzee Genome

Chimpanzees are humans’ nearest living relatives. The split between human and
chimp ancestors took place about 6.5–7.5 million years ago. The genetic
characteristics that differentiate us from chimps and distinguish us as humans are
still a source of fascination. Human and chimp genomes underwent several changes
after their ancestral lineages diverged, including substitutions of a single nucleotide,
duplications and deletions of DNA fragments of various sizes, addition of mobile
genetic elements, and chromosomal rearrangements.
The analogy of the genomes of chimpanzee and human demonstrates remarkable
resemblance, substantial discrepancies, and new insights for biomedical research:

1. It demonstrates unequivocally that humans and chimps share a similar and recent
evolutionary origin, as predicted by Charles Darwin in 1871.
2. It reveals essential properties of the human genome for human medicine, such as
the types of genes that have developed the fastest over thousands of years and
unique regions of chromosomes that have experienced significant positive selec-
tion throughout the history of moderns. Ever since some of these represent
reactions to current pathogens or evolutionary changes important to human
well-being, this reflects light on human biology and, in particular, human
diseases.
3. It shows that humans and chimps have been able to handle more genetic
mutations than many other animals, such as rats. This supports a significant
evolutionary assumption, and it could explain why primates have more creativity
than rats, as well as a higher rate of genetic disorders.

The major outcomes of the consortium league are as follows:

1. The genomes of chimps and humans are remarkably identical, and they encode
for proteins whose functions are highly similar. The DNA sequences of the two
genomes are almost identical and can be directly compared. Also, after account-
ing for DNA insertions and deletions, humans and chimps share 96% sequence
similarity. In chimps and humans, 29% of genes encode the exact amino
15 Genomics 747

sequences at the protein level. Since chimps and humans separated about six
million years ago from a common ancestor, the modern human protein has only
undergone one unique shift.
2. When compared to other animals, some groups of genes change extremely
rapidly in both chimps as well as humans. Genes involved in sound perception,
nerve signal transmission, sperm formation, and cellular ion transport are among
these groups. The expeditious course of evolution of the above genes might have
affected primates’ unique characteristics.
3. Throughout evolution, humans and chimps probably acquired more potentially
dangerous mutations throughout their genomes than rats, mice, and other rodent
species. Though such modifications can result in disorders that reduce the overall
fitness of a species, they might have enabled primates highly adaptable to drastic
changes in the environment and allowed them to develop specific evolutionary
acclimatization.
4. The mutual sections of the two genomes differ by around 35 million DNA base
pairs. There are an additional five million sites that vary due to an insertion or
deletion of one of the lineages, as well as a much smaller number of chromosomal
rearrangements. The majority of these discrepancies are thought to be nonfunc-
tional DNA. However, up to three million variations are present in important
genes encoding structural proteins that include other functional areas within the
genome. The biological foundation for the peculiar features of the human race,
comprising human-specific disorders such as Alzheimer’s, few cancers, and HIV,
can be found somewhere within these relatively few variations.
5. Despite the lack of statistical evidence, a few groups of genes seem to be
developing more quickly in humans than in chimps. Genes that encode transcrip-
tion factors, elements that control the function of few other genes and perform
important functions during embryonic development, are the single largest outlier.
6. Perhaps more drastic shifts have occurred in a limited number of other genes. The
chimp genome lacks or has partial deletions of more than 50 genes found in the
human genome. The exact number of gene deletions in the human genome is
unclear at this time. Three primary inflammation genes appear to be missing from
genome of the chimp, which may explain a few of the reported variations among
chimps and human beings in terms of immune responses. Humans, but from the
other hand, tend to already have impaired the role of the caspase-12 gene, which
expresses an enzyme which may well influence Alzheimer’s disease progression.
7. Over the past 250,000 years, six regions of the human genome have provided
strong signs of specific sweeps. (Particular sweeps appear when a mutation comes
up within a population and is so beneficial that it propagates across the population
to a few hundreds of generations and eventually becomes “normal.”) One region
includes as many as 50 genes, whereas others contain no known genes and are
referred to as a “gene desert” by scientists. Surprisingly, components in this gene
desert can regulate the transcription of a nearby protocadherin gene that has been
linked to nervous system patterning.
748 S. K. AVS et al.

Another study group looked at non-repetitive non-reference sequences (NRNR)

in 15,219 Icelanders’ genomes. An aggregate of 326,596 bp of NRNR DNA was
discovered, with only 244 inserts longer than 200 bp accounting for 84% of the total.
Notably, as compared to the chimpanzee genome, it was discovered that more than
95% of the NRNRs longer than 200 bp were also existing in the genome assembly of
the chimpanzee, implying that they were ancestral. As a result, misinterpretation of
polymorphic sequences may affect the total degree of human-chimp interspecies
divergence due to a lack of knowledge on genome populational diversity. It does not,
however, negate any of the assumptions and evidence presented in this study.
Nonetheless, these results eventually contribute to the notion that human and
chimp pan-genomes must be produced and compared.

15.6 Features of the Chloroplast Genome

Tobacco and Marchantia polymorpha, a liverwort, were the foremost plants in which
the chloroplast genome (cpDNA) was sequenced successfully. Ever since, the
cpDNA of 3721 species of plants, comprising green algae and that both terrestrial
and freshwater plants, has been discovered. The organelle genome database at the
National Center for Biotechnology Information (NCBI) has them. The advent of
high-throughput sequencing technologies has allowed for such substantial advance-
ment in the domain of chloroplast genetics in recent years. The completely
sequenced genome of Synechocystis sp. PCC6803, a cyanobacterial species,
represents a real advance in cpDNA science. It is a well-known primordial photo-
synthetic organism that is commonly used in research on photosynthesis, carbon and
nitrogen assimilation, and the evolution of plastids. Synechocystis was quickly
dubbed a model microorganism due to its unusual characteristics, which include a
fully sequenced genome and the ability to feed in both autotrophic and heterotrophic
modes. Finally, phylogenetic comparative studies among Arabidopsis thaliana and
different microorganisms reported that more than 4500 genes encoding plant
proteins are derived from Cyanobacteria. It is significant to note that the proteome
of the chloroplast is made up of about 3000 proteins, the major part of which is
transcribed by the genomic DNA in the nucleus.
However, the nuclear genome does not contain any of the chloroplast proteins.
The chloroplast genome encodes some of them. The number of genes coded by
cpDNA varies from 0 to 315 in different plant species (NCBI). Significantly,
proteins encoded by a few of these genes have an important role in chloroplast
operations, especially photosynthesis. The majority of cpDNA-encoded proteins are
generated by their internal expression mechanism. Furthermore, both chloroplast-
derived transcription factors and non-chloroplast-derived transcription factors con-
trol the translation of proteins that are being coded by cpDNA at different levels of
the process. Furthermore, when a plant is exposed to multiple forms of abiotic
pressures, the chloroplast proteome undergoes major changes. The majority of the
time, it’s about proteins involved in photosynthesis.
15 Genomics 749

15.6.1 Structure and Organization of the cpDNA

At the onset of the plastom organization studies, chloroplast DNA was expected to
represent just a circular molecule inside living plant tissue. Many angiosperm
species have multibranched linear structures of cpDNA, according to recent micros-
copy studies. However, it is still unknown in which form the cpDNA will exist as its
most common. It varies by plant species and is affected by several factors such as cell
growth stage, type of tissue, and experimental model. The DNA of chloroplasts is
circular and has a length of 120,000–170,000 bp. They usually have a contour length
of 30–60 micrometers and a mass of 80–130 million daltons. The genomes of most
chloroplasts are fused into a single wide ring, except for dinophyte algae, whose
genome is split up into nearly 40 tiny plasmids, each 2000–10,000 bp in length.
There are 1–3 genes in each minicircle, but empty plasmids without any coding
DNA have also been discovered.
A combination of the two inverted repeats divides a long single copy section
(LSC) from a short single copy section (SSC) in many chloroplast DNAs. The length
of the inverted repeats varies greatly, from 4000 to 25,000 bp. Inverted repeats of
plants, which are each 25,000 bp long, are at the upper end of this scale. Inverted
repeat regions typically have three rRNA genes and two transfer RNA genes, and
they can be modified or contracted to have as few as 4 or as many as 150 genes
(Fig. 15.27). Although any two inverted repeats are usually unidentical, they can
often be quite similar, suggesting that they evolved in concert. The inverted repeat
regions of land plants are strongly conserved and accumulate few mutations. Similar
inverted repeats have been found in the cyanobacterial genome and two other
lineages of the chloroplast (Rhodophyceae and Glaucophyta), while few DNAs of
the chloroplast, such as some red algae and peas, have lost them. Few members, such
as Porphyra, made the direct repeats by reversing one of their inverted repeats. The
inverted repeats probably help to stabilize the remainder of the genome because
chloroplast DNAs that have lacked a few of the inverted repeat segments appear to
get rearranged more.
Nucleoids—In young leaves, every chloroplast comprises about 100 copies of
the DNA, which decreases in aged leaves to about 20 copies. They’re generally
stuffed in the form of nucleoids that can have multiple similar rings of chloroplast
DNA. Each chloroplast contains a large number of nucleoids. Although chloroplast
DNA isn’t identified with authentic histones, red algae have been found with a
histone-like chloroplast protein (HC) encoded by the chloroplast DNA that firmly
packs every chloroplast DNA rings in the form of a nucleoid. The chloroplast DNA
nucleoids in primitive red algae are concentrated in the middle of the chloroplast,
while the nucleoids in green plants and green algae are scattered across the stroma.
Transfer of genes between chloroplast and cell nucleus—Most of the genes from
the ancestral chloroplasts have been passed into the cell nucleus from chloroplast
DNA during evolution. Endosymbiotic gene transfer is a mechanism that also took
place in the second semiautonomous organelle, mitochondria. Around 18% of the
genes in the nuclear genome of A. thaliana are thought to be of cyanobacterial origin.
A gene encoding the chloroplast translation initiation factor 1 has been “acquired”
750 S. K. AVS et al.

Fig. 15.27 A graphic representation showing the distances of two inverted repeat regions (IRs). A
long unique sequence (LSC) and a short unique sequence (SSC) are marked on the Arabidopsis
thaliana cpDNA map (Prof. Emmanuel Douzery). Small ribosomal subunit proteins are indicated
by yellow; large subunit ribosomal proteins, orange; hypothetical chloroplast open reading frames,
lemon; protein-coding genes involved in photosynthetic reactions, green, or other functions, red;
ribosomal RNAs, blue; and transfer RNAs, red and black. Introns are indicated by a gray tint

by the cellular genome from the chloroplast DNA is one example. The RAR genes, a
significant number of genes involved in DNA damage repair as well as recombina-
tion, were also transferred in large numbers from chloroplast DNA to the cellular
genome. Because there is a high degree of similarity seen between multiple genes
that are associated with recombination as well as repair of damaged DNA found in
Arabidopsis thaliana and a human whose mutations are believed to cause
abnormalities like many cancers including non-lipid colon cancer, or Cockayne
syndrome, breast cancer research in this area is particularly important to medicine.
15 Genomics 751

15.7 Features of Mitochondrial Genome

Mitochondria are cellular organelles with an extrachromosomal genome that is

derived from and distinct from the genome of the nucleus. Sylvan and Margit
Nass first described and isolated mitochondrial DNA (mtDNA) in 1963, when
they identified that few mitochondrial fibers appeared to be DNA-linked based on
their fixation, stabilization, and staining actions. Eighteen years later, in 1981, the
first mtDNA complete sequence was published and developed as the mtDNA
Cambridge Reference Sequence (CRS). The mitochondrial DNA molecule is a
circular dsDNA molecule consisting of about 16,569 bp and a weight of 107 daltons
that is 5 millimeters in diameter and free of histones. Due to differences in G + T
base composition, mtDNA strands have different densities. The strong (H) strand
contains genes for 2 rRNAs (12S and 16S), 12 polypeptides, and 14 tRNAs, while
the light (L) strand contains genes for only 8 tRNAs and 1 polypeptide. Both
13 protein products are components of the oxidative phosphorylation system’s
enzyme complexes. Intron less genes and small, or even absent, intergenic
sequences, except in one regulatory region, are other distinguishing features of
mtDNA. The D-loop of the mitochondria region is found in many mitochondrial
genome mainly in the noncoding region (NCR), created by the stable inclusion of a
third 680 base DNA strand, also known as 7S DNA. The D-loop or noncoding area,
a 1121 bp section situated between 16,024 and 576 positions in accordance with the
CRS numeration, is where replication begins (Fig. 15.28). There are two transcrip-
tion promoters in the D-loop region, one for each strand. The convention that was
originally proposed was moderately changed gradually, deciding the substitution of
CRS for rCRS (revised Cambridge Reference Sequence), which is used to position
the nucleotide numbers in the mitochondrial genome. More specifically, each base
pair’s numerical designation starts at an unspecified location on the H strand and
continues for roughly about 16,569 bp around the molecule.
A set of comparable haplotypes characterized by the mixture of single nucleotide
polymorphisms (SNPs) within mitochondrial DNA derived from a common origin is
known as a haplogroup, formed as a result of mutations that have been accumulated
simultaneously through maternal lineage. Every somatic cell might just have up to
1000 mitochondria, with each comprising up to 10 copies of mitochondrial DNA.
Mitochondrial DNA typing seems to be more likely to yield a DNA typing result
instead of a polymorphic region typing in the nuclear genome when the quantity of
the retrieved DNA is too little or dissolute. In contrast to genomic DNA, mitochon-
drial DNA is solely transmitted from the mother, which explains why, apart from
mutation, the mitochondrial DNA sequences of siblings, as well as maternal
relatives, are similar. In forensic investigations, such as in the analysis of a deceased
individual’s remains, identified maternal relatives could contribute some samples as
a reference for a comparative analysis with the mitochondrial DNA type, and this
functionality can be extremely beneficial. Due to the lack of recombination, refer-
ence samples may be taken from maternal families from many generations away
from the origin of proof. The fact that most people’s mtDNA is haploid and
monoclonal streamlines the procedure of deciphering the sequenced DNA data.
752 S. K. AVS et al.

Fig. 15.28 Representation of the human mitochondrial genome including the genes and their
control regions labeled. The mitochondrial genome, consisting of 37 genes that are crucial to
respiratory chain assembly and function

Even so, heteroplasmy may be discovered on rare occasions. If an individual has

more than one detectable mtDNA type, he or she is said to be heteroplasmic.
Heteroplasmy is classified into two categories: length polymorphisms and point
substitutions. For forensic human identification, only the latter is important. The
majority of forensic laboratories around the world do not record polymorphism
lengths, and the standards for the identification of humans with mitochondrial
DNA don’t require them.
Mitochondrial DNA in Forensic Human Identification—The evidence sample
(s) and the mitochondrial DNA sequences of a referral sample are allowed for
comparison in forensic analysis. If the two sequences are distinct, it is possible to
rule out the possibility that they came from the same source. Although any further
nucleotide differences seen between the two sequences are not specified in any
scientific report or advisory publication, criminal routine departments regard them
as an exemption situation. The specimens cannot be rejected out if their sequences of
mitochondrial DNA are similar because they must be from the very same maternal
lineage or have a similar origin. Whenever heteroplasmy is found in both cases
around the same nucleotide positions, they shouldn’t be ruled out. If one of the
samples is homoplasmic in contrast to the other which is heteroplasmic, yet both
share exactly one mitochondrial DNA species, the samples should not be ruled out
because they may have come from the same source. Many scholars have reported
15 Genomics 753

that samples with one-base differences in mtDNA be investigated again, especially

in terms of mutation rate.

15.8 Comparative Genomics

The analysis of the variations and resemblance within the framework of the genome
and the arrangement of various species is known as comparative genomics. How do
the distinctions between humans and other species manifest themselves in our
genomes? For example, how similar are the different kinds and the number of
proteins in different species like bacteria, yeast, worms, fruit flies, and humans?
Comparative genomics is essentially the application of bioinformatics techniques to
whole-genome sequence analysis to define biological concepts, i.e., biology in
silico. Two factors influence comparative genetics. The first is an attempt to increase
a far more in-depth comprehension of the macroevolutionary process (the root of all
main classes of organisms) and the other one on a local scale (factors that cause the
uniqueness of related species). The necessity to translate DNA sequences is the
second engine data into known-function proteins. The justification DNA sequences
encoding essential information can be found here. It’s more likely that cellular roles
will be conserved than sequences encoding nonessential information between spe-
cies noncoding sequences or functions.
The development of paralogs and orthologs is a crucial step in the evolution of
genes. It’s important to differentiate between orthologs and paralogs when compar-
ing genome organization in different species. Orthologs are homologous genes in
distinct species that code for the proteins with the exact functionality. They have
evolved through direct vertical descent. Paralogs are homologous genes that encode
proteins with similar but not identical functions within an organism. These concepts
mean that orthologs have simply arisen by the progressive mutation accumulation,
while paralogs originate from gene duplication followed up by the accumulation of
mutations (Fig. 15.29).
Many bacterial genomes are smaller than that of the minimal eukaryotic genome.
The genome of the obligate intracellular parasite Encephalitozoon cuniculi is the
smallest eukaryotic genome ever sequenced having a genome size of 2.9 Mb, while
in E. intestinalis, its closest relative may even have a smaller genome of only 2.3 Mb.
The length of intergenic spacers is reduced in these species, and most putative
proteins are shorter than their orthologs in other eukaryotes, resulting in genome
compression. Neurospora crassa, a multicellular fungus, has about 10,000 ORFs,
which is about 25% less than the fruit fly Drosophila melanogaster. Most of the
abovementioned genes have no homologs in either Saccharomyces pombe or Sac-
charomyces cerevisiae.
Genes and regulatory sequences can be identified using comparative genomics. It
can be difficult to accurately classify genes in a full genome sequence, and
identifying regulatory elements can be even more difficult. Aligning orthologous
genomic sequences from various organisms and searching for sequence conservation
regions is an effective tool for identifying functional components such as genes and
754 S. K. AVS et al.

Fig. 15.29 Example of paralogs are the protein superfamilies. The location of introns in different
globin superfamily members. Inside the inverted triangles, the proportion of the introns in base pairs
is mentioned. It’s worth noting that the sizes of the polypeptides and the locations of the various
introns are fairly consistent

the regions regulating them. The rationale behind this strategy is that causes of
mutations within the functional DNA are deleterious and therefore counterselected,
followed by a slower rate of evolution in those functional elements. The amount of
divergence captured and the phylogenetic scope of the aligned sequences are the two
most significant factors influencing the outcome of a comparative study. The
strength and resolution of the tests are affected by the amount of divergence. The
scope, which is defined as the narrowest phylogenetic category that includes all
interpreted sequences, has an impact on the potential application of insights and the
generalizability of the results. A dipteran scope, for example, could be utilized to
look for elements that are involved in their common ancestor along with those
present before metazoa, arthropods, and hexapods diverged (Fig. 15.30).
The driving force of the evolution of species can be studied at the genome level.
Saccharomyces paradoxus, Saccharomyces mikatae, and Saccharomyces bayanus
are thought to have split from Saccharomyces cerevisiae about 5–20 million years
ago. A comparative study has been conducted upon sequencing all four genomes.
15 Genomics 755

Fig. 15.30 The importance of scope and the impact of shared ancestry on comparative sequence
analysis. The light purple tree depicts the relationships between six genomes that are currently being
studied (not to scale). Every line that has been colored represents the scope of the phylogeny that
shall be applied to each pair of species at the terminal nodes: Gray represents placental mammals,
black represents teleosts, and dark purple represents dipterans. Colored line overlaps suggest
common heritage and catch characteristics that have been shared by the mentioned scopes and,
by extension, functional components.

Table 15.6 Genomic rearrangement in three yeast species in comparison with S. cerevisiae. S.B.
Species Reciprocal translocations Inversions Segmental duplications
S. paradoxus 0 4 3
S. mikatae 4 13 0
S. bayanus 5 3 0

They discovered a high degree of “genomic churning” near the telomeres, with gene
families showing major changes in the number, order, and orientation. Outside of the
telomeric regions, only a few rearrangements were observed and are listed in
Table 15.6. All the 20 inversions which generally belong to the same iso-acceptor
form were transcribed in the opposite direction that flanked by tRNA genes. The
importance of tRNA genes in genomic inversion has previously gone unnoticed.
756 S. K. AVS et al.

Seven of the nine translocations happened among Ty components, and two resulted
among closely related ribosomal gene pairs.

Box 15.1: A HapMap Harvest of Insights into the Genetics of Common

Disease—Teri A. Manolio et al.
In order to study the genetic variation in humans and the patterns associated
with it, as well as create a database of the same, the International HapMap
Project was designed. These patterns in genetic variation would be established
with the intent of studying common diseases and their genetic associations.
Forty common diseases were characterized, and 100 loci were identified for
these diseases. These preliminary results included genome-wide association
studies providing novel strategies for the development of several fields such as
pathophysiology. Undiscovered etiological pathways were identified for cer-
tain commonly occurring diseases suggesting that novel drug targets could be
recognized based on inheritance patterns and risks. The human genome is
often subjected to certain evolutionary pressures that have also been analyzed
through HapMap-based studies. These analyses have suggested that the
disease-causing pathogens and their adaptation to new environments is
influenced by multiple loci.
The goal of the International HapMap Project was essentially to provide a
foundation for genetic studies of human health and disease and to curate a
public, genome-wide database of patterns of common human sequence varia-
tion. The first draft of the human genome sequence was published in the year
2001, and the final draft was published in 2003. Immediately after this, the
HapMap Project came into picture as there was necessity to study and charac-
terize human genomic variation, including the millions of naturally occurring
single nucleotide polymorphisms (SNPs), and quantitate the frequencies of
these associations.
Genome-wide association (GWA) studies are advantageous because
genetic variations that occur in common diseases can be studied quickly
while also being cost-effective. The HapMap Project brought about a revolu-
tion in the field of GWA studies. There is a growing body of evidence that
suggests the need to investigate the role of evolutionary pressures on the
human genome and how it affects the inheritance of various diseases. It has
also led to important advances in estimation of genotypes specifically relating
to untyped SNPs in a nonredundant and unique fashion, while also aiding in
studies about population substructure (Fig. 15.31). Another major benefit of
HapMap is that the DNA and cell line samples are openly available along with
the consult and the process through which they were collected. This has
continuously contributed to genomic research and GWA studies.
15 Genomics 757

Fig. 15.31 In GWA studies, the various SNP-trait associations that were detected are depicted in
the figure. Involved neighboring genes, according to chromosomal location and associations that
showed a significance value of P < 9.9
107
758 S. K. AVS et al.

15.8.1 Building a HapMap for the Human Genome

In order to consider the ethical issues, carry out data analysis and genotyping,
formulate a scientific plan, select suitable diseases and SNPs that need to be typed,
and make the data publicly available, a consortium was created among researchers
hailing from various countries including the UK, the USA, China, Japan, Canada,
and Nigeria. This came to be known as the International HapMap Project. From four
populations with varying genetic ancestry, a human haplotype map was produced as
a product of this consortium by genotyping of 270 samples. These samples were
collected from people from whom specific consent was taken for the purpose of the
project and research on the same.
In the year 2005, after the completion of the phase 1 of the project, a description
of the one million SNPs that were sequenced was published. Subsequently, the
HapMap Project proceeded to phase II during which three million SNPs were
sequenced and the data published in 2007. Due to absence of polymorphism in
about 1.3 million SNPs out the 4.4 million originally selected SNPs, the former
could not be genotyped. Some did not pass quality control assessment. Centromeric
regions, telomeric regions, gaps in sequences, duplications, and insertions were
found to be quite challenging to study. These regions came to be known as “not
HapMap-able.”
This project ultimately led to the discovery of association patterns among SNPs in
the human genome, and the variation of these patterns across genomes was deter-
mined. In the four populations that were studied, the variation patterns showed a
level of similarity to a certain extent. Some populations such as the Yoruba popula-
tion (sampled from Nigeria) had relatively short haplotype blocks and less overall
LD. The regions that showed a higher LD value were similar in all the four
populations. The haplotypes displayed various degrees of diversity with the blocks
which also showed variation across the four populations.
Evidence from the data gathered strongly suggested that the selection of the tag
SNPs using HapMap played an important role and that they were transferrable across
other populations.
Exceptions to this include rarer SNPs. Due to small sample sizes, there were
various levels of differences in the determination of LD and allele frequency which
also proved to be a major limiting factor for the transferability of the HapMap-
derived tag SNPs. To avoid these errors and increase the accuracy, HapMap is being
developed with seven additional populations.

15.9 Summary

• The process of identifying functional elements along a genome’s sequence and

thus giving it meaning is known as genome annotation. It’s required because
DNA sequencing generates sequences with unknown functions.
• The study of how genes and intergenic regions of the genome contribute to
various biological processes is known as functional genomics. A researcher in
15 Genomics 759

this field studies genes or regions on a “genome-wide” scale (i.e., all or multiple
genes/regions at the same time) in the hopes of narrowing down a list of candidate
genes or regions to investigate further.
• The entire genetic composition of an organism constitutes to form its genome
which is classified into prokaryotic and eukaryotic genome based on the type of
organism and features of the genome. A prokaryotic genome is considerably
smaller in size than a eukaryotic genome and has a less defined nucleus. A
prokaryotic genome can be classified into several categories and are physically
diverse. The genome of prokaryotes is composed of a single replicon which
consists of its own replicator and initiator. Several terms have been used to
establish the classification of DNA molecules which might be present in the
genome that is multipartite in nature. Repeats in a prokaryotic genome are
known to influence important functions and are of high biological significance.
• The eukaryotic nuclear genome can be divided into two or more linear DNA
molecules, each of which is stored in a chromosome which is distinguished. All
eukaryotes are known to also contain shorter mitochondrial genomes that are
generally circular in nature. Plants are observed to have an additional genomic
structure, i.e., present in their chloroplast known as the chloroplast DNA. This is
unique to the eukaryotic genome of plants alone and cannot be identified in any
human or animal eukaryotic model.
• The smallest eukaryotic genome has been found to be shorter than 10 Mb in
length, and the largest eukaryotic genome is larger than 100,000 Mb. The
complete absence of corresponding link between the organism’s genome com-
plexity and its genomic size led to the theory of the C-value paradox.
S. cerevisiae is a common illustration that depicts this point.
• The presence of DNA-binding proteins known as histones is very crucial in
packaging the DNA molecules in a chromosome. The histone protein was
found to bind to the DNA molecule in a manner similar to the presence of
“beads on a string.”
• The Human Genome Project aims to interpret the human genome sequence,
define all of the genes found within it, and develop the research insights to
explore all genetic data that has been generated. As genomics technology
advanced, this three-billion-dollar, 15-year initiative progressed significantly
with the initial goal to create a human genetic map, followed by a physical map
of the human genome, and eventually a sequence map.
• Human genome maps have been generated on a variety of dimensions and
resolution levels among which genetic linkage and physical maps are utilized to
order the genes on each chromosome.
• The effectiveness of using end sequences from long segments (18 to 20 kbp) of
DNA cloned into bacteriophage lambda in the assembly of microbial genomes led
to the suggestion of using end sequences from 150-kbp bacterial artificial
chromosomes to simultaneously map and sequence the human genome (BACs).
• The human haploid genome comprises approximately 30,000 genes and is and
around three billion bp in length. Protein-coding sequences are the most
researched and well-understood part of the human genome. Genes that encode
760 S. K. AVS et al.

for noncoding RNA (e.g., transfer RNA and ribosomal RNA), untranslated
regions of mRNA, pseudogenes, introns, repetitive DNA sequences, regulatory
DNA sequences, and sequences linked to transposons are all examples of
noncoding DNA.
• The International HapMap Project’s mission is to identify the general
characteristics of variations of a DNA sequence within the human genome and
enable this knowledge publicly accessible. The HapMap will aid in the identifi-
cation of genetic variations that influence common diseases, as well as the
development of screening methods and the selection of targeted therapies.
• Human and chimp genomes underwent several changes after their ancestral
lineages diverged, including substitutions of a single nucleotide, duplications
and deletions of DNA fragments of various sizes, addition of mobile genetic
elements, and chromosomal rearrangements. As compared to the chimpanzee
genome, it was discovered that more than 95% of the NRNRs longer than 200 bp
were also existing in the genome assembly of the chimpanzee, implying that they
were ancestral.
• Mitochondria are cellular organelles with an extrachromosomal genome that is
derived from and distinct from the genome of the nucleus. The mitochondrial
DNA molecule is a circular dsDNA molecule consisting of about 16,569 bp and a
weight of 107 daltons that is five millimeters in diameter and free of histones.
Mitochondrial DNA studies can be employed in forensic human identification.
• The analysis of the variations and resemblance within the framework of the
genome and the arrangement of various species is known as comparative geno-
mics. The development of paralogs and orthologs is a crucial step in the evolution
of genes. It’s important to differentiate between orthologs and paralogs when
comparing genome organization in different species.

References
Collins FS, Fink L (1995) The human genome project. Alcohol Health Res World 19(3):190–195
Liao X, Li M, Zou Y, Wu F-X, Pan Y, Jianxin W (2019) Current challenges and solutions of de
novo assembly. Quantit Biol 7:90–109. https://doi.org/10.1007/s40484-019-0166-9
Application of Molecular Genetics
16
Dhruti Patwardhan and Nidhi Sharma

16.1 Biotechnology to Study Human Gene

Biotechnology plays an important role in the study of human genes. Techniques

from molecular biology can be used to identify genes associated with diseases and
the mutations involved in them. These techniques can be used for prenatal testing as
well. A sample can be taken from the amniotic fluid or fetal tissue to obtain DNA
which can be screened for mutations using recombinant DNA technology. This is
especially useful in cases where the affected protein cannot be detected in the early
stages because it is either produced in lower quantities or not expressed in fetal
stages. This allows us to predict if the newborn will be affected by certain diseases
that it may be susceptible to on the basis of family history.

16.1.1 Huntington’s Disease

Huntington’s disease (HD) is a neurodegenerative disorder which involves loss of

motor control, jerky movements, and decline in cognition accompanied by change in
personality and psychiatric symptoms. It is a rare disease, and symptoms of the
disease appear later in life between 35 and 40 years of age. It is inherited in an
autosomal-dominant manner. George Huntington was the first to describe this
disorder in detail in 1872. Since the symptoms appear later, many affected
individuals already have children and have passed on the genes to their offspring.

D. Patwardhan
Indian Institute of Science, Bangalore, India
N. Sharma (*)
La Sapienza University of Rome, Rome, Italy

# The Author(s), under exclusive license to Springer Nature Singapore Pte 761
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_16
762 D. Patwardhan and N. Sharma

There is a 50% chance of the offspring getting affected with the disorder as it shows
autosomal-dominant inheritance.
Huntington (HTT) was the first disease-associated gene to be mapped to the
human chromosome in 1983. It started with a group of scientists who worked on
finding a DNA probe that showed a specific restriction fragment length polymor-
phism (RFLP) pattern for HD. They tested 12 probes on Southern blot of chromo-
somal DNA digested with HindIII. One of the probes showed a specific RFLP
pattern for DNA from two families which had a history of HD. A large amount of
effort over 20 years was devoted to identifying these families and obtaining their
pedigree and medical histories. To identify where in the human DNA this
HD-specific probe was binding, researchers made use of a series of mouse cell
lines called human-mouse somatic cell hybrids. These cell lines were engineered to
contain a specific subset of human chromosomes. On hybridizing the probe to a
number of these cell lines, it was found that the probe recognized a region on the
fourth chromosome. They therefore concluded that the gene responsible for HD was
present on the human chromosome 4 and in the region to which the probe was
binding.
Over the next 10 years, efforts were put in to identify this gene and the nature of
its mutations. It was found that the gene had a trinucleotide repeat of CAG at the
beginning of the gene. In normal individuals, number of this repeat varied from 6 to
21. In affected individuals, this number was found to be greater than 40, even up to
100. The trinucleotide expansion was identified as the cause of the disease. Today,
molecular genetic approaches can be used to detect the number of CAG repeats in
the HTT gene. We can therefore predict if an individual will suffer from the disease
later in life and the chances of passing on the disease to their offspring.

16.1.2 Cystic Fibrosis

Cystic fibrosis is caused by a mutation in the cystic fibrosis transmembrane conduc-

tance regulator (CFTR) gene. Cystic fibrosis affects cells that produce mucous,
sweat, and digestive juices. It causes the fluids to become thick and blocks ducts
and passageways adversely affecting the lung and digestive system. It may cause
difficulty in breathing and persistent lung infections. It is inherited in an autosomal-
recessive pattern.
One of the most common mutations in CFTR is a 3 bp DNA deletion known as
508. To detect mutations in CFTR for the purpose of diagnosis, allele-specific
oligonucleotides (ASO) are used. These are probes constructed as an exact match
against the mutated allele or the normal allele. This probe will only bind to the allele
which is an exact match and will not bind even if there a single nucleotide mismatch.
To do the test, DNA is isolated from the white blood cells of the individual to be
tested. This is spotted on a nylon membrane and allowed to hybridize with both the
ASOs against normal and mutated allele under specific conditions which do not
allow a mismatch. In unaffected homozygotes, only the ASO against the normal
allele will hybridize. In case of carriers, both ASO against normal as well as mutated
16 Application of Molecular Genetics 763

Fig. 16.1 Genetic diagnosis of cystic fibrosis. Allele-specific oligonucleotides are prepared for
both normal allele (normal ASO) and allele having Δ508 deletion (D508 ASO). DNA is extracted
and blotted for parents and three children. Both ASOs hybridize for parents both of whom are
heterozygous. The affected child (child 1) shows hybridization only with D508 ASO. Child 2 is
homozygous for the normal allele and shows hybridization with only normal ASO. Child 3 is a
carrier and shows hybridization with both ASOs

allele will hybridize. In case of affected individuals, only the ASO against mutant
allele will hybridize. This is illustrated in Fig. 16.1. Although a powerful technique,
it suffers from an obvious limitation. The CFTR gene may have mutations other than
508, which will not be detected by this technique. Thus, a negative result on this does
not necessarily mean that the individual has no mutations in this gene. However, as
more mutations are identified and more genomic data is available, we will be able to
identify most if not all mutations that are present in the population and provide a
screening that has a better coverage.

16.1.3 Sickle Cell Anemia

Sickle cell anemia is a disorder in which the shape of erythrocytes is affected due to a
substitution mutation in the β globin gene. The abnormal erythrocytes become
elongated and curved resembling a sickle due to polymerization at low oxygen
764 D. Patwardhan and N. Sharma

tension. The normal erythrocytes are disc shaped. Aggregation of red blood cells
leads to oxygen deprivation to many tissues which might severely damage them.
The mutation in β globin gene eliminates restriction site for the enzymes MstII
and CvnI. These differences lead to a different restriction pattern seen on the
Southern blot for mutated allele and normal allele. This distinguishing feature can
be used to diagnose individuals having the mutated allele. The MstII restriction
enzyme has three sites in the normal β globin gene cleaving the gene into two
fragments. In the mutated allele, the site in the middle is lost cleaving the gene into a
single fragment. DNA can be extracted from an individual exposed to MstII diges-
tion, and the fragments can be separated by gel electrophoresis. This can be
transferred to a nylon membrane and visualized by Southern hybridization using
probes that recognize the fragments of β globin gene. Two small fragments indicate
that the individual is homozygous for the normal allele. A single large fragment
denotes that the individual is homozygous for the abnormal allele. A large fragment
and two small fragments indicate that the individual is heterozygous, having one
normal and one abnormal allele.
The difference in pattern of restriction digestion fragments produced can be
utilized here to diagnose individuals having sickle cell anemia. It can be used to
perform prenatal screening to test the genotype of the fetus and determine if he/she
will suffer from the disease. However, not all mutations will eliminate or create a
restriction site. The use of this technique is therefore limited.

16.2 Biotechnology to Study Plants

Plant breeders often select plants having favorable characteristics and use their seeds
for further breeding. This is nothing but manual selection of traits to ensure a more
robust crop with desirable traits. Biotechnology can be used for the same. Genes that
confer the desirable trait from different organisms can be isolated and introduced in
the genome of the plant of interest. We can increase nutritional value of crops, obtain
insect- and herbicide-resistant crops, and also increase their yield.

16.2.1 Transgenic Crops

Plants whose genome have been modified by genetic engineering techniques to

introduce a desirable trait are called transgenic plants. Vitamin A deficiency is a
major health concern especially in Africa and Southeast Asia. Vitamin A deficiency
may lead to blindness and also weakens the immune system. In these countries,
vitamin A-rich foods like milk, eggs, and fish are generally expensive and not within
the reach of the poorer sectors. Rice is relatively cheaper and is a staple diet in these
parts. Though rice is a good source of carbohydrates, it lacks micronutrients like
vitamins and minerals. Golden rice was created in 1999 with an intention to fortify
rice crop with vitamin A. This is called biofortification. The technology was
16 Application of Molecular Genetics 765

provided free of cost by its investors for use in the public sector rice varieties without
any limitations on use of recombinant crops and seeds.
Genetic engineering in rice plant allows it to biosynthesize β carotene which is a
precursor of vitamin A. β carotene is produced in the endosperm, giving the rice its
characteristic golden-yellow color. For this, two genes were introduced in the rice
plant, Phytoene synthase (psy) from daffodil and phytoene desaturase (crtl) from soil
bacterium Erwinia uredovora. These genes are placed under endosperm-specific
promoter. The introduction of these gene leads to the synthesis of lycopene which is
naturally converted to β carotene by the plant’s endogenous enzymes. In 2005,
golden rice 2 was announced which produced significantly higher amounts of β
carotene than the original golden rice. Golden rice received its first approval in 2018
from Australia, New Zealand, Canada, and the USA. Golden rice is a cost-effective
way to provide essential nutrients to a large population. It is also safe for
consumption.

16.2.2 Herbicide Resistance

A large portion of the crop yield is destroyed due to weed infestation. These
unwanted weeds can be removed by herbicides. The herbicides, however, may
also affect the crop plants. They may get washed into the water and deposited in
the soil which also adversely affects the environment. Herbicide-resistant plants will
protect plants from herbicides while allowing surrounding weeds to be destroyed.
Glyphosate is an herbicide which has the ability to kill plants by inhibiting an
enzyme called EPSP synthase which is present in the chloroplast. This enzyme is
important for the synthesis of essential amino acids, and without this enzyme, plants
are unable to survive. This herbicide does not affect humans and is effective at low
concentrations. It is also rapidly destroyed by soil microorganisms. EPSP synthase is
also present in bacteria and essential for their survival. There is a strain of E. coli that
is however resistant to glyphosate. The EPSP synthase gene of this resistant strain
can be used to confer resistance against glyphosate in crop plants. To do this, the
EPSP synthase gene from glyphosate-resistant E. coli was cloned into a vector under
a plant viral promoter sequence and upstream of the plant’s transcription termination
sequence. This recombinant vector was introduced into the bacterium
Agrobacterium tumefaciens.
Discs were cut out from plant leaves and infected with Agrobacterium
tumefaciens carrying the vector. Due to infection with bacteria, the plant tissue
developed calluses which consists of unorganized plant parenchymal cells. These
callus cells were tested for their ability to resist glyphosate. The calluses which were
able to survive were further cultured and grown into transgenic plants. These plants
were exposed to high concentration of glyphosate, and only those plants which were
able to produce the resistant EPSP synthase gene in high quantities were able to
survive and the others died.
Glyphosate-resistant corn and soybean have been created in this manner and are
available in the USA and other countries since its introduction in 1996.
766 D. Patwardhan and N. Sharma

Unfortunately, persistent use of herbicides due to resistant crops have now led to the
evolution of herbicide-resistant weeds. Weeds are developing resistance
mechanisms for a large number of herbicides. We therefore need more studies
pertaining to understanding evolution of resistance and sustainable solutions
addressing these issues.

16.2.3 Insect Resistance

Just like weeds, insects also impact crop production to a large extent. With growing
human population, it is essential to optimize our food production to meet the
demand. Creating insect-resistant crops have led to an increase in overall yield of
crops like corn, potato, and cotton. It has also cut down on the use of insecticides
which may have harmful effects on humans.
Genetically modified crops containing δ-endotoxin also known as Cry proteins
from Bacillus thuringiensis (Bt) were introduced in the mid-1990s. Most of the
recently produced insect-resistant strains contain multiple Cry proteins which are
toxic to Lepidoptera and Coleoptera species. The Bt toxin when ingested by the
insect is solubilized in the midgut where it gets proteolytically cleaved at the N
terminal to an active form. The active molecules bind to a receptor in the epithelial
cells of the midgut. This induces formation of pores in the membrane resulting in
osmotic lysis and cell death which eventually kills the insect. In the transgenic
plants, Bt toxin is expressed directly in its active form. Transgenic insect-resistant
crops have had a major beneficial impact on agriculture by improving crop yield and
reduction of pests.

16.2.4 Production of Biofuels

With rapidly depleting sources of fossil fuels and its negative impact on the
environment, it is essential to look for alternate sources of energy. Biofuels are
one such source which can fully or partially replace the use of fossil fuels. The first
generation of biofuel utilized sugarcane or sugar beet for production of ethanol
through fermentation. Different alcohols, for example, butanol, can be produced by
applying different fermentation process. The first generation of biofuels utilized
crops which can also be used as food or feed, thereby increasing their demand and
creating shortage of food crops. With the advancements in biotechnology, second
generation of biofuels were developed which reduced reliability on food crops.
Lignocellulosic biofeedstock was used for this which was a less expensive biomass.
The cellulose and hemicellulose present in this biomass is broken down into its
constituent simple sugars through enzyme-catalyzed hydrolysis. These are then
fermented with the use of microorganisms to produce alcohol. These fuels are also
known as cellulosic ethanol or cellulosic biobutanol. Newer biofuels now referred to
as the third generation of biofuels are produced by using algae biomass with
microbial enzymes. Microalgae offer many advantages because they are able to
16 Application of Molecular Genetics 767

rapidly double their biomass. They also are rich in oil, and some of them have oil
content about 80% of their dry weight. They can also be grown in waste or
non-potable water. This solution also has its own problems. It requires a high density
of algae culture over large surfaces which is not very optimum. Apart from algae,
other prokaryotes or eukaryotes which show high accumulation of oils are also being
considered for production of biofuels.
There are multiple stages at which use of biotechnology can increase efficiency of
production of biofuels. Microbial enzyme activity can be enhanced and improved via
biotechnology to achieve better microbial digestion and fermentation of biomass.
The use of genetically modified organisms for pretreatment or conversion to ethanol
can boost productivity. The cell wall and composition of lignocellulose in plants
being used for production of biofuels can be modified using biotechnology to
increase the yield of ethanol. Using herbicide- and insecticide-resistant plants can
improve the biomass produced. Creating plants that can survive and grow in harsh
soil or weather conditions will allow plants meant for biofuel production to be grown
on nonarable land. This will allow arable land to be used only for crop production.

16.3 Biotechnology to Study Pharma Product

Biotechnology can make use of the fact that all organisms contain the same
nucleotides and follow the same code for its conversion to proteins. This allows a
human gene to be inserted into a plant or a bacteria to produce the protein of interest.
The bacteria or plants can then be cultured on a large scale, and protein of interest
can be isolated and purified. Thus, the bacteria are acting as molecular factories
producing the protein of interest on a large scale.

16.3.1 Recombinant Insulin

Insulin became one of the first human protein to be produced using recombinant
DNA technology and licensed for therapeutic use in 1982. Insulin is required in the
body for glucose metabolism, and lack of insulin leads to diabetes. Insulin can be
given to diabetics externally which allows them to maintain their blood glucose
levels. Insulin is produced by cells in the pancreas in the form of a precursor peptide
known as preproinsulin. This gets cleaved, and some amino acids are removed from
its center and at its end. This leads to formation of two polypeptide chains called A
and B which are held together by disulfide bonds.
Before the use of rDNA technology, insulin was produced by extracting it from
porcine or bovine pancreas. There were two issues associated with this method. One
was that although animal insulin was chemically similar to human insulin, it is not
identical. This difference led to immune reaction from patients causing inactivation
of insulin and inflammation in many patients. Secondly, production of insulin from
animals was very expensive and difficult to obtain in large quantities. Both these
issues were prevented by using bacteria to produce human insulin. Since
768 D. Patwardhan and N. Sharma

posttranslational modifications differ between bacteria and humans, the insulin gene
was not inserted as is in E. coli. Instead, the polypeptides were synthesized sepa-
rately using two different plasmids. Polypeptide chain A has 21 amino acids, and
polypeptide chain B has 30 amino acids. The genes for these two chains were
constructed using oligonucleotide synthesis (Fig 16.2). The genes were inserted in
the vector adjacent to a lacZ gene to produce fusion proteins. The fusion proteins
consisted of polypeptide A or B fused to β galactosidase (product of lacZ gene). The
vector also contained a gene for antibiotic resistance which was useful in selection of
bacteria containing the vectors. These recombinant bacteria were cultured in large-
scale fermenters. From the bacterial extracts, fusion proteins were isolated and
treated with cyanogen bromide to remove the β galactosidase (Fig 16.2). The insulin
chains were then purified and mixed. The chains were able to spontaneously unite to
form the active molecule. This insulin was capable of being purified, packaged, and
used in therapy.

16.3.2 Recombinant Growth Hormone

Human growth hormone is produced in the body by the pituitary gland and released
into the blood. It performs a host of biological functions like metabolism of proteins,
carbohydrates, and lipids as well as cell proliferation and immune regulation.
Growth hormone is essential to ensure proper growth and stature, and its deficiency
may lead to dwarfism. Dwarfism can be treated by administering growth hormone. It
is also used in the treatment of burns, bone fractures and disintegration, and gastric
burns. Till mid-1980s, the only source of human growth hormone was human
cadaver tissue. The supply for this hormone was therefore limited. There were also
reports that associated pituitary-derived growth hormone with Creutzfeldt-Jakob
disease. Recombinant DNA technology provided a means of safely producing
abundant amounts of human growth hormone (hGH).
hGH is produced by the pituitary as a prehormone. It contains a hydrophobic
leader peptide of 20 amino acids. During secretion, this leader peptide is removed to
produce the mature hormone of 191 amino acids in length. To facilitate the direct
expression of mature hormone, cDNA coding for the leader peptide was removed.
The cDNA for growth hormone was partially chemically synthesized and partially
derived from the actual mRNA of human pituitary. This was significant because
unlike insulin, which is only 51 amino acids long, chemically synthesizing entire
mRNA for such a large protein like hGH would have been difficult. This cDNA was
cloned into a plasmid which was introduced into a strain of E. coli. Since
nonglycosylated form of the hormone was active, prokaryotic system was preferred
for its production. The recombinant bacteria is grown in large quantities, and growth
hormone produced was isolated and purified.
16 Application of Molecular Genetics 769

Fig. 16.2 Production of recombinant insulin. The genes coding for polypeptide chains A and B of
insulin are inserted in a bacterial plasmid fused to the lacZ gene. The plasmid is transformed into
E. coli and cultured in large-scale fermenters. The fused lacZ/insulin A or lacZ/insulin B fusion
protein accumulates in the cell from where it is extracted and purified. It is further treated with
cyanogen bromide to separate insulin from β galactosidase. The A and B chains are purified and
mixed to form the active insulin protein

16.3.3 Recombinant Vaccine

Traditionally, vaccines have been produced by either killing or attenuating the

pathogen. This inactivated pathogen is no longer able to cause the disease but
stimulates the immune system to produce antibodies against it, providing protection
against future infections from the pathogen. Using recombinant DNA technology,
only the peptide that acts as the antigen can be produced which can elicit an immune
response. These are called subunit vaccines. They provide protection against
770 D. Patwardhan and N. Sharma

possible risk of live or attenuated vaccines like reversal of attenuation and virulence
in susceptible hosts. Additionally, the recombinant proteins can be produced in large
quantities.
Recombinant protein vaccine currently in use is against hepatitis B. Hepatitis B
virus (HBV) infects liver cells causing chronic infection and cirrhosis. The hepatitis
B surface antigens (HBsAg) are produced in yeast expression system. Yeast cells are
capable of making posttranslational modifications in proteins. Protein products that
require glycosylation can therefore be produced in the eukaryotic yeast. It also
secretes the HBsAg into the supernatant of the culture allowing for easier purifica-
tion. The HBsAG when administered assemble into viruslike particles which are
highly immunogenic and capable of eliciting an immune response. Recombinant
vaccine against human papillomavirus (HPV) has also been developed which
contains the L1 major capsid protein. Many subunit vaccines, however, have weak
immunogenicity on their own and need to be administered with an adjuvant to
promote long-lasting and strong protective immune response.
Genetic engineering can also be used to create live recombinant vaccines. The
idea is to use a live recombinant vector containing heterologous antigen encoding
genes. The live vector can elicit a strong immunological reaction against its own
antigens as well as toward the heterologous antigens being expressed. An example is
the work being done on recombinant BCG vaccine. The vector M. bovis BCG
provides many advantages. It is safe and can elicit T-cell-mediated immunity.
Recombinant BCG (rBCG) expressing foreign antigens for various diseases like
malaria, tuberculosis, and HIV is being developed. For example, rBCG expressing
HIV antigens has been shown to produce specific antibodies against HIV, produce
interferon γ, and induce T helper and cytotoxic T cells. Efforts are also being used to
utilize viral vectors for expression of heterologous antigens.
Direct injection of DNA plasmid into the muscle to induce immune response is
also another approach that is being studied as a vaccine system. In a DNA vaccine
system, the antigen can directly be expressed by host cells in a manner similar to
viral infection. They have been shown to elicit both humoral and cell-mediated
immunity. DNA vaccines avoid problems associated with producing recombinant
proteins like inaccurate folding of protein and purification costs. DNA vaccines,
however, have their own set of problems like low efficiency of transfection of cells
in vivo, production of anti-DNA antibodies, and possible integration into host
genome. Although successful in animal models, DNA vaccines have shown limited
immunogenicity in primates. Ongoing efforts in increasing its effectiveness include
strategies like augmenting gene expression, co-expression of cytokines and other
molecules that boost immune response, and formulations to protect DNA from
degradation.
Even after production of purified vaccine, there are challenges involved in
administering the vaccines especially in developing countries. Absence of facilities
for manufacturing, transportation, and storage pose challenges for vaccination in
remote places. To circumvent these issues, creation of edible vaccines was proposed.
Edible vaccines are transgenic plants or animals that express the antigen of a
pathogen and, when consumed, can elicit an immune response in the body. These
16 Application of Molecular Genetics 771

vaccines would provide the advantage of being inexpensive, not require special
storage conditions, as well as not require trained medical personnel for administra-
tion. Transgenic tobacco plants having leaves expressing antigenic subunit of
hepatitis B virus have been produced. This is just a model system, and for actual
use, the gene for HBV would be transferred into a food plant. In another example,
rabies antigen was expressed in spinach and fed to volunteers. Eight of the fourteen
volunteers showed high expression of rabies-specific antibodies. Edible vaccines are
undergoing further studies and clinical trials.

16.3.4 Recombinant Protein

The proteins mentioned above including recombinant insulin, recombinant growth

hormone, as well as the vaccines are examples of recombinant proteins. The first
recombinant protein was somatostatin produced in 1976. From there, a host of other
proteins have been cloned and expressed in heterologous systems from bacteria to
plants which are widely used in therapy today. Recombinant proteins provide the
advantage of producing large number of products at lower cost. It is also safer as it
avoids transmission of infection from animal- or cadaver-derived products.
The X-linked disorder hemophilia occurs due to a lack of clotting factors.
Hemophilia A is caused by a defect in factor VIII, and hemophilia B is caused by
a defect in factor IX. For a long time, plasma-derived clotting factor concentrates
were used for the treatment of hemophilia. However, due to lack of proper screening
methods, they were discovered to be also transmitting blood-borne viruses including
HIV and hepatitis. In 1984, the gene for factor VIII was successfully cloned, paving
the way for its production using recombinant technology. By 1992, factor VIII was
commercially produced and licensed for therapeutic use. Factor IX was commer-
cially available for people with hemophilia B in 1997. These recombinant proteins
provided a safe and effective method for treatment of hemophilia. Table 16.1 lists the
recombinant proteins used in clinical treatments.
A range of recombinant proteins including hormones, antibodies, enzymes, and
vaccines have been produced which have been used for therapy in certain diseases.

16.4 Biotechnology to Study Animals

Applying novelty of molecular genetics in the field of biotechnology, the study of

animals has become one of the important branches of study that includes a vast area
of related topics such as animal health, animal in research, transgenic animal, gene
pharming, etc. Animal biotechnology is an important branch of biotechnology that
includes a wide range of topics such as use of animal in research, clones, transgenic
animal, gene pharming, and animal health. In the past history of human beings,
animals have been bred to the enormous purposes over 1000 years, such as breeding
of (i) working dogs to flock pasture animals, (ii) cows produces more tender and
tasty meat, (iii) horses that are easier to tame, and so on. All these things have been
772 D. Patwardhan and N. Sharma

Table 16.1 List of recombinant proteins and their therapeutic applications

Proteins Therapeutic application
DNase I Cystic fibrosis
Coagulants factors Hemophilia A and B
Erythropoietin Anemia in chronic renal disease
Glucocerebrosidase Gaucher disease
Growth hormone Pituitary dwarfism
Insulin Diabetes
Alpha interferon Some leukemias, Kaposi’s sarcoma, and hepatitis B
and C
Gamma-1b interferon Chronic granulomatous disease
Interleukin-2, interleukin-3, and Immunotherapy of cancer
interleukin-4
Tissue-type plasminogen activator Acute myocardial infarction and massive pulmonary
embolism
Antibodies for cellular immunotherapy Neoplastic processes
Vaccines Influenza and hepatitis A and B
Monoclonal antibodies anti-antibodies Lupus and rheumatoid arthritis

possible by the use of biotechnology. Notably, animal biotechnology is the core

result of genetic engineering which includes recombinant technology for transferring
exogenous or foreign DNA into germ line. Genetic engineering is a modification of
an organism’s characteristic by adjusting its genetic material.

Use of Animals in Research

Animal models are an essential part of primary research. The need of medical
assessment of various drugs or products produced for disease treatment is supported
by the use of animal models. It has been noticed that working on computer models
and in vitro cell studies would not reproduce the result as an organism does. Thus,
working with computer models and cells will be a supplement to animal research.
Regardless of initial failure, recent development in animal biotechnology has
changed the fate of agriculture, medicine, and animal breeding, and a prompt effort
to conserve the endangered animals to save the flora and fauna is remarkable.
It’s very well known that for any new product to be approved for human use, the
manufacturer must first demonstrate that it’s safe, and therefore, trials on cell culture,
in live animals, and on human subjects are facilitated by virtue of this field. The most
required animal used for testing is a pure-bred rat, mice, primates, etc., prior to trial
on human subjects. Zebra fish, a hardy aquarium fish, is extremely used for valuable
research, while dogs are enormously used for study of cancer, heart disease, and lung
disorders. Importantly, HIV and AIDS like lethal studies are always conducted on
monkey and chimpanzees.
16 Application of Molecular Genetics 773

16.4.1 Transgenic Animals

Transgenesis (transfer of significant genes) or transgenic animal is one of the most

significant and exciting research tools in biotechnology. Through this advanced
technology, inserting new genes in livestock for economical important
characteristics such as fertility, resistance, or tolerance to the environmental stress
has become a major revolution in animal breeding. Another significant application
that transgenic animal brought to us would be producing clotting factors in the milk
of domestic livestock.
To elaborate the literal definition of transgenic animal is a deliberate modification
of genome of one animal by introducing DNA through recombinant DNA technol-
ogy and then must be transmitted to the germ line that homogeneously spread to
every cell and pass over through the next generations. It means that gene can be
altered artificially so that some characters of animals are changed and can pass to all
offspring.
In modern history, fundamental to these techniques is an ability to culture early
embryos in vitro which allowed a variety of manipulations in the genome to be
performed. One of the first genetic manipulations of the embryo is the production of
chimeric mice achieved by mixing an early stage of development (eight cells) to
develop a single form of embryo carrying chimeric characteristics in adult. Likewise,
knocked-out mouse is also another great achievement in the history of biotechnol-
ogy. First ever knocked was created by Mario R. Capecchi, Martin Evans, and Oliver
Smithies in the year 1989 who won the Nobel Prize in 2007 for introducing knockout
mice technique. Knockout generally refers to a deletion (knockout) of a gene to
inactivate its function.

Reasons that Promote the Production of Transgenic Animal

1. Some transgenic animals are being produced, keeping the goal in mind for
specific economic traits which can produce milk containing particular human
proteins like protein C (a potential coagulant) and fibrinogen (plasma
glycoprotein).
2. The primary goal was to produce advanced cattle breed which is subsequently
followed by the second goal of production of disease model. This disease model
shows disease symptoms as the result of transgenesis. This model has been
manufactured to study several disease studies. For example, OncoMouse® and
3
Tg AD mice are produced to study various human cancer and Alzheimer’s
disease. These animals successfully show the symptoms of the disease alike
humans.
3. One ultimate purpose is to study the interaction between environment and
genome. This can be possible with transgenesis that provides a unique opportu-
nity to design unique models for such study.

Methodology for Producing Transgenic Animal

To date, there are several methods in used, but the most efficient methods are as
follows:
774 D. Patwardhan and N. Sharma

(a) Recombinant retrovirus.

(b) Embryonic stem cell.
(c) Pronuclear DNA microinjection.

Recombinant Retrovirus: Recombinant retrovirus technique is mostly useful for

eukaryotic genome. Retrovirus is a family of virus in which genetic material is RNA
and transcribed into DNA by specific enzyme “Reverse Transcriptase (RT).” Of this
significant characteristic, RT becomes an essential tool for cloning or transgenesis of
eukaryote genome. Eukaryote genome cannot clone in directly bacterial cell because
it contains additional introns, which make the genome heavy and lengthy which
cannot transfer to the bacterial cells. Thus, mRNA from the desired gene will transfer
to this virus which later will be transcribe into single-strand complementary DNA
(cDNA) by RT. DNA polymerase will transcribe the second strand, complementary
to the first strand to complete the DNA amplification. A direct gene transfer to
retrovirus will be followed by injecting virus to the host cell and cultured to maintain
the cell line, and cultured cell will develop to a mature embryo. Retrovirus method is
mainly used to transfer genetic material into host cell to produce the chimeric
characteristics of transgenic animal containing diverse genetic constitution from
the donor.

Embryonic Stem Cell

This is one of most common techniques and often being used to produce successful
transgenic animal. This method is first initiated with isolation of totipotent cell (a cell
that has a capacity to develop into any specialized cell) from the embryo. The gene of
interest is being inserted in vitro into these cells (totipotent) and then incorporated
into the host embryo resulting in a chimeric animal (Fig. 16.3).
DNA of interest is isolated and injected to the embryonic stem cells by gene
delivery method, and cells were cultured and maintained carefully. This embryonic
stem cell containing DNA is injected to the blastocysts and implanted to the uterus of
foster mother. The first generation will be heterozygous, and mating with wild type
will produce the homozygous transgenic strains.

Pronuclear DNA Microinjection

First time, this technique was described by Gordon et al., and mice were the first
transgenic animals produced through this method. Male and female pronuclei
(nucleus of sperm or egg cell) are microscopically spotted after immediate hours
of sperm entry into the cell.
The gene of interest or cluster of recombinant genes in a construct will be
subjected to microinject into either of these pronuclei (Fig. 16.4). This manipulated
cell is first needed to be cultured in vitro (in a lab, not in an alive organism) to
develop to a specific embryonic phase, which is further transferred to the recipient
female. The recipient female is always a pseudopregnant that promotes the hormone
stimulation in the body to make her uterus receptive to the embryo that has to be
transplanted in it. However, transfer of gene by DNA microinjection would not be
16 Application of Molecular Genetics 775

Fig. 16.3 Embryonic stem cell-based transgenic method

homogeneous since it is a random process, so it’s not necessary that all pups will be
born with expressed desired gene.

Application of Transgenic Animals in Human Welfare

Disease Model: For ages, mice model has been depicted as potential source for
studying human disease since mice shares physiological, anatomical, and genomic
characteristics with human. To study most effective disease such as cancer, AIDS,
and Alzheimer, transgenic animals have been produced to exhibit similar symptoms
in order to understand the basic fundamentals of these disease. In short, transgenic
animals enable scientists to understand the role of genes in specific diseases.
Growth. Transgenic pigs and sheep are being produced with increased growth
and heavy body composition. This attempt had been achieved by transfer of gene
that regulates growth hormone. Delivery of proteohormone (peptide hormone) made
it possible.
Quality of Animal Products. Improved quality and composition of animal
products can be achieved by respective gene transfer. A model for this hypothesis
is being proposed by Mercier in the year 1987. He proposed a model that is lacking
lactose content in the milk on purpose. Practically, sheep and cattle had been
produced carrying lactose gene but combined with udder specific promoter. This
construct results lactose degradation into the end product. Thus, milk lacking lactose
will be useful for a large population that are suffering with lactose intolerance.
776 D. Patwardhan and N. Sharma

Fig. 16.4 DNA microinjection in pig embryo to inject transgene (image taken on scale bar 20 μm).
A DNA construct or recombinant construct of transgene was prepared and injected to the pronuclei
(nucleus either egg or sperm cell) with needle or microinjector. This is further followed by
cultivating into matured embryo and implantation to the foster mother

Researchers also have produced transgenic animals that promote cysteine synthesis
(an essential amino acid) in the animals that enhance the woo growth particularly.
Gene Pharming. Pharming seems a misspelled word for farming, but it isn’t at all.
This word comes from two different word “farming” and “pharmaceutical.” Thus,
pharming denoting here is the production or farming of significant genes or proteins
by means of secretion in the transgenic animal’s blood, milk, saliva, eggs, etc.
Altering the gene makeup (modifying its own DNA or splicing) of an animal through
transgenesis or transfer of a particular gene for production of valuable proteins for
human purpose leads to the idea of gene pharming. In this direction, tissue-specific
promoter inducing the protein production in domestic animal is a reliable source for
human needs. Therefore, remarkable efforts have been made by scientists, in partic-
ular direction to use animals as bioconversion system.
In the year 1987, Gordon et al. and Simons, McClenaghan, and Clark success-
fully demonstrated that human T-PA (tissue-specific plasminogen activator, to treat
the blood clotting) and sheep beta-lactoglobulin were, respectively, expressed in the
milk of transgenic mice.

Industrial Applications
Two scientists from Nexia Biotechnologies, Canada (2001), had spliced a spider
gene into the cells of lactating goats. Eventually, they observed that goats started to
16 Application of Molecular Genetics 777

produce silk in a form of tiny strands from their body along with their milk. The
amount of silk was quite enough to commercialize it. This strand was subjected to be
extracted and weaved into thread that would be useful for manufacturing objects like
military uniform, tennis racket strings, etc.
In 1997, the first cow “Rosie” was produced as a transgenic cow that secreted
protein-enriched milk at 2.4 grams per liter. This cow was more nutritional than
normal bovine milk. The milk was containing human gene “alpha-lactalbumin.”

Ethical Issue
Beside its application in human welfare, transgenic animals had faced many bioethi-
cal issues raised by environmentalist and activist and cannot be ignored by scientist,
biotechnology industry, policy maker, and public domain. Those ethical issues and
doubts we tried to sum up here are as follows:

1. Why transgenesis does not have any universal or standardized protocol?

2. Should only promising research permit to demand such protocol?
3. Why transgenic animals have to be taken into consideration of human
welfare only? What about lab animal welfare or other forms of life?
4. Should transgenic method first needed to be examined on in vitro (cultured cells
in the laboratory) before using live animals to reduce animal surfing?
5. Do transgenic animals provide an evolutionary benefit in the right direction or
instead will result in a drastic consequence for nature and humans?
6. Should patent policies be legitimate to restrict the free exchange of scientific
research?

16.4.2 Improved Reproductive Rate

Animal reproduction through animal biotechnologies have endowed many

improvements in agriculturally important traits and livestock in which production
of development of genetically improved animals for farming is the primary concern
for researchers. The very primitive and first biotechnology tool applied to improve
the production was artificial insemination as it is well known that reproductive
success is of topmost importance for economic efficiency of cattle production in
animal pharming. High productive efficiency is required for efficient milk produc-
tion and meat production and therefore has an influence on herd profitability. Among
the most recent of these emerging technologies, reproductive cloning and production
of transgenic animals are the best choice to improve the reproductive rate in animals.
Nevertheless, the other novel techniques include synchronization of estrus, in vitro
fertilization, multiple ovulation (female released more than one egg in a month by
intrauterine insemination), embryo transfer, and cloning, and they are all important
potential tool for improvement of livestock reproductivity rate. Researchers have
found that it is important to avoid the risk and challenges that impede productivity,
reproduction rate, and health with adverse environment conditions. This concern has
778 D. Patwardhan and N. Sharma

been prioritized in animal reproduction system that results in encouragement of the

practice of techniques mentioned below.

Estrus Synchronization
This is a technique related to regulate the estrus synchronization of female. Estrus
synchronization is basically a manipulation of heating time to reduce for a short
period (36 to 96 hr). Such synchronization can be achieved by using of one or more
hormones. This technique is one of the competent methods that increase the possi-
bility of animals to breed at the beginning of breeding season.

Artificial Insemination (AI)

AI has been practiced worldwide on large scale from more than half of the century.
This is one of the most common and efficient method among all. The purpose of this
technique is to determine an efficient bull with maximum fertility rate. It has been
proven by several studies that seminal plasma contains fertility-associated antigen
which defines the differences in fertility rate between males whose seminal compo-
sition is the same though. To this reason, even every bull comprised the same
amount and composition of semen but may affect the sperm capacitating, fertiliza-
tion, or related events. Because of technology boom, in recent years, sperm collec-
tion and AI have been improved by the advent of sperm sexing or selection which
here refers to a possibility to select the sperm carrying X chromosome or Y
chromosome with 85–95% of accuracy. This method is common in some domestic
animals such as buffalos. At practical ground, this technique is facilitated by using
cutting-edge flow cytometric principle, where fluorescent-labeled X chromosome-
bearing spermatozoa can separate from the fluorescent-labeled Y chromosome-
bearing spermatozoa. In addition to this, the production of more male progeny and
the reduction of sex-linked disease are the most beneficial outcomes of this
approach. However, this technique has some limitations including reduced number
of sexed sperms and variety of damage to the sperm cells, viz., sperm membrane
destabilization. Changes in the competence, thereby, life span of spermatozoa would
reduce after fertilization in female genital tract.

Embryo Transfer
This technique is one of the tools which provides a faster rate to livestock and an
opportunity in which both male and female has contributed equally. This method
involves superovulation which is an important step to increase the oocyte number
from the superior donor. The first mammalian embryo transfer was reported by
Walter Heape in 1890, while the first birth of calf through this method was reported
by Betteridge. The first live calves developed from bubaline embryos (using embryo
transfer method) were born in 1983 in the USA and later in India.
The essential stages for this method are as follows:

• Donor cow of good pedigree animals and treated with hormones (FSH and LH) to
stimulate ovulation and release eggs in large number—multiple ovulation (MO).
• Insemination is performed using semen of a chosen bull.
16 Application of Molecular Genetics 779

• Embryos are flushed nonsurgically after 6–7 days of insemination.

• Collected embryos are implanted to the recipient cows whose estrus cycle is at the
correct receptive stage due to hormone manipulation.
• Embryo may be frozen and stored for a long time.

In India, embryo transfer and protocols of ET technology are being standardized

for cattle, buffalo, sheep, goat, camel, and other species of animals. Embryo transfer
is an effective method, and genetic improvement is higher in embryo transfer than AI
alone.

In Vitro Fertilization (IVF)

In vitro fertilization is an artificial technique to fertilize the oocytes outside and
transfer the developed embryo to the recipient mother. The oocytes can be collected
either from slaughterhouse or live animals. This oocyte undergoes the maturation
and fertilization in vitro and later will develop into mature and viable embryo.
“Pratham” was the first IVF buffalo calf produced in India in 1990. Through this
technique, it is now possible to study development process, gene expression, epige-
netic modification, and cytogenetic disorder in various species and also provide a
model for study of embryogenesis in human. In this method, unfertilized eggs are
first fertilized in the laboratory and cultured for a few days until they have success-
fully developed into early embryos. This embryo is now ready to be implanted in the
recipient uterus through a long syringe. It is important to notice that the recipient
mother should be in receptive stage of estrus cycle before the implantation. The first
live offspring achieved from IVF technology was a rabbit.

Cloning
The literal meaning of clone is an identical copy of any organism or tissue or cell
which contains identical copies of genetic information. In cloning, it is possible to
reproduce an entire organism from any cell taken from parent organism, and resulted
clone is an identical copy of parental organism in every means. The genetic
composition of clone is identical to its donor (or parental organism). The main
objective of cloning is to increase the number of identical copies of superior
livestock to produce high-quality end product although cloning does not change
the genetic makeup of the animal.
In nature, cloning is quite usual and frequent. For instance, naturally occurring
asexual reproduction in some lower eukaryotes and prokaryotes is similar to those
twins reproduced from one fertilized egg.
The major breakthrough in cloning world had appeared in 1996 when Ian Wilmut
and his colleagues have successfully produced a clone of sheep named “Dolly”
(Fig. 16.5). Dolly was produced from fertilization between cultured adult somatic
cell as a donor and enucleated oocyte recipient cell (lacking chromosomal DNA).
The cloning process includes the following steps: (i) Chromosomal DNA will be
removed from a mature oocyte. (ii) This egg nucleus will be replaced by the somatic
cell nucleus from the donor which is supposed to be cloned. The donor cell now will
be fused with enucleated oocyte, and reprogramming of somatic cell genome will be
780 D. Patwardhan and N. Sharma

Fig. 16.5 Cloning method to produce “Dolly” sheep. Enucleated oocyte is fused with somatic cell
by electric shock. Fused cell developed into fertilized egg and matured embryo, followed by
implantation into foster mother. This embryo has a genetic information identical to the donor cell
and appeared into Dolly sheep which is a clone

activated as of embryonic genome. Activation of reprogramming can be induced by

chemical or electrical pulse. Reconstructed cloned embryo is further cultured, and
this viable embryo is transferred to synchronized recipient which carries the live
cloned offspring until parturition.
In addition to the genetic cloning of valuable animals, it is also possible to
generate the genocopies of exceptional animal from those who are incapable of
reproduction. For example, in the case of steers or animals that are dead, cells can be
stored.

16.4.3 Improved Health

At the global level, increasing figures of population simultaneously are adding to the
requirements of overproduction of high-quality protein, meat, milk, and eggs as
essential needs for life, but sustaining a good health and protection from adverse
environmental factors which can affect the livestock’s longevity are quite question-
able and the primary concerns to be taken. Apparently, biotechnology plays a
significant role in the diagnosis of livestock diseases and genetically transmitted
conditions which reduce animal’s health and productivity drastically. Nowadays,
16 Application of Molecular Genetics 781

advanced biological technique produces cheaper and more efficient drugs because
natural drugs from natural source materials are excessively expensive. In this
scenario, drug production utilizing applications of genetic engineering in either
microbial or tissue culture system has become a wise decision eventually in favor
of human and animal’s health. Largely produced human insulin, human growth
hormone, and plasminogen activator (used in treating heart disease) are the biggest
success in animal biotechnology.

16.4.3.1 Vaccines
Immune system of animals is induced by arrays of vaccine to produce antibodies
targeting disease or infection. Emerging recombinant DNA technology has
introduced the possibilities to develop a recombinant antibody and vaccines com-
mercially available at low cost. Empirical knowledge in vaccine development and
relationship with immune system makes scientist and industries to produce massive
range of vaccine that can perform better to boost the body’s immune system than the
conventional vaccines. Data from the trial history suggested that these engineered
vaccines are way safer than the traditional vaccines which may develop the “revert
effects” (inactive non-virulent can revert into virulent and cause disease). Therefore,
such genetically engineered vaccines have been developed to eliminate this threat to
animal health.
Biotechnology industry is booming day by day to produce entirely new
engineered vaccines and their new ways of uses. Vaccines have been developed
for many purposes, i.e., modulator for growth hormone to increase the growth rate,
additives as a feed conversion, stimulator in milk production, enhancer to improve
animal carcass and meat quality, and modulator in reproduction system to enhance or
to suppress the reproduction rate.
Recombinant or engineered vaccines are useful for those diseases for which
vaccine has been not developed. These vaccines do not contain the dangerous
infectious agent unlike the traditional vaccines, and this property makes these
vaccines efficiently safer. Production of vaccines are considered as less expensive
with mass production, and maintenance cost is negligible since it can be stored even
at room temperature.

16.4.3.2 Diagnosis
Examination of poor health in cattle, pets, and other domestic animals is an addi-
tional responsibility for farmers and biotechnologist. Improvement in diagnosis
methods or in tools makes the situation under control for many poultry firm running
around. Nearly a decade, scientists from Japan and Taiwan became in spotlight when
they invented the DNA to detect the hereditary weakness in poultry pigs during
transportation or in slaughterhouse. This test has identified the gene expression
associated with “porcine stress syndrome” in pigs. They observed that pig with
this gene in active state produce pale and poor quality of meat. Now, it has become
an easy job for poultry people to use DNA testing to identify the pig with this active
gene and can eliminate during the breeding program to reduce the risk in the
offspring.
782 D. Patwardhan and N. Sharma

16.4.4 Feed Additives

The main objective to use feed additives is to enhance the quality of feed for the
animal to improve animal’s performance and health. Feed additives can be available
in many forms, relatively concentrated form, such as vitamins produced by animal or
vegetable origin, amino acids, enzymes, minerals, antibiotics and probiotics, and
single-cell proteins. For example, yeast products high in protein have been used as a
feed additive for many animals: cattle, pigs, and poultry. Rich in nutrition and highly
edible, these products also help in creating a healthy balance of bacteria in the
digestive tract and prevent bacterial diarrhea. A beneficial bacterial product
“phytase,” commercially named as “TRANSPHOS,” had been used as an inexpen-
sive feed additive. The wide use is to substitute the costly mineral phosphate used as
an additive in the feed of monogastric animals. Similar to this, bacteriocin is another
feed additive that had been produced and used to fight against livestock pathogen
like Listeria monocytogenes, Staphylococcus aureus, etc. Lysine is the most essen-
tial supplement for animal growth, and in routine life, animals hardly get this
supplement in enough amount. This L-lysine monohydrate is safe, stable, and edible,
being produced in many countries from bacteria through fermentation and added to
the feed material to increase the quality of nutrition in feed.
Feed additives are categorized as follows:

16.4.4.1 Antibiotics
Antibiotics contain antimicrobial and antifungal properties, usually of plant or
fungal origin produced for pharmaceutical purposes, and can be synthesized in
laboratories. Antibiotics are meant to be used for the treatment of infections, but
there are a few antibiotics available in the market that can improve the growth of
animals and increase the feed conversion efficiency. The most common antibiotic
used as feed additive is “ionophore.” The function of ionophore includes metabolic
role in improvising the production efficiency. These ionophores have general meta-
bolic role within the animal to improve the production efficiency.

16.4.4.2 Enzymes
Applying advance biotechnology, many enzymes are produced at large scale and
relatively inexpensive (McDonald et al. 2010). These enzymes are widely being
used as a feed additive in a nonruminant and ruminant diet. The primary goal was to
improve the nutrition value when poor quality and inexpensive ingredients are
incorporated during feeding the animal. Many enzymes are available commercially
including phytase (phosphorus digestion), hemicellulose (plant cell wall digestion),
and cellulase/xylanases as a feed additive. Also, digestibility of amino acid can be
improved with phytase supplementation.

16.4.4.3 Probiotics
While antibiotics are designed to be involved as a feed additive to treat any bacterial
infection, on the other hand, probiotics are being used to improve the strength of
certain strains of bacteria in the gut. Probiotics basically are a microbial population,
16 Application of Molecular Genetics 783

which enhance the activity of the digestive system. Apart from all, these probiotics
(microbial population) also have been observed to produce vitamin B complex and
many digestive enzymes, for protection against toxins, to increase intestinal mucosa
immunity, etc.

16.4.4.4 Beta-agonists
Beta-agonist is a natural or synthetic organic compound that shares a common
chemical structure with phenethanolamines. Therapeutically, this compound is
involved in massive use to maintain smooth muscle mass. Beta-agonist is a type of
metabolic modifier which means such compounds modify the metabolism in specific
and directed way. These compounds show overall effect on productive efficiency
(weight gain or milk production), improving carcass composition (lean vs. fat ratio),
increasing milk yield in lactating animals, and decreasing animal waste per produc-
tion unit. Two main compounds that are popular and commercially available are
somatotropins and beta-adrenergic. Such compounds are widely used as feed addi-
tive to improve the nutrient amounts in feeds and the productivity of livestock.

16.5 Genetically Modified Organisms (GMOs)

Genetically modified organisms (GMOs) are completely transformed animals whose

genetic material has been commodified in a way that it could not occur naturally. It is
now possible by cutting-edge application of genetic engineering, and molecular
biology together has been involved in modification of genetic makeup of any
organism.
In the history of GMs, Europe is the leading place that was producing cloned and
GM animals throughout the 90s. The most famous example of genetically modified
sheep named “Dolly” had been developed through the cell nucleus transfer from the
differentiated cell. This first genetically modified animal was produced at Roslin
Institute in Scotland. Afterward, a genetically modified bull named “Herman” was
produced by a Dutch biotechnology company “Gene Pharming Europe.” The pur-
pose of producing genetic modification was aimed to generate a subsequent trait of
female offspring that would be able to produce milk proteins like lactoferrin in their
milk and would be an excellent source for food, nutraceutical, and pharmaceutical
purposes. Other examples including some experimental animals like genetically
modified mouse, pig, fish, and chicken have been developed within European
institutions. These animals have been produced with specific advantages and
benefits to food production and other areas of application.
Genetic modification of organisms can be understood by the following
categories:

• Green genetic engineering (or agrogenetic engineering): Established to develop

genetically modified plants in agricultural firms and food sector.
• Red/yellow genetic engineering: Aim to develop medical diagnostic tool, gene
therapy, and essential drugs like insulin and vaccines.
784 D. Patwardhan and N. Sharma

• Gray/white genetic engineering: Involved in production of enzyme or chemicals

by utilizing genetically modified microorganism.
• Genetically modified animals: Produced genetically modified organism to the
purpose of improvement in the quality of food, milk, meat, and dairy product.

The list of few commercially available genetically modified organisms (GMOs) is

as follows:

16.5.1 Glowing Fish

Glowing fish, commercially known as GloFish, is a genetically modified fish and

initially had been developed for pollution detection instead for consumption. Natu-
rally bioluminescent fishes are found in the sea and inhabitant of darkest part of sea.
Scientists from Singapore adapted this idea and developed a fluorescence transgene
that is sensitive to sea pollutant. They had transferred the naturally derived fluores-
cence gene-incorporated pollutant-sensitive biosensor that will detect the environ-
mental pollutant. Other than its scientific role, GloFish also had captured the public
interest for decoration purpose, and so it has become commercial for public to keep
in home aquarium. Since 2003, it has become commercial under the license of
Yorktown Technologies.
Currently, there are 12 lines of GloFish in the market, including tetras, zebra fish,
and barbs, in such colors as electric green, moonrise pink, and cosmic blue.

16.5.2 GM Salmon

Besides insects, fishes had been genetically modified to provide good source of
dietary consumption to the consumers. The first genetically modified fish in the
market is the AquAdvantage salmon. After three decades of its production, in
August 2017, it has become available in Canada. This GM salmon is produced by
AquaBounty Technologies which is twice in size and grown in same period of
non-GE salmon. GE salmon is produced by recombinant gene containing growth
hormone gene from Chinook which is activated by adjacent gene from ocean pout
(a fish). AquAdvantage had been approved by US FDA and declared safe to eat. This
fish also contains same nutrition as other non-GE Atlantic salmon. There had been
no biological side effects observed according to the FDA reports.

16.5.3 GM Mosquito

Mosquitoes are worldwide known to be vector for detrimental diseases such as

malaria, dengue, chikungunya, and Zika. In earlier past, to combat these epidemic
diseases, scientists were on the mission to reduce the female population.
16 Application of Molecular Genetics 785

Scientists at Imperial College London had successfully reduced female popula-

tion by means of genetic engineering. An enzyme known as I-PpoI is an endonucle-
ase that cut the ribosomal gene sequence (rDNA) in the mosquito. This enzyme
specifically destroys the rDNA cluster located on X chromosome. Thus, scientist at
Imperial College London had developed the mosquito strain in which sperm cell
possesses this enzyme to cut the rDNA cluster located on X chromosome and
produced only male offspring. Applying this technology, they were successfully
able to produce 95% male offspring which will further inherit this enzyme to the
generations.
Another example of GE mosquito is Friendly™ Aedes that had been
manufactured at an Oxford-based biotechnology company, Oxitech. This mosquito
had been produced by inserting the gene that kills the insect at larval stage. So when
male Aedes, which does not consume human blood, mate with wild-type Aedes
female, then progeny will be unviable and killed at larval stage before the adulthood.
These mosquitoes had been tested in fields located in different places. First time,
Friendly™ Aedes were launched in Brazil (Piracicaba) in April 2015, and a 91%
reduction in dengue fever were observed in Dorado district of Brazil after immediate
launching in 2016. In total, 12 cases were observed in 2015/2016 that was a huge
reduction from 133 in previous years.

16.5.4 Eco-friendly Pig

GM pig had been produced at University of Guelph in Ontario, Canada, in 1999.

They develop GM pig that contains phytase enzyme in their saliva. Phytase enzyme
digests the plant phytate into phosphorus which is useful for algal growth when the
phosphorus is released from the manure of GM pig into the water stream. The origin
breed was Yorkshire pigs that genitivally modified for phytase enzyme.

16.5.5 Bird Flu-Resistant Chicken

To fight against most devastating disease for chicken such as bird flu, scientists in the
UK had developed transgenic flu-resistant chicken. Thereafter, in another attempt,
scientists from University of Cambridge had developed GM chicken with short
hairpin RNA. This structure somehow blocks the spread of the influenza virus
(mechanism is unknown). Thus, this technology had improved the poultry chicken
and environment as well as human health which is prevented by flu infection.

16.5.6 Human Gene Therapy

Gene therapy is referred here as an incorporation of gene to replace a defective gene

that might have been involved in many genetic disorders. This is a technique to
correct the genetic error which occurs in some genetic disorder like cystic fibrosis,
786 D. Patwardhan and N. Sharma

cancer, ADA, immunodeficiency, etc. First-time gene therapy in human history was
performed successfully by William French Anderson, Michael Biase, and Ken
Culver in 1990. These guys showed that a severe immunodeficiency, adenosine
deaminase (ADA) deficiency, also known as “boy in a bubble disease,” can be
treated with gene therapy. To spot a bit light over here, ADA is a recessive disease
carrying two copies of recessive allele of ADA gene. Normally, two copies of ADA
gene promote the production of adenosine deaminase in cells throughout, but error in
even one gene will inhibit the conversion of deoxyadenosine (a waste product) into
inosine and thus will lead to heavy buildup of deoxyadenosine in the body. This
accumulated buildups later undergo phosphorylation, convert into toxic triphosphate
responsible for killing T cells, and eventually result in failure of immune system and
early death.
Gene therapy can be done in two possible ways:

1. Somatic gene therapy is taking into account that gene transfers into the body cell
by means of somatic cell rather than to germ cell (egg or sperm cell). The aim to
somatic gene therapy is not to let the gene pass to the offspring in the future but
just stay in patient’s body till its effective state. Study and trials on somatic
therapy has prevailed its success as clinically effective. Gene therapy for ADA
that has been discussed above was the first somatic gene therapy in 1990 and
1991 with two patients of ages 4 and 11 years old. Both kids are growing well
with the continuity of the treatment. Later in 1992, a 29-year-old woman
experiencing familial hypercholesterolemia, a genetic condition (defect in the
chromosome 19) that is associated with increased cholesterol in the blood due to
defective LDL receptor on the liver, was treated with somatic gene therapy. This
woman was treated with homozygous FH ex vivo delivery to the liver. This
treatment was carried out for 18 months, and liver biopsies demonstrated no
discernible abnormalities.
Consequently, five more patients had been successfully treated with gene therapy
since then. After the success of above stories, scientists are focusing on the
clinical trial for many other diseases especially chronic genetic disorder and
cystic fibrosis.
2. In addition to somatic gene therapy, gene transfer would have been done with
germ line cells (eggs and sperm) as well. Gene delivery to the germ cells would
modify the genetic makeup of germ line and would definitely pass on the future
generations. Germ line gene therapy would be capable to vanish the risk for
inherited genetic disorder from the family forever. This type of assurance could
be achieved by another method like diagnosis during the IVF if there is any
known risk before the implantation. Germ line therapy is a distant prospect and
have negative opinion; such therapy is illegal in most of the Europe. However,
germ line gene therapy and somatic gene therapy raise different issues. Only
somatic gene therapy brings the effective prospect of treatment and have provided
a promising cure for few genetic disorders although treatment is complex and
success rate is uncertain.
16 Application of Molecular Genetics 787

Fig. 16.6 Gene delivery system through a viral vector used in cancer therapy

Despite that techniques are not advanced in gene therapy, researchers are still
attempting to develop the methods for gene transfer into the cells in the culture,
animals and humans. Within the effect, viral genome was first ever reported as an
efficient method for gene delivery into the mammalian cells in the culture. In the
beginning of 1980 with the development of retroviral vectors, gene delivery into
cultured mammalian cells became widely accepted (Fig. 16.6).
Genome editing has been established as a powerful and efficient tool as a part of
gene therapy. Compared to the earlier, current techniques are much more efficient
and advanced for modification of DNA. These days, researchers are able to investi-
gate the gene editing in plants, insects, zebra fish, mice, and human cell line in vitro.
In theory, gene editing is capable to introduce point mutation to investigate tran-
scription regulation and epigenetic modification. Hence, this technique has a
promising contribution in medicine. In recent years, genome engineering is
advanced by introducing a powerful and efficient tool: cluster regulatory interspaced
short palindromic repeat (CRISPR) nuclease Cas 9. CRISPR chops the DNA
sequences identified by guide RNA. CRISPR technique is undergoing a widespread
use in the research and has already been used for genome engineering of more than
dozen species.
Interestingly, a recent study from Chinese research group have demonstrated
human embryo genome editing by using CRISPR/Cas9 system. Unfortunately, this
experiment arises significant questions from the scientific and technical point of
view upon the risk of these technology over the future. The Chinese research team
claimed that the embryo was “mosaic” in nature, meaning that only few cells had
788 D. Patwardhan and N. Sharma

desired gene editing but there was enough number of off-target effects or mutation in
nontargeted genes that can be harmful if embryo had been viable. Thus, in terms of
human welfare, their work further points out a significant concern on social and
ethical policies of genome editing especially in human embryos.
Gene delivery system through viral vector in human gene therapy. A viral vector
had been used for packaging of gene of interest being absorbed by the cell membrane
or through the endocytosis method followed by delivery of gene to the nucleus
which is the place of target gene.
This mechanism has been widely used for treatment of cancer, lung cancer,
immune deficiency, cystic fibrosis, etc.

16.5.7 Complications and Issues in Gene Therapy

Besides its great success in various disease treatments, gene therapy has rose many
ethical issues in the society. One of the great accomplishment of gene therapy is gene
editing is now no more an obstacle for scientists. World’s advanced technique
CRISPR has made this opportunity possible for biologist to edit any gene required.
Thus, it had become a grave concern that the day is not far for parents to achieve
when they will be desiring a customized baby, and they can decide a list of new
features such as redhead, blue eyes, and extrovert to be added in their child’s
genome. While everyone is in the race to make their kids smarter, so why
wouldn’t you?
It’s ethically and economically a dilemma, questioning upon fate and fairness,
about vanity and values. Considering all the issues of grave concern, a vigilance
committee including not only scientist but also lawyers, doctors, religious, and
ethicist have decided to permit somatic cell gene therapy to cure genetic disease
but should not extend to the germ line gene transfer which will cause the gene editing
for preferred child appearances, which would further pass on to offspring. This
action may lead to breach the policies of availing gene therapy and will reason for
harmful outcome for human society.
There is also sensitive concern about safety highlighted in 1999 after an incident
when a patient was participating as volunteer for gene therapy trial through viral
vector, but shockingly, this patient had a fatal immune reaction after injecting viral
vector for treatment of his metabolic disorder.

16.6 Molecular Markers

A molecular marker is referred to as a tool to detect variation at DNA or gene level

among the individuals. Variation occurs due to base pair changes, rearrangements/
translocation/inversion of sequence, insertion or deletion of sequence, and variation
in the number of tandem repeats. Molecular markers can be categorized into protein
markers such as allozymes, DNA marker such as mitochondrial DNA (mtDNA), and
nuclear DNA marker such as microsatellites, tandem repeats, RAPD, RFLP, AFLP,
16 Application of Molecular Genetics 789

etc. Molecular markers have been widely used to study genetic variation because of
its dynamic properties such as ubiquitous, stably inherited, contain multiple alleles
for each marker, devoid of pleiotropic effects, detectable in all tissues, and long shelf
life of DNA sample.

16.6.1 Types of Nuclear Molecular Marker

16.6.1.1 Restriction Fragment Length Polymorphism (RFLP)

RFLP represents a molecular marker that utilizes the information about restriction
enzyme recognition sites in different individual. However, this technique is not so
often being used nowadays, but it was the only technique being used for DNA
analysis in forensic science widely. The principle of this technique is that any small
mutation like base substitution, insertion, deletion, duplication, and inversion within
the whole genome can remove or incorporate new restriction site. Thus, any two
individuals will show differences by a great as many as RFLPs.
RFLP analysis distinguishes that where the disease gene lies on the chromosome.
In RFLP assay, a cloned DNA fragment is used as a probe and must bind to one site
alone which reveals a different-sized restriction fragments as an RFLP locus on
Southern blot.
In model organism, a set of strain or individual that provides standard RFLP loci
has been utilized for RFLP mapping of that particular species. Human RFLP
mapping has been established on a set of individuals in 61 families worldwide and
an average of eight children per family. Mapping of human RFLP loci was taken into
consideration that disease allele may be linked to the restriction site, and this linkage
information of each individual may provide a sustainable approach to assess the
probability of a person getting disease in next generation. RFLP mapping is a useful
tool to predict any genetic disease linkage in pedigree analysis. Despite the fact that
RFLP is one of the primitive and efficient method, it is still a very time-consuming
and tedious method compared to the new advanced DNA analysis techniques.
Moreover, it requires comparatively large sample size to determine RFLP mapping.
Remember that RFLP is always considered as a “single locus marker” since we
investigate the single restriction enzyme recognition site on a DNA fragment which
reflects similar to the single locus on an allele.

16.6.1.2 Random Amplification of Polymorphic DNA (RAPD)

RAPD is a PCR-based molecular technique to develop DNA markers. This tech-
nique has been established by Welsh and Maclelland in 1991. RAPD was designed
with the concept of the DNA sequence-based polymorphism at a very large number
of loci among the individual. RAPD is a pre-DNA sequencing free technique that
makes it more conceivable. This technique has been beneficial for certain purposes
in animal breeding such as to identify and classify the accessions of the breed, for
identification of the breed, and to study genetic diversity.
RAPD principle works on the virtue of a set of primers, a short oligonucleotide
sequence that binds to many different loci. This binding results in the amplification
790 D. Patwardhan and N. Sharma

of random sequence in the complexed DNA sample. Amplification is done in the

PCR and amplified PCR product influenced by the size of both primer and template
DNA. Let’s consider that two different genomic DNA from two different individuals
create a different pattern of the amplified PCR product. A specific fragment found in
individual 1 not present in individual 2 will represent a DNA polymorphism, and this
difference can be used as a genetic marker. An advantage of RAPDs is that it is
technically simplified irrespective of demanding any prior DNA sequence informa-
tion. It has been observed that this method is more advanced and constructive
compared to fingerprinting and RFLP though this technique has a significant
disadvantage of the fact that polymorphism can only detect the presence or absence
of a particular amplified DNA sequence in the sample that appears a band of a
specific molecular weight. This information is regardless of homozygosity or het-
erozygosity of that particular DNA fragment. Also, reproducibility of the data is a
technical demerit in RAPD.

16.6.1.3 Amplified Fragment Length Polymorphism (AFLP)

AFLP is another PCR-based molecular marker and very specific to the species and
subspecies. The common use is determining the close relationship between species
or subpopulation in mostly plants, humans, animals, fungi, and bacteria. This marker
prevails the application in investigation of genetic variation, population structure,
and differentiation. AFLP is capable to generate hundreds of replicable markers from
small size DNA sample. In this way, AFLP provides high-resolution genotyping
quality. Pertaining its advantageous features, AFLP is known as a substantial genetic
marker, used in many broad applications, for example, molecular systematic studies,
phylogenic studies, DNA fingerprinting, quantitative trait loci (QTL) mapping, and
population genetics. AFLP analysis is more convenient because it generates large
numbers of marker fragment for any organism without having prior knowledge of
genomic sequence. Additionally, AFLP requires only small fraction of starting
template, and it shows much higher reproducibility compared to RAPD. On the
basis of factors like time consumption, cost-effectiveness, resolution, and reproduc-
ibility, AFLP is equal to the other markers, i.e., RAPD, RFLP, and microsatellite.
Template fragments are generated by (1) digestion with combination of two
restriction enzymes and (2) ligation of restriction enzyme-specific adaptors (red
and blue) to each end (3). After adaptor ligation, a preamplification step is completed
by using combination of primers that matches any of these adapter sequences, and
each primer carries selective nucleotide that is represented here with N. (4) Selective
PCR amplification step completed by adding additional nucleotides to the primers
(specific to two restriction sites) (5). Gel electrophoresis image shows amplified
AFLP product.
However, the major drawback of this technique is that AFLPs cannot distinguish
dominant homozygous individuals from dominant heterozygous individuals as it is a
dominant biallelic marker (i.e., SNP, two possible nucleotide variation for a single
position). AFLP is widely used in population genetics and genome typing and
subsequently more suitable for detecting genetic polymorphism and tracing animal
genetic resources.
16 Application of Molecular Genetics 791

Fig. 16.7 Schematic representation of AFLP method

For a high-impact AFLP, researcher should choose an appropriate primer combi-

nation that will generate the sufficient polymorphic marker to study. Literature
suggested that at least minimum combination of three primers from the dozens of
combinations (that have been used for examining polymorphism in a few
individuals, and the results enable them (primers) to identify optimal pairs) will be
used for a sufficient gain. AFLP success from the practical point of view requires
four major factors including (i) standardized reaction condition, (ii) optimized
reagents, (iii) a robust and reliable electrophoresis platform, and (iv) accurate analy-
sis software (Fig. 16.7).

16.6.1.4 Sequence-Tagged Microsatellite

Microsatellites or simple sequence repeated (SSR) loci are also known as variable
number of tandem repeats (VNTRs) and simple sequence length polymorphisms
(SSLPs). These repeats are very common in eukaryotes genome and, to some extent,
in prokaryotes genome. Microsatellites vary from one to six nucleotides in length.
Depending upon the numbers of nucleotide in the repeats, they are categorized as
mono (A), di (AA), tri (AAA), tetra (AAAA), penta (AAAAA), and hexa
(AAAAAA). Di-, tri-, and tetranucleotide repeats are so often used for genetic
studies. These repeats are present in repeated manner at least 5–20 times with the
minimum length of 12 nucleotide.
792 D. Patwardhan and N. Sharma

Microsatellite provides ease to perform high score, high polymorphism, and a

strong automation for thousands of markers at a time and makes this technique
suitable for a number of genetic studies including (i) DNA fingerprinting, (ii) genetic
mapping, and (iii) paternity analysis and genetic diversity. Microsatellite sequence at
specific loci can be easily detected by PCR. Primer for PCR should be specific to
conserve flanking region of repeats. A sequence tag is used as a primer to amplify the
corresponding SSR and resulted to amplified sequences known as sequence-tagged
microsatellite (STMs). These sequence tags must have been taken from the group of
enormous amplified restriction fragments or from the genomic DNA. Primer for
STM amplification is designed by combination of universal primers that have been
used for microsatellite repeats and anchored at 50 end of the targeted microsatellite
repeat. Thus, primer designing for STM is cost-effective and cut down the cost of
developing SSR marker (constructing a microsatellite marker) direct to the synthesis
of a single primer specific to a conserved flanking region (conserved DNA sequence)
of SSR.
When we have to apply STMPs in large complex DNA, complications have been
reported in isolation of restriction fragments containing target SSRs from the pool of
amplicon which has been used to construct a sequence tag profile. This problem has
been solved by enriching the pool of SSR-rich restriction fragments available from
the same DNA template which were used to construct the sequence tag profile,
reported in Hayden and M. J. et al. In spite of many advantages, STMSs also possess
some limitations including extensive use of high cost of cloning, sequencing, and
primer synthesis. Still standard protocol uses radioisotope, and primer selection
procedure suffers from a number of problems like redundancy of clones and the
occurrence of artificial chimeras.

16.7 DNA Fingerprinting

This technique is also known as DNA profiling and DNA typing. DNA fingerprint-
ing is usually collections of fragmented DNAs from individuals to be compared for
particular purpose and generate a DNA-specific profile in a term of fingerprinting.
DNA fingerprinting is nonetheless but a distinctive pattern of DNA fragments
according to the length isolated by gel electrophoresis. In a forensic field, DNA
sample is first isolated and purified from the suspects and victim in order to suspect a
crime scene. These samples are further digested by restriction enzyme, amplified by
PCR, and profiled using electrophoriesis. DNA fingerprinting is first invented by
Alec Jeffreys in 1985 in England. He used restriction enzyme to cut the DNA into
fragments because PCR had not been developed that time. Initially, fragments were
used with radioactivity-labeled DNA, but now this technique is improvised with the
discovery of PCR and fluorescent dye. Routine fingerprinting testing is
accommodated with repeated sequence or short tandem repeats which allow to
distinguish DNA fragments more effectively. Nowadays, DNA fingerprinting is
always performed by PCR assay.
16 Application of Molecular Genetics 793

To understand the analysis result of fingerprinting, we can consider the DNA

fragments on autoradiograph image below. This radiograph contains five essential
lanes describing (i) markers, a known size of DNA fragments; (ii) control lane (TS),
a sample from positive source and expected to bind with DNA probes favorably; (iii)
experimental lane which denoted the blood collection from defendant, defendant’s
shirt, and jeans; and (iv) blood sample from victim’s shirt and victim’s blood.
The given example shows that blood on defendant’s shirt and jeans are similar to
the blood of victim. Defendant was lying here because his own blood doesn’t match
with victim’s blood and blood on his clothes (Fig. 16.8).
DNA analysis has become the routine job in many forensic labs for solving the
crime scene and identifying the suspects. In the beginning of DNA technology,
scientists have realized that DNA database from the crime scene would provide a
criminal justice in no time, and with more efficiency also, some local DNA database
have been created on purpose. Before the DNA fingerprinting, protein assay was
being used for several years for solving the cases, but this technique was not reliable
because of less availability of technical support and protein degradation was the
major issue. DNA collection was much easier, and DNA is more resistant to physical
damage compared to the protein. DNA analysis can be performed from any tissue,
i.e., blood, hair, saliva, semen, skin, and bones, while protein markers are restricted
to the cells where these proteins are expressed. Besides, this technique has also been
used to analyze the relationships and source of another organism in the population.
For example, DNA fingerprinting has revealed that anthrax has been transmitted to
the population having same source of origin.

16.8 Fluorescent in Situ Hybridization (FISH)

If a part of genome is possible to clone, then it can be used to make a labeled probe
for hybridization to chromosome in situ. The logic of this approach is identical to
Western or Southern blot just in case this probe does not bind with any DNA or
protein instead binds to largely intact chromosome since the probe is cloned for
chromosome specific. This method involves a few steps to be performed such as
isolation of chromosome by tearing cells chemically or mechanically and spread on
the microscopic glass. The chromosome on slide is supposed to be denatured so that
the double-stranded long DNA can convert into single stranded. Thereafter, dena-
tured labeled probe is added to this mixture. In result, the probe will be hybridized to
homologous sequence in situ with the chromosomal DNA on the slide and appar-
ently location of hybridization on the chromosome will be detected by bright
fluoroscent spot on the chromosome DNA under the fluoroscent microscope
(Fig. 16.9). With the advance technology, FISH can also be used to localize and
detect various RNA target (mRNA and miRNA) within the cells and tissue.
Now, the probe sequence will be used to map the position of hybridization on the
chromosome by observing the banding pattern related to centromere or any other
cytological feature. Unfortunately, this technique does not allow to observe recom-
binational mapping due to low resolving power, as in example of two genes that
794 D. Patwardhan and N. Sharma

Fig. 16.8 DNA fingerprinting/profiling from a crime scene. The DNA samples collected from the
victim (V) were found on defendant’s (D) clothing (jeans/shirt). First lane shows DNA ladder (λ),
and second lane shows positive control (TS). This profile shows a successful DNA fingerprint assay
from a crime scene resulting in prediction of criminal

positioned 5 cM apart to each other on the human chromosome, but in situ

hybridization mapping technique would not be able to distinguish this
recombinational-like feature.
In advance in situ hybridization technique, probe can be a cDNA, cRNA, or
synthetic oligonucleotide. While choosing the probe, researchers should consider the
16 Application of Molecular Genetics 795

Fig. 16.9 FISH image of chromosome shows locus-specific fluorescent signals. (a) Cytogenetic
bands (gray) with a hybridized probe with chromosome spotted in red. (b) A clone selected from
patient suffering with multiple congenital malformations and mental retardation. FISH analysis is
used for locating the break point of a translocation on chromosome 11 or 19, and FISH was able to
show the red signals split between chromosomes 11 and 19 where the translocation took place

binding sensitivity, specificity, production facility probe penetration strength, and

stability of hybrid. These probes are basically conjugated with fluorescein, biotin, or
digoxigenin.
FISH probes can be categorized (Fig. 16.9) as (i) locus-specific probe or
(ii) chromosome paint probes. Locus-specific probes are particular to detect a
distinct gene or chromosomal area, and such probes help detect any deletion or
amplification of DNA sequence (if any occur), while chromosome paint (Fig. 16.10)
probes are completely specific for detecting structural abnormalities within the
chromosome structure or rearrangement of chromosome. The optimal size of
probe should be of length 50–300 bases.

Box 16.1: Production of Recombinant Human Proinsulin in the Milk

of Transgenic Mice (Qian X et al.)
Diabetes is the third most worldwide health issue among the people and
characterized with high blood sugar which can lead to a number of serious
complications such as heart disease, stroke, kidney failure, blindness, nerve
damage, Alzheimer’s, etc. The main causes of diabetes are lacking insulin
production (Type I) and failure of its use after production (Type II). After
success of medical trial, insulin treatment has become one of the promising
treatments for diabetes. At molecular level, human insulin gene in vivo is first
transcribed and translated in single-chain precursor known as “proinsulin”

(continued)
796 D. Patwardhan and N. Sharma

Fig. 16.10 FISH image of human metaphase chromosome painting. (a) Chromosomes 1, 2, and
4 were labeled yellow and rest painted red. (b) Image shows reciprocal translocation between
chromosoms appeared in bicolor chromosom indicated with white arrow. FISH technique used to
detect translocation between chromosomes is stained yellow, while chromosome is stained red

Box 16.1 (continued)

which is 110-amino acid (aa)-long peptide in the ß cells of the islet of
Langerhans in the pancreas. This signal peptide, first 23–24 aa at the N
terminus, is removed during the protein folding in the endoplasmic reticulum
which results in proinsulin (86 aa, 9.5 kDa). Molecular structure of protein has
three domains: an amino terminal B chain of 30 aa, 3.4 kDa, a carboxy
terminal A chain (21 aa, 2.4 kDa), and a connecting C chain (34 aa,
3.0 kDa). This structure is generally considered as a premature protein struc-
ture, but a cut by neuroendocrine cell-specific prohormone convertase (PC1
and PC2) in order to remove C chain and remaining A and B chain together is
bound with a sulfide bond known as mature insulin (5.8 kDa). With an
alarming need of insulin production for diabetes treatment, recombinant
DNA technology has become a boom for the medical field. Human insulin
now biosynthetically can be possible to manufacture at large scale for clinical
use worldwide. Primitively, chain A and chain B successfully were produced
in two separate bacterial strains and followed by purification and sulfide bond
formation by air oxidation. Furthermore, insulin and its analogues were
produced in a yeast as inactive proinsulin that further goes for enzymatic
cleavage of C chain by using enzymes such as trypsin and carboxypeptidase

(continued)
16 Application of Molecular Genetics 797

Fig. 16.11 Transgene construction and the identification of transgenic mice. (a) Schematic
representation of the transgene construction. The full length of insulin cDNA in the pCMV6-
XL5-INS-cDNA was amplified by PCR and inserted into the pBC1 vector at the Xho I site,
generating the pBC1-INS construct. Before microinjection, the pBC1-INS construct was excised
with Sal I and Not I. From left to right, the linearized pBC1-INS comprises the 2
β globin
insulator; the goat β-casein promoter and untranslated exons E1 and E2; human insulin cDNA;
untranslated goat b-casein exons E7, E8, and E9; and 39 genomic DNAs. Pr1F, Pr1R, Pr2F, and
Pr2R primers were used in PCR for the identification of the transgenic mice. (b and c) Identification
of the transgenic mice by PCR using the Pr1 primer pair (b) and Pr2 primer pair (c). Non-transgenic
wild-type (WT) mouse DNA was used as a negative control, and the DNA used for microinjection
served as a positive control. b-actin was amplified to show the same amount of DNA used in each
PCR reaction
798 D. Patwardhan and N. Sharma

Table 16.2 List of proinsulin concentration in the collected milk of transgenic

mice at the midlactation period

Box 16.1 (continued)

B to obtain full potential mature insulin. However, this production method has
some limitations on the net production rate, and according to diabetic federa-
tion, in 2030, one out of ten people will suffer with insulin deficiency or
diabetes which gives rise to the possibility of increased demand of insulin
production. Current insulin production methods are insufficient to cover the
targeted demands to cure the diabetes. Thus, the biopharmaceutical protein
production in the mammary glands of transgenic animals is one of the exclu-
sive and extensive trial to increase the proficiency of insulin production for
medical uses. This approach has achieved the heights of current expectations
in the medical field as it promises the production of high-quality therapeutic
medicine for human at very efficient cost. In the current scenario, production
of human proinsulin in the milk of transgenic mice provides a possibility and
feasibility for scaling up the potential proinsulin production by using trans-
genic dairy animals.
To generate the transgenic mice expressing the human proinsulin milk, full
length of human insulin cDNA was amplified in the pCMV6 vector
andinserted into the mammary gland-specific expression vector pBC1

(continued)
16 Application of Molecular Genetics 799

Box 16.1 (continued)

(Fig. 16.11a). pBC1 vector contains goat ß-casein promoter and flanked by 50
to 30 untranslated sequence of goat ß-casein gene (Fig. 16.11a) This transgene
was first linearized in the vector and injected into the fertilized mouse eggs
followed by transfer into the recipients. Recipient female mice were crossed
with wild type male, which gives F1 transgenic mice pups. However, qPCR
for identification of copy number of transgenes in the founder and offspring
reveals a slight difference in the numbers of transgenes in the offspring from
its founder (Table 16.2) which can be due to the loss of transgene transfer
efficiency. However, loss of transgenes is a common phenomenon that
explains that transgenes are usually exogenous fragmented DNA supposed
to be introduced to some random sites into the host genome and will not result
in 100% transfection capability of vectors (Fig. 16.11)
Secretion of proinsulin is targeted only in the milk product of transgenic
mice, but to confirm the negligible secretion of insulin in the blood or plasma
of these animals, further ELISA test was performed. ELISA test confirmed that
there is no difference between the insulin levels in the blood of transgenic and
non-transgenic mice. Also, no glucose amount was observed in the plasma of
non-transgenic mice which was similar to the transgenic mice. To further
obtain the bioactivity of proinsulin or conversion into mature insulin, this
proinsulin which was collected from the milk was further digested with
enzymes. Digested product was targeted to the CHO cells which express the
enormous insulin receptor on its surface. Treated CHO cells were harvested
and analyzed for tyrosine phosphorylation of insulin receptor. Levels of total
phosphorylation were compared with positive control, undigested transgenic
milk, and digested and undigested non-transgenic milk. Results from these
samples do not show any detectable activity compared to the digested trans-
genic milk which confirms the bioactivity of proinsulin produced from the
milk of transgenic mice. This observation further clarifies that mammary gland
does not produce the mature insulin since the digestion of proinsulin was done
exogenously, and therefore, no insulin bioactivity was observed in undigested
proinsulin. This mammary gland may not express PC1 and PC2, enzymes
required to release the C chain from the proinsulin, but mammary gland can
recognize the insulin signal peptide and able to secret the proinsulin protein in
the milk. This proinsulin production is empirically favorable in the human
health since it does not affect the blood glucose and blood insulin levels that
reduce the systemic side effects on diabetic patients.
800 D. Patwardhan and N. Sharma

16.9 Summary

• Molecular genetics is being used for a long time, which has been engaged in
enormous applications in the field of animal biotechnology, transgenic animal,
production of genetically modified organisms, human gene therapy, development
of molecular markers, and forensic science.
• The most advantageous contribution of molecular genetics is in development of
transgenic animal. Transgenic animal is produced by injecting DNA into fertile
egg that contains foreign DNA (of desired requirement or trait) that is integrated
into a chromosome. Knockout mice is a transgenic mouse in which genes are
disabled of particular role.
• In molecular genetics, it is now possible to improve the reproduction system and
health of cattle and domestic animals by enrichment of diet, improved diagnosis
tool, providing feed additives, adding nutrient supplements, etc.
• In human gene therapy, detrimental diseases are now possible to treat by altering
the disease-associated gene in human cells.
• Variation in DNA sequence of individuals or studying polymorphism in a
population can be assessed by analyzing molecular markers such as RFLP,
RAPD, AFLP, and microsatellite.
• DNA fingerprinting and in situ hybridization are mostly used in forensic science.
Both techniques are being used in understanding the crime scene and analyzing
the samples through these methods to identify criminals.

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Genetic Analysis of Development
17
Tapodhara Datta Majumdar and Atrayee Dey

17.1 Model Organism for Genetic Study

17.1.1 Yeast (Saccharomyces cerevisiae) (Fig. 17.1)

Yeast is a saprophytic and unicellular fungi. It has a wide application in food

production especially brewer’s yeast or baker’s yeast. Yeasts are eukaryotes and
are characteristically different than prokaryotic bacteria. They have a dimension
much greater than bacteria which are 5
10 μm and 0.5
5 μm, respectively. They
have many defined features that make them unique and specific, like they have an
innate resistance toward numerous antibiotics, sulfamides, and also antibacterial
agents. It’s a property which cannot be transferred to other organisms. Yeasts are
widely found in soil, water, plants, animals, and insects. Plant tissues (i.e., leaves,
flowers, and fruits) are very common yeast habitats, but a few species are also found
in animals. Some yeast varieties can also be isolated from extreme environment
conditions like high salinity, low water potential, low temperature, and also low
oxygen availability (Table 17.1).

17.1.1.1 Saccharomyces cerevisiae

Saccharomyces cerevisiae (S. cerevisiae), widely known as brewer’s yeast, can
survive either as single-celled or as pseudo-mycelia. S. cerevisiae differs from

Both authors have contributed equally.

T. D. Majumdar (*)
Indian Statistical Institute, Kolkata, India
A. Dey
Department of Zoology, Banwarilal Bhalotia College, Asansol, West Bengal, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 803
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_17
804 T. D. Majumdar and A. Dey

Fig. 17.1 The yeast culture.

High-resolution microscopic
image showing yeast culture
under high magnification
depicting both budding and
vegetative yeast cells

Table 17.1 Yeast habitats

Habitat Yeast
1. Plants Debaryomyces hansenii, Hanseniaspora uvarum, Wickerhamomyces
anomalus, Metschnikowia pulcherrima, Magnaporthe oryzae, Roesleria
subterranea
2. Animal and Sugiyamaella mastotermitis, Saitozyma flava, Apiotrichum
insects mycotoxinovorans, Candida albicans, Cryptococcus neoformans
3. Soil Lipomyces, Schwanniomyces, Apiotrichum dulcitum, A. porosum,
Cyberlindnera saturnus, Lipomyces starkeyi
4. Water Rhodotorula, Debaryomyces hansenii, Candida lipolytica, Candida
tropicalis
5. Extreme Glaciozyma, Leucosporidium (extreme cold), Methanopyrus kandleri
environment (extreme hot), Picrophilus torridus (acidophile), Halobacterium, and
Natronobacteria (high saline conditions)

other yeasts mainly in their ability to ferment sugars. Due to this it finds wide
application in food and dairy industry (Table 17.2).

17.1.1.2 Saccharomyces cerevisiae: Structure and Components

(Fig. 17.2)
A budding cell of S. cerevisiae shows the cellular components under electron
microscope. Being a eukaryote S. cerevisiae has membrane embedded distinct
organelles like mitochondria, nucleus, Golgi, and endoplasmic reticulum. The dia-
gram is a part of vegetative reproduction where a budding cell arises from the mother
cell and separates as daughter cell under normal growth conditions.

1. Cell wall: Yeast falls under the category of eukaryotes that possesses an external
cell wall. It is the outermost cell organelle and provides external protection and
maintains the osmotic balance of the cell. It has an outer and an inner layer. It
protects the cellular components from foreign particles and other cell wall-
17 Genetic Analysis of Development 805

Table 17.2 Taxonomy of Domain Classification

S. cerevisiae
1. Kingdom Fungi
2. Division Ascomycota
3. Subdivision Saccharomycotina
4. Class Saccharomycetes
5. Order Saccharomycetales
6. Family Saccharomycetaceae
7. Subfamily Saccharomyetoideae
8. Genus Saccharomyces
9. Species cerevisiae

Fig. 17.2 S. cerevisiae cell structure and components

degrading enzymes of different hosts like plants. The outer layer is build up of
chains of densely populated glycosylated mannoproteins and functions in cell-
cell communication process. The inner layer of the cell wall provides firmness to
the wall and is 70–100 nm to 200 nm thick depending on the growth conditions.
The main building components of the cell wall are L1,3-glucan and chitin and
contribute to about 50–60% of the wall dry weight. The glucan-chitin network
provides elasticity to the wall. Cell wall proteins are covalently linked to this
network either indirectly through a L1,6-glucan moiety or directly. Some proteins
806 T. D. Majumdar and A. Dey

are bonded to cell wall via disulfide bonds. The phosphodiester bridges of the
carbohydrate chains of the cell surface proteins make the wall hydrophilic and
help in water retention during drought conditions. An estimated 1200 genes of
S. cerevisiae build up the cell wall. Morphological and compositional changes in
the wall occur depending on the phase of the cell cycle, nutrient availability, and
environmental conditions such as pH, temperature, and the availability of oxygen.
2. Cell membrane: The next organelle after cell wall is the cell or plasma membrane,
which is 7.5 nm wide. It is composed of polar lipids and proteins which, by their
interactions, construct the famous lipid-protein bilayer of eukaryotes. The
proteins are located asymmetrically and are either intrinsic (present all throughout
the membrane) or extrinsic (partially embedded in the membrane). The proteins
are involved in transport of solutes, signal transduction, anchoring of the cyto-
skeleton, and synthesis of outer membrane components. The inner side consists
mainly of phospholipids like phosphatidylethanolamine, phosphatidylinositol,
and phosphatidylserine. The major lipid classes are glycerophospholipids,
sphingolipids, and sterols. Glycerophospholipids consist of two fatty acid acyl
chains ester-linked to glycerol-3-phosphate; various substituents such as choline,
ethanolamine, serine, myoinositol, and glycerol are linked to the phosphoryl
group. Cardiolipin is also widely present. Sphingolipids have a ceramide back-
bone which is composed of a long-chain base phytosphingosine that is N-acylated
with a hydroxy C26 fatty acid. S. cerevisiae contains three sphingolipids: inositol
phosphate ceramide, mannosyl-inositolphosphate-ceramide, and mannosyl-
diinositolphosphate-ceramide. The membrane also constitutes sterols (hydropho-
bic molecules with a polar hydroxyl group) mainly ergosterol and some
zymosterol.
3. Nucleus: The genetic assembly of a eukaryote is present in its membrane-bound
distinctive nucleus. Due to this it is considered as one of the most essential
organelles of the cell. The DNA-containing chromosomes are present in the
nucleus. It endorses formation, expression, and functioning of chromosomes. It
also is the site of transcription of DNA to mRNA and aids in the formation of
rRNA in the nucleolus. After formation, mRNA and rRNA export to cytoplasm
and proceed for protein synthesis. Large pores are present in the nuclear mem-
brane that regulates the traffic of macromolecules into and out of the nucleus. The
DNA is held together in the chromosome via histone proteins, which are present
in the nucleus and help in chromatin folding. In budding yeast, the main structural
elements are the nuclear envelope, the nuclear pore complex, and the nucleolus.
The nuclear envelope consists of chromatin anchorage sites, spindle pole body,
heterochromatin, and the ribosomal DNA (rDNA). The nuclear pore complex
takes part in repairing damaged DNA. As a eukaryote the yeast nucleus is
functionally compartmentalized. The formation and functioning of these
compartments and subcompartments are not due to the intranuclear membranes
but depend instead on sequence elements, protein-protein interactions, specific
anchorage sites at the nuclear envelope or at pores, and long-range contacts
between specific chromosomal loci, such as telomeres. Finally, long-range inter-
action of loci in trans, such as the clustering of telomeres or of transfer RNA
(tRNA) genes, influences the nuclear order.
17 Genetic Analysis of Development 807

4. Vacuole: The vacuole of S. cerevisiae plays an important role in the physiology of

this organism. Vacuoles are the largest compartment in yeast cells, occupying
about 25% of the total cell volumes. Vacuoles contain mainly low molecular
weight solutes and ions, but quite small amount of proteins. Among the many
roles of this organelle are pH homeostasis and osmoregulation, protein degrada-
tion, and storage of amino acids, small ions, and polyphosphates. It also plays
precious role in sporulation, protein turnover, and ion transport. Besides, it
functions in maintaining homeostasis of cytosol in many respects.
5. Golgi apparatus and mitochondria: The Golgi apparatus is responsible for the
proper functioning of secretory proteins in eukaryotic cells. It helps in modifying
and ultimate sorting of the newly synthesized secretory proteins, which are called
cargo proteins. The cargo proteins are synthesized in endoplasmic reticulum
(ER) and transported to the Golgi apparatus into cisternae (membrane sacs).
After the cargo proteins enter the Golgi apparatus, they are modified and
processed by Golgi enzymes. These are then transferred to the trans-Golgi
network and transported to their destinations via transport proteins. Mitochondria
are the other essential components of eukaryotic cells and are endosymbiotic in
origin. They have their own genetic machinery. Mitochondria consist of two
membranes, the inner and outer membrane. The intermembrane space lies
between the two membranes, and together they guard the mitochondrial matrix.
They consist of tubular networks. Mitochondria are known as the powerhouses of
eukaryotic cells and are one of the most important organelles in respiratory active
cells as they take an active part in synthesis of cellular ATP and also play an
important role in tricarboxylic acid (TCA) cycle and oxidative phosphorylation.
Its function also includes amino acid and lipid metabolism as well as synthesis of
iron-sulfur clusters and heme.
6. Endoplasmic reticulum (ER): The ER originates from the nuclear envelope and
forms a membranous tubular network that exudes and branches all along through
the cytoplasm. It mainly takes part in protein exportation. An amino-terminal
signal peptide targets the secreted proteins to the ER. The signal peptide gets
removed upon entry into the ER lumen, and the protein assumes its final, folded
conformation for the first time. Those proteins that do not fold properly or fail to
form correct three-dimensional structures usually get associated with an
ER-binding protein known as BiP. BiP may also serve to catalyze the folding
or assembly of normal protein structures. Besides this, synthesis and assembly of
lipid bilayers are also initiated in ER. The ER also helps in assembly of dolichol-
linked oligosaccharides and their transfer.

17.1.1.3 Life Cycle and Reproduction (Fig. 17.3)

Life cycle of S. cerevisiae can be divided into two phases: (1) mitotic phase in which
the mother cell undergoes budding and goes through G1, S, G2, and M phases to
form an identical daughter cell and (2) meiotic phase, which forms four haploid
spores held together in ascus. The spores come out of ascus under favorable
conditions and undergo mitosis to form new diploid.
808 T. D. Majumdar and A. Dey

Fig. 17.3 Life cycle and reproduction of S. cerevisiae

The genome size of S. cerevisiae is approximately 1.2 x 106 base pairs comprising
of 16 chromosomes, with about 5700 protein-coding genes. The yeast cell divides
vegetatively under normal conditions and gives birth to one daughter cell. Being
eukaryotes it has four phases of life cycle, i.e., G1 (gap 1), S (stationary), G2 (gap 2),
and M (mitotic) phases. At the end a genetically identical daughter cell is formed.
But before the daughter is released, each sister chromatid is duplicated and
segregated by mitosis. The G1 phase consists of round yeast which still remains
unbudded. It then develops a small bud from one side and reaches the S phase. The
chromosomes which remain diffused and indistinguishable in the nucleus during the
G1 gets duplicated, yielding pairs of sister chromatids in the S phase. In the G2
phase, the bud grows, and the nucleus is found adjacent to the bud. The pair of sister
chromatids remain attached to each other and are still diffused in the nucleus. In the
M phase, chromosome goes through a twofold condensation and separates, as a
result of which the sister chromatids are segregated between the mother and bud.
These chromatids are pulled apart by spindle fibers. Finally, cytokinesis divides the
cytoplasm and leads to the formation of two genetically identical nuclei, one mother
and one daughter cell. Both of them again return in G1 phase, thus completing the
mitotic life cycle.
When two haploid cells mate and fuse to form a diploid cell, it leads to formation
of homologous chromosomes. The diploid cell contains two of each chromosome.
Diploid grows either by budding (vegetative mitosis) or undergoes meiosis. There
17 Genetic Analysis of Development 809

are two mating types of the yeast, termed as a and α. Haploids of a can only mate
with that of α and vice versa. The haploids produce pheromones to induce mating,
which attracts the haploids of the opposite mating type. This stimulates fertilization.
During fertilization the cells move toward each other by growing projections, a
process called shmooing. The cells then fuse to form a diploid cell, which is α/a and
cannot mate. The newly formed zygote can divide either mitotically or meiotically.
Mitotically they generate diploid cells, whereas meiotically they generate four
haploid cells of which two will be α and two will be a.
Yeast usually divides vegetatively. Under conditions when diploid budding
yeasts are starved of nutrition, meiosis takes place. Meiosis leads to production of
four haploid spores. Spores are more resistant to the environment than vegetatively
dividing cells. The four spores of a single meiosis are held together in an ascus, or
tetrad, surrounded by a thick wall. When favorable conditions return, the haploids
get released from the ascus. They then divide by mitosis to again form new diploid.
Two closed nuclear divisions characterize meiosis in yeast. The first is meiosis
1, where homologous chromosome associate lengthwise and then segregate into
daughter nuclei. In meiosis 1, chromosome number gets halved in the daughter
nuclei. The maternal and paternal genes get mixed together through independent
assortment of the individual chromosomes. So it is also referred as reductional
division. Here crossing over takes place between the paired homologues by homol-
ogous recombination. The second meiosis or meiosis 2 is similar to mitosis and gives
rise to four haploids. The meiosis of yeast makes it one of the most widely studied
model organisms for eukaryotes. Researchers can easily control the process and
mediate crosses between strains.

17.1.1.4 S. cerevisiae as an Effective Model Organism

S. cerevisiae was the first eukaryotic organism whose whole genome was sequenced.
Various mammalian homologues were found in it. About 31% of important mam-
malian protein sequences have homology to potential protein-encoding genes of
yeast. Two close homologues of mammalian ras proto-oncogene, RAS1 and RAS2,
were studied to be present in it. Among the best matches are the human genes that
cause hereditary nonpolyposis colon cancer (MSH2 and MLH1 in yeast), neurofi-
bromatosis type 1 (IRA2 in yeast), ataxia telangiectasia (TEL1 in yeast), and
Werner’s syndrome (SGS1 in yeast). It has been successfully used as a model in
regulation of gene expression, signal transduction, cell cycle, metabolism, apoptosis,
and neurodegenerative disorders. S. cerevisiae, being a facultative anaerobe, can
satisfy its energy requirements with adenosine triphosphate (ATP) generated by
fermentation. So it doesn’t need much amount of other mitochondrial proteins for
its survival. Due to this various knockout mutations can be done in yeast
mitochondria for studying mitochondrial functions. This makes yeast an ideal
organism for identifying the molecular processes required for biogenesis of
respiratory-competent mitochondria. The budding yeast of S. cerevisiae has been
nowadays extensively used in aging research. Two major approaches, “budding life
span” and “stationary phase,” have been used to determine “senescence” and “life
spa” in yeast. Role of genes like Sir2, histone deacetylases, gene silencing, rDNA
circles, and stress genes has also been extensively studied in the aging process in
810 T. D. Majumdar and A. Dey

yeast. Even, integrated analysis of existing regulatory and metabolic networks, by

studying about 55 transcription factors regulating 750 metabolic genes, and novel
regulatory mechanisms was also put forward in a recent study.

17.1.1.5 Uses and Threats

The widest use of S. cerevisiae is probably in the bakery industry in baker’s and
brewer’s yeast, used in bread and other bakery products. The enzymes produced
during the fermentation process are used in formation of different alcohols, organic
acids, and esters. These impact flavor and vary from strain to strain. In addition yeast
may be utilized as food. They contain a good concentration of protein as well as most
of the B complex vitamins. At present days S. cerevisiae is also used in producing
biodiesel and biofuel.
S. cerevisiae is usually non-harming to humans. But recently a certain fungemia
has been detected which is caused by the yeast. Besides, a very rare and an acute case
of pyelonephritis was also diagnosed.

17.1.2 Fruit Fly (Drosophila melanogaster) (Fig. 17.4)

Drosophila melanogaster (D. melanogaster), the common fruit fly, has its ancestral
home in Africa. It was first put forward by Thomas Hunt Morgan as a model
organism, over 100 years ago, when he discovered the white gene in the fly. Since
then it has been used as a model organism in both medical and scientific research for
over a century. One of the main advantages of the fly is that it shares 60% homology
to humans and has 75% genes homologous to human diseases. Due to this it finds
ample application in studies including gene biology, cell biology, developmental

Fig. 17.4 Fruit fly (Drosophila melanogaster)

17 Genetic Analysis of Development 811

Table 17.3 Taxonomy of Domain Classification

D. melanogaster
1. Kingdom Animalia
2. Phylum Arthropoda
3. Class Diptera
4. Family Drosophilidae
5. Genus Drosophila
6. Subgenus Sophophora
7. Species melanogaster

biology, population genetics, and most importantly neuroscience. Drosophila has a

simple nervous system, consisting of around 100,000 neurons that can carry out
complicated neuronal tasks, similar to that in humans, such as learning and memory.
Due to this it has find immense popularity in the study of a wide range of human
neurodegenerative diseases including Huntington’s, Alzheimer’s, and Parkinson’s
disease. Drosophila has minimal generation time, low maintenance costs, and well-
developed genetic structure. They produce large numbers of externally laid embryos,
and they can be genetically modified in numerous ways. Due to the above properties,
the fruit fly can be used to study complicated cycles relevant in biomedical research,
including cancer. Another emerging use of the fly is in the field of regenerative
biology (Table 17.3).

17.1.2.1 Drosophila melanogaster: Structure and Components

(Fig. 17.5)
D. melanogaster’s body is divided primarily into three segments, namely, the head,
thorax, and abdomen.
Some characteristic features of Drosophila play an important role in its taxonomy
classification and species identification. Likely, the shape of its head and face, the
number of relative size of oral setae, and compound eye are some most relevant
distinguished features. The head is complex in structure and has a different mor-
phology in different species. Next to the head comes the thorax. Prothorax, meso-
thorax, and metathorax are the three main segments of thorax. Humeral callus of
prothorax bears a pair of setae. This pair of setae is very useful in identifying the
species. Mesothorax is the largest segment of fruit fly and provides the wings the
required strength for flight. The number and disposition of acrostichals or bristles on
the mesonotum (the dorsal portion of the mesothoracic integument of insects) are
important characteristics for species identification. Each thorax segment consists of a
pair of legs, which are further segmented into coxa, trochanter, femur, tibia, and
tarsus. The tarsus region is further divided into five small tarsal segments.
Chaetotaxy (arrangement of bristles on the leg of the flies), body coloration, length
of the leg segments, and sex combs are widely used in species identification and
classification. The abdomen of Drosophila is covered by chitin. The dorsal region of
the abdomen segment is known as tergite, while the ventral region is called sternite.
These two regions are widely useful in taxonomy studies and also help in
differentiating between male and female. The pigmentation patterns of tergites and
812 T. D. Majumdar and A. Dey

Fig. 17.5 Anatomy of D. melanogaster

the six quadrilaterally shaped sternites in female compared to only four sternites in
male are widely used for identifying the species and gender.
The anatomy of the fly is very conserved and interesting. Having homology to
human genome, it has analogy to various developmental process and signaling
pathways of humans. Over the years it has helped researchers tremendously in
getting a picture of the development and signaling processes in humans by serving
as a very efficient model organism. Some selected functional anatomy are the brain
and nervous system, muscle, kidney, gastrointestinal tract, and cardiovascular
system.
Ectodermal brain of Drosophila and its development provide an immense under-
standing of neurobiology. Neuroblast is first formed in the embryo which then grows
into the ventral nerve cord. The brain turns into a much mature and complex
structure from larvae to pupa. It develops defined lobes having distinct functions.
Fly muscle tissue is much conserved in evolution and provides the main support
during flight. The kidney consists of three parts, i.e., the garland cells, the
nephrocyte, and the Malpighian tubule. Drosophila nephrocytes share evolutionarily
conserved domains of the vertebrate glomerular slit diaphragm. Nephrocytes and
garland cells of fruit fly are a classic example of primitive reticuloendothelial and
renal system. Fluid balance is maintained by the Malpighian tubule. The
17 Genetic Analysis of Development 813

gastrointestinal tract of Drosophila is a very complex structure and over the years
has been used to study host-pathogen interactions. It’s heart is primitive and is
morphologically different from the multichambered heart of vertebrates, though
the functioning and genetic paradigm are very much similar. Detailed studies of
the fly’s heart have provided insights into studies regarding cardiac aging and also
diabetes-related cardiac diseases.

17.1.2.2 Life Cycle and Reproduction (Fig. 17.6)

The life cycle of Drosophila is divided into egg, larval, pupal, and adult stages. The
first larva comes out in 24 h and completes the instar stages by the next 72 h,
reaching the pupal stage. Within the next 3–4 days, adult fly comes out and can live
up to 70–90 days under favorable conditions.
D. melanogaster has a very short life cycle. A female fly lays huge number of
eggs and will continue to do so for weeks. Larva hatches out from eggs in a span of
24 h at 25  C and passes through three “instar” stages of development by the next
72 h. Pupa is formed at the end of the third instar stage. At this time various organs of
the body provide the required energy for growth and metamorphosis into adult fly,

Fig. 17.6 Life cycle of D. melanogaster

814 T. D. Majumdar and A. Dey

for instance, the imaginal discs that ultimately develop into adult structures such as
wing, eye, and limbs and consist mostly of undifferentiated epithelial cells. After
3–4 days eclosion occurs and the adult fly emerges. Female flies do not lay eggs in
the first days of life. After the completion of a life cycle of 10 days, adult flies can
live up to 70–90 days.
During the growth cycle, a number of genes play their part. The collaborative
expression of these genes produces various proteins which lead to the entire larval
setup of the fly. These proteins form the anterior-posterior axis and the dorsal-ventral
axis called the bicoid and the dorsal, respectively. The synchronization of these
proteins activates the transcription of specific cascades of genes. These genes include
gap genes, pair-wise genes, segment polarity genes, and hox genes that ultimately
divide the embryo into structural segments and functional regions. Once the embryo
is completely developed, the first instar larva hatches out and starts eating. The larva
eats a lot this time, and this food not only helps for its growth but also as future
storage food (fats and sugars) during the later stages of metamorphosis, mainly
during pupal stage. During the growth process, molting takes place when the larvae
shed the exoskeleton. This process involves the chain-wise functioning of a number
of hormones like ecdysone, juvenile, and most importantly the prothoracicotropic
hormone (PTTH). PTTH, released from neurosecretory cells in the brain, stimulates
the release of the molting hormone ecdysone from the prothoracic glands into the
hemolymph. Ecdysone then forms a new cuticle or exoskeleton. Ecdysone is
complemented by another hormone, eclosion hormone, which helps in initiating
the molting process by allowing the larva to shed the exoskeleton and enter the next
instar stage. Larvae wander for a place to pupate at the end of the third instar stage.
At this time the larvae are appropriately referred to as “wandering larvae.” Once the
flies reach the final stage of larva, they attain a critical size, where the organs should
be properly developed before pupation starts. During the pupal stage, most of the
larval structures are lost. During this transition time, autophagy, a “self-eating”
signaling mechanism, takes place. This converts the stored nutrients (fats and
sugars), uptaken during the larval stage, into necessary food to provide energy
required for the animal survival.

17.1.2.3 Drosophila melanogaster: An Efficient Model Organism

Drosophila melanogaster has served for years as a very important and efficient
model organism for various genetic studies by researchers. The genome of the fly has
been manipulated to study the function of the genes by generating transgenes by
various methods. Some of them are mentioned below.

1. P-element transposons—Transposons are either linear or circular DNA that can

be inserted into chromosomal DNA at specific sites with the help of transposase
enzyme. Plasmid DNA are primarily constructed with the modified P-element
inserts and are injected into the Drosophila embryo germline cells together with
plasmids encoding for the P transposase. P transposase functions to insert the
P-element transposon into a random point in the genome of Drosophila. The fly
then produces a progeny that will carry the transgene. To determine the successful
17 Genetic Analysis of Development 815

integration of the transgene in the genome, sometimes a marker DNA is linked

to it.
2. Homologous recombination—P-element transposons have many disadvantages,
including lack of target site specificity. Therefore homologous recombination was
put forward. This method uses the cell’s own genetic makeup to insert foreign
DNA or even for knockout genes by the process of homologous recombination.
3. Bacterial artificial chromosome (BACs)—BACs and recombination engineering
are gaining attraction in introduction of transgenes in Drosophila. The technique
uses recombination to insert genes or other DNA fragments into BACs. The
process is becoming more popular because it is faster and easier than using
restriction enzymes and ligases to insert DNA.
4. CRISPR/Cas 9—Small sets of repetitive nucleotide sequences with interspaced
non-repetitive sequences are present in the bacteria. These are called Clustered
Regularly Interspersed Short Palindromic Repeats (CRISPR) and function in
bacterial immunity against viruses. When a phage attacks the bacteria, these
CRISPR transcribe into shorter CRISPR RNA (crRNA) and noncoding trans-
activating crRNA (tracrRNA). Cas enzymes help the bacteria to keep viral DNA
copies in the form of CRISPR arrays, after the virus attacks the first time. These
DNA copies get transcribed and processed into crRNA and tracrRNA when the
virus attacks again. They together target the virus to be cleaved by other cas
enzymes like Cas9. This technique of cleavage of viral DNA by cas 9 in associa-
tion with crRNA and tracrRNA is used to insert artificial constructs into the fly
genome. The cleavage caused by cas 9 is then repaired by techniques such as
homologous recombination.

Besides transgenesis, mutagenesis is also widely used to study altered genetic

functions in Drosophila. There are many ways to generate mutants, two most
important being P-element mutagenesis (where P-element transposons are used to
disrupt gene functions by inserting or removing them from certain positions in the
genome) and chemical mutagens like ethyl methanesulfonate (produces a form of
guanine, O6-methylguanine, that incorrectly base pairs with thymine during DNA
replication).
Also Drosophila is used nowadays for genetic screening, which is aimed in
identification of noble genes that are important to understand new and important
biological cycles and mechanisms.
Outside the importance in studying the gene functioning, Drosophila also finds
application in studying other fields like behavioral scoring, signaling pathways, and
also metabolic cycles.

1. Behavioral scoring—Complex behavior like visual learning, courtship, and olfac-

tory learning are well-established behavior in the fly and can be well assayed by
studying their behavioral pattern.
2. Signaling pathways—Drosophila has helped in studying various signaling
pathways, which play important role in humans. The most important being the
Notch pathway, which is involved in many diseases like alagille syndrome, aortic
816 T. D. Majumdar and A. Dey

valve disease, cerebrovascular dementia, and leukemia. Other signaling pathways

which are well studied in Drosophila includes JAK/STAT, TGF-β, Wingless,
Hedgehog, Toll, Hippo, and MAPK.
3. Metabolism—Drosophila has also gained recent importance in putting light into
the mechanisms underlying complex conditions like diabetes, obesity, hyperlip-
idemia, and inborn errors of metabolism.

Due to the abovementioned uses of Drosophila, a variety of applications in

disease research have been found. Below are examples of some such diseases
where the fly has given significant contribution.

1. Cancer—Over the years the fly has implied a lot in cancer biology. Hippo
signaling, which controls organ size, proliferation, and apoptosis, was first
characterized in Drosophila. Other human cancerous genes had also find homo-
logue in Drosophila.
2. Insulin signaling peptides—Drosophila insulin-like peptides (Dilps) were
identified in the flies and were found to act as sensors that regulate energy balance
and growth.
3. Neurodegeneration—This is one field where Drosophila has an immense contri-
bution. It has been used in the study of a wide range of human neurodegenerative
diseases including Huntington’s, Alzheimer’s, and Parkinson’s disease.

17.1.2.4 Threats
Drosophila melanogaster till date is not known to cause any harm to humans.
However in two of very recent articles in Nature journal, the researchers have
found a change in aggressive and courtship behavior of the fly with change in
environment and climate. The other group of researchers investigated a primitive
cannibalism in the fly behavior, where the younger larvae attacked and consumed the
later-stage larvae. Though these are not threats to the humans, they can affect the
laboratory preservation of the flies.

17.1.3 Nematode Worm (Caenorhabditis elegans) (Fig. 17.7)

An electron microscope image showing the thread-like structure of free-living

nematode, Caenorhabditis elegans, under high magnification.

17.1.3.1 Introduction
Discovered by Sydney Brenner in the mid-1960s, Caenorhabditis elegans
(C. elegans) is a free-living nematode found mainly in soil and compost heaps and
has since become one of the most established model organisms. It has advantages of
a very short life cycle which lasts only for 3 days, is small size (1.5 mm in adults),
and can be easily handled under laboratory conditions. Due to these properties, this
soil nematode offered great potential for genetic analysis. The population mainly
consists of self-fertilizing hermaphrodites (XX) with a rare and very small
17 Genetic Analysis of Development 817

Fig. 17.7 Caenorhabditis elegans

Table 17.4 Taxonomy of Domain Classification

C. elegans (ITIS Report)
1. Kingdom Animalia
2. Phylum Nematoda
3. Class Chromadorea
4. Order Rhabditida
5. Family Rhabditidae
6. Genus Caenorhabditis
7. Species elegans

percentage of males (X0), who have a distinct morphology. The other distinguished
features of C. elegans are the nematode’s small but compact genome which is only
20 times that of E. coli and a simple cellular system which consist slightly greater
than 1000 cells, of which 302 cells make the nervous system (Table 17.4).

17.1.3.2 Caenorhabditis elegans: Structure and Components (Fig. 17.8)

The body of C. elegans is divided into anterior, posterior, dorsal, and ventral regions.
They are primarily hermaphrodites having both male and female reproductive organs
in the same body.

1. Feeding—They are predators and possess grasping mouth parts and spines to
catch and eat their prey.
2. Respiration, circulation, and excretion—They exchange gases and excrete meta-
bolic wastes through their body wall. The nutrients and waste are carried out
throughout their body by diffusion.
818 T. D. Majumdar and A. Dey

Fig. 17.8 Structure and components of C. elegans

3. Nervous system—They have a very simple nervous system. Nerves start from
ganglia in the head and run through the whole body. They have several sense
organs which can sense hosts and preys.
4. Movement—The muscles extend throughout the entire body. The pseudocoelom
has a fluid that works together with the muscles as a hydrostatic skeleton.
5. Reproduction—C. elegans has a well-developed and simple reproductive system.
Being maximum hermaphrodites they undergo self-reproduction; in very rare
cases male progeny is found. A special mention is the presence of the organ
vulva, which is required during mating, as males inject sperm through it and for
deposition of embryos after internal fertilization. A further detail about the
reproduction in C. elegans is given in the next section.

17.1.3.3 Life Cycle and Reproduction (Fig. 17.9)

The life cycle of C. elegans is divided into egg, larval, pupal, and adult stages. There
are three instar larval stages. Improper food and environment leads to these larval
stages entering the diapauses (e.g., the dauer stage shown in the diagram above,
which instar larva L2 can enter under inappropriate growth conditions). After the last
instar L4, C. elegans enters the pupal stage and finally the adult stage.
C. elegans adults lay embryos that pass through stages like gastrulation, comma
stage, and two- to threefold changes in embryo formation before hatching into the
first larval stage. The first-stage larvae, L1, comes out of the eggshell and begins
feeding. When adequate amount of food is there with low population density of
maturing larvae, the larvae go through a definite genetic clock and produce four
larval stages, namely, L1, L2, L3, and L4, before molting into adults. The cycle takes
about 3.5 days at 20  C under rich nutritional conditions. But harsh and unfavorable
environmental conditions with little food and crowded population can trigger L1s to
17 Genetic Analysis of Development 819

Fig. 17.9 Life cycle of C. elegans

enter diapause state, blocking further development. When the scarcity of food
reduces, they regain development and reach the L2 stage. If in the L2 stage the
larvae again face inappropriate growth conditions, they enter into a stage called
dauer, where development of the reproductive system is arrested. If the dauer find
food, then the third larvae, L3, come and proceed to adulthood. The larvae can enter
the third diapause when L4 adults are again left without food. At this diapause the
reproduction is halted by the degradation of most of the germline, except
proliferating zone cells which remain cell cycle arrested. Upon refeeding, worms
in adult reproductive diapause resume germ cell proliferation, meiotic development,
and oogenesis and can become fertile.
C. elegans primarily disseminate as hermaphrodites, with two X chromosomes
and a diploid set of autosomes (2X, 2A). Up to L2 the nematode lives as
hermaphrodites. In the L3 stage, some of the hermaphrodites produce the male
germ cells. Nondisjunction occurs in about 0.1–0.2% cases which lead to male
progeny (X0) production. On the other hand, 50% of cross-progeny becomes male
if mating occurs. These male germ cells then get differentiated into sperm between
L3 and L4 stage. The sperms are produced both by the hermaphrodite individuals
and from mating. They stay in the spermatheca and wait for the ovulation of the very
first oocyte. Female germ cells are produced in the L4 stage which finally differenti-
ate into oocytes. Once the ovulation is over, the sperm meets the oocyte and
fertilization occurs marking the initiation of embryogenesis. When the hermaphro-
dite is unmated, it can produce about 300 embryos, but in the mated condition the
820 T. D. Majumdar and A. Dey

Fig. 17.10 Adult C. elegans highlighting the reproductive system

hermaphrodite can produce up to 1000 embryos. This shows that the main factor on
which self-fertility depends is not oocyte production but rather the amount of self-
sperm formed by the hermaphrodite (Fig. 17.10).
The reproductive system of C. elegans carries the major organs like the uterus,
vulva, spermatheca, and distal tip cell along with embryos and oocytes.
Gonadogenesis or formation of gonads in C. elegans depends on the presence of
four gonad precursors Z1, Z2, Z3, and Z4. It begins at L1 stage of larval develop-
ment. Z2 and Z3 are the germline progenitors, and Z1 and Z4 are the somatic
precursors. The germline progenitors are present in between the somatic precursors.
The germline precursors give rise to germline gonad cells, whereas the somatic
precursors give rise to distal tip cell (DTC) and the other somatic gonad cells. From
the L4 stage, the hermaphrodite takes the shape of the adult, with the gonad arm on
the dorsal side of each U-shaped tube capped by the DTC. Spermatogenesis
completes within a short span of 24 h in both the hermaphrodite and male. Four
spermatids are formed from spermatogenesis which produces motile and amoeboid
sperms. The spermatids are generated by two sequential meiotic divisions of meiotic
germ cells. The prophase arrest stage is absent in the cell cycle of spermatogenesis.
Oocyte, on the other hand, is large and nutrient-rich. In contrary to prophase of
sperm formation, meiotic prophase of oogenesis takes a considerable elongated time
span of about 54–60 h. The oocyte is then released for fertilization. There is an
extended pachytene in oogenesis, where germ cells synthesize RNAs and proteins
that are donated to the oocyte. The oocyte most adjacent to the spermatheca
(proximal oocyte) undergoes ovulation into the spermatheca and takes part in
fertilization. Before this it undergoes meiotic maturation which includes breakdown
of nuclear envelope, progression to metaphase of meiosis I, and rearrangement of the
oocyte cortex and cytoplasm. In the absence of sperm, oocytes get arrested in
diakinesis and ongoing oogenesis is inhibited. Sperms are not available when adult
hermaphrodites are depleted off their self-sperms or when the female undergoes any
mutation. Thus, oocyte production and utilization only occur in the presence of
sperm.
17 Genetic Analysis of Development 821

17.1.3.4 Caenorhabditis elegans: An Efficient Model Organism

The nematode C. elegans has emerged as an important animal model in various
fields including neurobiology, developmental biology, and genetics. The
characteristics of the nematode that have made it a highly acceptable model organ-
ism include its well-characterized genome which can be genetically manipulated,
vividly described developmental program, ease of maintenance, short life cycle, and
small body size. It has been widely accepted as a model organism, with a number of
toxicological, host-pathogen interaction and metabolic pathways studies being car-
ried out on it over the years. Some of them are described below. Since reverse
genetic and transgenic experiments are much easier and less expensive to conduct in
C. elegans as compared to many other model systems, it is a useful model for
studying effect of chemical exposures on metabolic pathways. C. elegans can act as
both in vitro and in vivo mammalian models in toxicology. C. elegans also provides
a good platform for the study of host-pathogen interactions. It has been discovered
that the nematode has an inducible defense response and that the defense mechanism
shares similarity to that of other organisms. This makes the worm a potential model
for the study of innate immunity. The interaction between prokaryotes and
eukaryotes is pervasive and has important medical and environmental significance.
But despite this importance, the existing data on the mechanisms and pathogenic
consequences of bacterial-fungal interactions within a living host is very scarce.
C. elegans was utilized as a host to study the interactions between two ecologically
related and clinically troublesome pathogens. One is the bacteria, Acinetobacter
baumannii, and the other is the fungi, Candida albicans. It was observed from the
results that Acinetobacter baumannii inhibits filamentation, a key virulence determi-
nant of Candida albicans. The nematode has also proved efficient in studying
substrate-inhibitor interaction mechanisms. It was found in another study that the
interaction of a lead compound (named lead compound-76) had an inhibitory effect
on MPK-1, needed by the worm in vulva development and egg laying and the
established homologue of the human ERK-2 (extracellular signal-regulated kinase-
2). Compound 76 showed selectivity in inhibition of phosphorylation of LIN-1, a
MPK-1 substrate essential for vulva precursor cell formation. Thus C. elegans
served as a model organism to study specificity of compounds targeting the ERK
protein. It also helped in studying mammalian bacterial pathogenesis, as it was also
shown to be killed by Pseudomonas aeruginosa. Besides these C. elegans has also
proved to be a useful organism for investigating molecular and cellular aspects of
numerous human diseases.

17.1.3.5 Threats
There are no known threats caused by C .elegans, till date, to humans. Except it can
sometimes be host to pathogenic bacteria and fungi, which should be handled and
taken care of.
822 T. D. Majumdar and A. Dey

Fig. 17.11 Xenopus frog. Female Xenopus laevis (left) and female Xenopus tropicalis (right)

17.1.4 Western Clawed Frog (Xenopus tropicalis) (Fig. 17.11)

Xenopus tropicalis (X. tropicalis), also known as Silurana tropicalis or the Western
clawed frog, is a small, aquatic frog that is found all along the west coast of
equatorial Africa. It is a close neighbor of the widely used model organism Xenopus
laevis (X. laevis). It complements X. laevis in taxonomy, anatomy, and life cycle.
X. tropicalis bears a diploid genome and a high conservation of gene synteny with
the human genome. It was first used as a model organism for genomic research in the
early 1990s. After this it has been used as an acceptable and interesting model for
biomedical studies, developmental and cell biology, biochemistry, functional geno-
mics, and immunology especially those metabolic cycles and systems that can
influence vertebrate development from embryonic stages through adulthood. The
embryos of the frog develop externally and the tadpoles are transparent. These
features facilitate experimental manipulation and post-factum analysis of animals.
Another important feature which makes X. tropicalis more acceptable model organ-
ism than X. laevis is its short life span of 6 months as compared to 18 months in
X. laevis. Raising and culturing them are also relatively easy as they can be easily
grown in water tanks or in recirculating aquatic systems (Table 17.5).

17.1.4.1 Xenopus tropicalis: Structure and Components (Fig. 17.12)

The anatomy of frog has similarity with that of humans but is much simpler.
The body of the frog is divided into three portions, i.e., head, short neck, and
trunk. The neck is short and rigid, permitting only limited head movement. The frog
body consists internally of a single cavity called the coelum. The trunk is thick and
17 Genetic Analysis of Development 823

Table 17.5 Taxonomy of Domain Classification

X. tropicalis
1. Kingdom Animalia
2. Phylum Chordata
3. Class Amphibia
4. Order Anura
5. Family Pipidae
6. Genus Xenopus
7. Species tropicalis

Fig. 17.12 Anatomy of Xenopus tropicalis. The structure and components of Xenopus tropicalis.
(a) Overall structural components of the frog. (b) Dorsal view of the frog anatomy

forms walls of the coelom. All the frog’s internal organs are held in the coelum, and
it is a continuous hollow space with no such partition as diaphragm in man.
The frog skull is flat with an expanded area that encloses the brain. The skeleton is
bony and provides support and protection to the frog’s body. The skeleton consists
of nine vertebrae in the vertebral column and no ribs. The forearm and hind legs of
frog perform important functions in the frog’s locomotion. They have similarity to
that of humans in terms of structure and nature of bones. Radio-ulna is the only
forearm bone in frogs, whereas humans have two forearm bones, the radius and the
ulna. But both have a single upper arm bone, the humerus. The hind legs of the frog
are highly specialized for leaping and consist of a single leg bone, the tibiofibula, in
contrast to man who has two lower leg bones, tibia and the fibula. Femur is the single
thigh bone found in both man and frog. Frog’s leg also consists of two elongated
824 T. D. Majumdar and A. Dey

anklebones, or tarsals. These are the astragalus and the calcaneus. The astragalus and
calcaneus correspond to the human talus and heel bone, respectively. The frog does
not have a tail, except ruminents of a primitive tail, called urostyle. The skeleton
movement is supported by muscles, mainly striated muscle, whereas internal organs
contain smooth muscle tissue.
The frog’s heart is three chambered and protected by the pericardium. It is made
up of two upper chambers, the right and left atrium and a one single ventricle as the
lower chamber. In contrast men contain two lower compartments, the right ventricle
and the left ventricle. The pure (oxygen-laden) and impure (oxygen-poor) blood are
always present together in the frog ventricle, but the blood however never mixes.
The right atrium passages toward the bottom of the ventricle and allows the impure
blood that enters into it from the body, to pass to the bottom. On the other hand, the
pure blood from the left atrium also enters the same single ventricle. But as the
oxygen-poor blood is present toward the bottom of the ventricle, it holds up the
oxygen-laden blood and protects it from flowing to the bottom. This mechanism
prevents both the blood from coming into contact. The pure blood then moves out of
the heart along with impure blood, when the latter leaves the ventricles and enters the
vessels leading to the lungs. The lung vessels, however, are filled with oxygen-poor
blood, blocking the oxygen-laden blood and forcing the latter to detour into the
arteries. This carries the oxygen-laden blood to the tissues.
Frog principally respires through its skin. It has a soft, thin, and moist skin having
an extensive network of blood vessels running throughout. The skin is divided into
two layers, an outer epidermis and an inner dermis. Oxygen freely passes through the
membranous skin and enters into the blood. Other than skin, frogs also have paired,
simple, saclike lungs to breathe. The mechanism of breathing in frog is different
from that in man. It has no ribs or diaphragm like men, and neither its chest muscles
are involved in breathing. The frog simply opens its mouth to breathe and let the air
flow into the windpipe. It can also breathe when its mouth is closed. It does this by
lowering the floor of the mouth keeping the nostrils open. This causes the air to enter
the enlarged mouth. Then, with nostrils closed, the air in the mouth is forced into the
lungs by contraction of the floor of the mouth.
The process of digestion in frog starts in the mouth. The teeth in frog have no
function in digestion and are present only in the upper jaw. The tongue is highly
specialized and helps in catching prey. Whenever the frog encounters a prey, it can
flick open the tongue from its folded position in the throat. The prey gets attached to
the tongue due to its sticky texture. The food then moves to the stomach through the
esophagus. From here the food moves into the small intestine, where most of the
digestion occurs. Large digestive glands, the liver and the pancreas, are attached to
the digestive system by ducts. Ureters carry the liquid wastes from the kidneys to the
urinary bladder, and the solid wastes from the large intestine pass into the cloaca.
Both liquid and solid wastes leave the body by way of the cloaca and the
cloacal vent.
The nervous system in frog is well developed and is divided into the brain, spinal
cord, and nerves. The brain is made of medulla, cerebellum, and cerebrum. The
automatic functions like digestion and respiration are controlled by the medulla,
17 Genetic Analysis of Development 825

whereas body posture and muscular co-ordination are regulated by cerebellum. The
cerebrum is very small as compared to human. The brain consists of ten cranial
nerves and ten pairs of spinal nerves. Olfactory lobes, present in the forepart of brain,
monitor the sense of smell in frogs. The eye has fixed lens that cannot change its
focus. The eyelids are poorly developed and cannot move, so to close its eye, the
frog draws the organ into its socket. A third eyelid called the nictitating membrane is
present but is almost rudimentary. Only in some instances that it may be drawn over
the pulled-in eyeball. External ear is absent and both eardrums (tympanic
membranes) are exposed. There is only one bone in the frog’s middle ear, and
semicircular canals help to maintain body balance.

17.1.4.2 Life Cycle and Reproduction (Fig. 17.13)

There are different stages of development in Xenopus frog. The fertilized egg from
ovum and sperm goes through zygotal, blastulation, and gastrulation stages and
finally reaches the adulthood. Xenopus tropicalis completes the life cycle in
6 months, whereas Xenopus laevis takes 12 months.

Fig. 17.13 Life cycle of Xenopus

826 T. D. Majumdar and A. Dey

Like any other amphibians, X. tropicalis starts its life as an embryo. The embryos
can be used for various biomedical studies as they are able to tolerate considerable
manipulation like single cell, germ layer dissections, and tissue transplantations. The
eggs and the embryos are widely used in targeted gene knockout, knockdown, and
overexpression studies and also serve as a source for high-throughput biochemical
studies. Besides, the cell-free extracts made from Xenopus oocytes are used as a
coherent in vitro system for cell and molecular biological studies. Oocytes are also
widely used for studies of ion transport, channel physiology, and environmental
toxicology. Due to all the above properties, the eggs and the embryos can serve as an
outstanding tool in biomedical research.
The egg is composed of an animal and a vegetal region, which are covered by a
vitelline membrane. After fertilization, the cortex determines the future dorsal region
at a position opposite to the site of sperm entry. The blastulation and the gastrulation
phases start soon after fertilization within a few hours. The blastula follows a radial
symmetry. The three germ layers mesoderm, endoderm, and ectoderm are next
formed. Mesoderm and endoderm are formed from the marginal zone and are then
internalized at the blastopore. Ectoderm spreads to cover the embryo, a process
called the epiboly. Archenteron (future gut cavity) is formed from the dorsal
endoderm after it separates from mesoderm. The lateral mesoderm then spreads
ventrally to cover inside of archenteron. By the end of gastrulation, the archenteron
formation is completed, and mesoderm completely covers the gut internally. Epiboly
gets accomplished by this time, with ectoderm covering the embryo. Yolk cells are
internalized and serve as food source. The dorsal mesoderm during this time
develops into notochord and somites. The somites, from mesoderm, at this stage
form dermatome, vertebrae, and trunk muscles. The dermatome turns into the future
dermis and the limbs are formed by the vertebrae and trunk muscles. Lateral
mesoderm becomes heart, kidney, gonads and gut muscles and ventral mesoderm
form blood forming tissues. The endoderm gives rise to the lining of gut, liver and
lungs. Part of the ectoderm forms the neural plate, which further forms the neural
tube. The anterior neural tube becomes the brain, whereas mid- and posterior neural
tube becomes spinal cord. Neurulation is followed by early tail bud stage. In this
stage the brain is divided, ears and eyes are formed, and three bronchial arches are
formed. The tail is also formed as an extension of notochord, somites, and neural
tube. Lastly, neural tube, from the edges of the neural folds, forms neural crest cells.
This crest cell then develops the sensory and autonomic nervous system, skull,
pigment cells, and cartilage.
X. tropicalis and X. laevis follow the same life cycle except that laevis takes about
a year to reach the adult stage, whereas tropicalis reaches adulthood in about
5–6 months. This is the reason why tropicalis is more preferred nowadays as a
model organism than laevis.
17 Genetic Analysis of Development 827

17.1.5 Xenopus tropicalis: An Efficient Model Organism

As mentioned earlier X. tropicalis is a close relative of the widely used model

organism X. laevis, with the advantage of a short life span as compared to the latter.
So, X. tropicalis is nowadays gaining rapid importance as a model organism. It has
an established genome sequence which can be manipulated for various biomedical,
cell biology, genetics, developmental biology, and also drug discovery and delivery
studies. Some of its applications as an emerging model organism are enlisted below.
Systematic chemical genetics screens (a term used to describe the difference in
chemical manifestation between mutant strains of various organisms) had been
successfully carried out in X. tropicalis and X. laevis. X. tropicalis is a fast-breeding
and diploid relative of X. laevis but much smaller in size. It is now successfully
adopted for research in developmental genetics and functional genomics. Its
embryogenesis cycle is almost similar to that of X. laevis but has relatively simpler
genome and shorter generation time. This makes it more convenient for use in
genomic and transgenic practices. Its embryos closely resemble those of X. laevis,
except for their smaller size. Therefore, assays and molecular probes developed in
X. laevis can be readily adapted for use in X. tropicalis. It can also be a key model to
host-pathogen interaction studies as it was found in a recent study that Chlamydia
pneumoniae (a human pathogenic bacteria) is pathogenic to X. tropicalis. It is also
used for transcription, translation, and targeted mutagenesis studies by applying
various recent and advanced technologies like transcription activator-like effector
nucleases (TALENs), zinc-finger nucleases (ZFNs), and CRISPR/Cas system. The
usefulness of X. tropicalis as a model species to investigate endocrine disruption and
developmental reproductive toxicity was assessed. X. tropicalis is reported to have
similarity in reproductive system with that of fish, birds, and mammals after devel-
opmental exposure to estrogens. This makes X. tropicalis a promising model for
research on endocrine disruption and developmental reproductive toxicity.
X. tropicalis has also entered the world of cancer, as it was very first studied as a
test organism for blood cancer. Vertebrate biology, particularly the genetic, bio-
chemical, and environmental factors that influence the development of a vertebrate
from embryonic stages through adulthood, can be well studied in X. tropicalis. It is
also finding uses as an important test species for estimating the impact on the health
of rare and endangered species of amphibians, which are exposed to environmental
toxins and disease due to waterborne pollutants and infectious agents such as the
chytrid fungus. It is also finding wide applications in functional genomics. Success-
ful understanding of the activity of genes in this easy model system will be of a
significant help in understanding of similar genes in other vertebrate systems.

17.1.5.1 Threats
There are no known threats caused by the Western clawed frog to humans, till date,
except the hygiene issues. It should be handled with care and aseptically.
828 T. D. Majumdar and A. Dey

Fig. 17.14 Mouse (Mus musculus)

17.1.6 Mouse (Mus musculus) (Fig. 17.14)

The house mouse, Mus musculus, was established in the early 1900s as one of the
first genetic model organisms. The major advantage of it is being a mammal, so it
shares genetic and physiological similarities with humans, therefore efficiently
serves as models for human phenotypes and disease. Other than this it has a short
generation time, produces comparatively large litters, is easily handled and grown,
and shows visible phenotypic variation. There are many biological processes in the
development process of rodent and primate lineages, which are conserved during the
years of evolution, and mice had served invaluable in studying these processes.
Besides this over the years mice had also helped in investigating the developmental
mechanisms by which the conserved mammalian genome develops and
differentiates to give rise to a variety of different species. Today, Mus musculus is
widely known as an excellent mammalian model for studying a wide variety of traits
and diseases, including those involved in metabolism, development, neurological
disorders, immunity, male sterility, adaptive evolution, comparative genomics, and
non-Mendelian inheritance. Mouse (2.5 Gb) has almost 99% similarity in the coding
region genes with humans (2.9 Gb). Despite this they can differ strikingly in
experimental results from humans. The experimental results and the knowledge
developed during a century of research on the mouse present an opportunity of
interpretation of human genes and their functions with experimental studies of
corresponding mouse genes (Table 17.6).
17 Genetic Analysis of Development 829

Table 17.6 Taxonomy of Domain Classification

Mus musculus
1. Kingdom Animalia
2. Phylum Chordata
3. Class Mammalia
4. Order Rodentia
5. Family Muridae
6. Genus Mus
7. Species musculus

17.1.6.1 Mus musculus: Structure and Components (Fig. 17.15)

Mice being a mammal has similar anatomy to humans. The diagram depicts the
internal organs of the mice starting from the esophagus to the anus.
Mus musculus is a mammal and has an anatomy like any other mammal. The
anatomical characteristics of Mus musculus are as follows.
Mammals are endothermic and require necessary insulation to maintain their
body temperature. This is provided by the body hair, which is also a signature of
being a mammal. These animals are warm blooded and have mammary or milk-
producing glands. Oil glands or sebaceous glands are located almost all over the
body. They produce sebum (a lipid mixture) that is secreted onto the hair and skin
and provides water resistance and lubrication. Sweat glands or sudoriferous glands
are absent in mice.
Mice have the dental formula as follows: incisors 1/1, canine 0/0, premolars 0/1,
and molars 3/3. Mice have a total of 18 teeth, including 4 incisors, 2 premolars, and
6 molars. Incisors are very important for mice. Mice have a natural yellow pigment
in their teeth. Mice are monophyodont, i.e., they have only one set of teeth
throughout life.
The skeletal structure can be classified into axial and appendicular. The axial
skeleton consists of the bones which make up the skull, vertebral column, sternum,
and ribs, whereas the appendicular skeleton consists of bones which make up the
appendages and girdles. The dorsal portion of the skull is made up of a series of
bones, namely, unpaired occipital and interparietal, the paired parietals, frontals,
nasals, premaxillae, maxillae, zygomatics, and squamosals. The occipital,
interparietal, and parietals form the cranium of the skull. Its posterior wall is made
up of the occipital, interparietal forms a part of the top dome, and the parietals form
the rest of the top and a portion of the sides of the cranial cavity. Frontals and nasals
form the anterior portions of the brain and along with premaxillae, maxillae,
zygomatics, and squamosals form the face and upper jaw. The vertebral column or
the spinal cord is formed as an extension of the occipital bone. Occipital continues
anteriorly as the spinal cord and forms a support of the thoracic and abdominal
cavities, further extending up to the tail. The spinal cord consists of the following
vertebrae: 7 cervical, 12–14 thoracic, 5 or 6 lumbar, 4 sacral, and 27 to 30 caudal.
The most anterior of the seven cervical are the atlas and axis. Each thoracic vertebra
articulates with a pair of ribs and some sacral articulates with the innominate bones
of the pelvic girdle. The appendicular skeleton consists of the paired pectoral and
830 T. D. Majumdar and A. Dey

Fig. 17.15 Anatomy of Mus musculus

pelvic girdles and the bones of the limbs. Each pectoral girdle consists of dorsal
scapula and ventral clavicle. The adult pelvic girdle is made up of right and left
innominate bones. These bones are further attached dorsally to one or more sacral
vertebrae and ventrally at the pubic symphysis. The bones of the forelimb are
humerus, radius and ulna, eight carpals, five metacarpals, five first phalanges, four
second phalanges, and five third phalanges with their tips. The bones of the hind
17 Genetic Analysis of Development 831

limb are femur, tibia and fibula, seven tarsals, five metatarsals, and the same number
of phalanges as in the forelimb.
The heart of the mice is a closed circulatory system, i.e., blood remains within the
vessels. It is enclosed by the pericardial cavity, which is a division of the thoracic
cavity. Being a mammal a mice’s heart is four chambered. The chambers are
muscular-walled and are divided into the left and right atria and the left and right
ventricle. The right side (right atria and right ventricle) receives the impure blood
from the veins and pumps it to the lungs for oxygenation. The left side (left atria and
left ventricle) receives oxygenated blood from the lungs and pumps it to the body via
the arteries. There are two principal vessels that carry blood from the heart. They are
the pulmonary artery from the right ventricle and the aorta from the left ventricle.
Branches of these arteries supply all parts of the body. There are three principal veins
entering the atria of the heart: the pulmonary veins from the lungs; the superior vena
cava from the head, neck, chest, and forelimbs; and the inferior vena cava from
regions of the body posterior to the diaphragm.
The lungs are large and fill the entire thoracic cavity along with the heart.
Muscular diaphragm controls the volume and movement of air to and fro the lungs.
The lymphatic system consists of vessels which transport the lymph to the blood,
from lymphatic tissues. The lymph gets distributed, on its way to the blood channels,
partially to the nodes that lie in the course of the lymphatic vessels and also to the
peripheral nodules present at the beginning of lymph channels in the digestive tube.
Mice do not possess palatine and pharyngeal tonsils.
The spleen is present in the left anterior quadrant of the abdominal cavity. It is the
largest secondary immune organ in the body and is slightly curved, elongated, and
oval. It initiates immune reactions against blood-borne antigens and purifies the
blood of foreign and toxic matters. It also removes old or damaged red blood cells
from circulation. It is composed chiefly of lymphatic tissue but has no lymph vessel
connections. The thymus was regarded as a vestigial organ with little or no function
in the adult animal. But now it has been categorized as an endocrine gland made up
of reticular tissue. In rodents the thymus has mainly two functions. It serves as the
site for lymphopoiesis in the embryo and newborn. The formed lymphocyte migrates
from the thymus to the spleen, lymph nodes, and other lymphoid organs. In addition,
the thymus produces an immunotrophic hormone, one which helps in enhancing the
immunological potential of cells.
Mice contain three pairs of salivary glands which are located in the subcutaneous
tissue of the face and neck: the parotid, submandibular, and sublingual. Each of them
is connected with the oral cavity through a single excretory duct. They are not mixed
glands and each has only one type of secretory cell. Parotid and submandibular
secrete serous and sublingual secretes mucous. The digestive tube extends from the
pharynx to the anus and includes the esophagus, stomach, small intestine, and large
intestine. The small intestine consists of duodenum, jejunum, and ileum. The large
intestine consists of caecum, colon, and rectum. The liver is a large gland occupying
the anterior third of the abdominal cavity. It touches the arch of the diaphragm
anteriorly and partially meets the stomach and duodenum posteriorly. The liver
functions include producing bile which digests fat, storing glycogen and
832 T. D. Majumdar and A. Dey

transforming wastes into less harmful substances. The mouse have a gall bladder
which is absent in rats. The pancreas is a diffuse gland with a pinkish color. It is
suspended in the mesenteries between the stomach, duodenum, and ascending and
transverse colons. It has both endocrine and exocrine functions. As an exocrine
organ, it secretes enzymes or digestive juices into the small intestine. There, it
continues breaking down food that has left the stomach. As an endocrine organ, it
produces the hormone insulin and secretes it into the bloodstream, where it regulates
the body’s glucose or sugar level. The urinary system includes the kidneys, ureters,
urinary bladder, and urethra. The principal function of the urinary system is the
maintenance of water and electrolyte homeostasis. The female genital system is
composed of ovaries, oviducts, uterus, and vagina. The male genital system consists
of testis, excretory ducts, accessory glands, urethra, and penis.
The reproductive system is well developed. Both ovaries are equally functional,
and the ova are fertilized in the oviducts. Embryo develops within the uterus and lies
within a fluid-filled amniotic sac, which provides necessary nourishment to the
embryo and also protects the embryo from any friction. It has a well-built and
connected placenta which connects the embryo with the mother’s system and
provides nourishment and immunity to the embryo. Male testes are contained within
the scrotum outside the body cavity.
The mouse has a complex brain like other mammals, smaller in size then rat.
Mouse brain consists primarily of water, protein, and lipids. It consists of enzymes
like alkaline phosphatase and glutamic acid decarboxylase, but their expressions
vary with development. Alkaline phosphatase is present in large amounts during
fetus stage, whereas glutamic acid decarboxylase is present all throughout but its
amount increases with maturation up to 30 days. Thus maturation to adulthood
involves shifts in enzyme activity which presumably is related to morphological
and functional changes.

17.1.6.2 Life Cycle and Reproduction (Fig. 17.16)

Mice reach sexual maturity in about 5 weeks and can mate within 2 months of birth.
Mice have a short gestation period of 21 days, and a female can give birth to about
seven litters in a year.
The mouse life cycle will vary depending on their habitant but typically lasts
about 2 years indoors. The first stage of a mouse’s life cycle lasts about a week. It
then reaches its adolescence which lasts a few weeks and is marked by big personal-
ity changes. Over the next several weeks, it eats plenty of food to provide fuel to his
growing body. The male becomes sexually mature once he is 5 weeks to 10 weeks
old, and females are 8–12 weeks old before they’re sexually mature adults. Once
they reach maturity, they undergo mating. Once mating completes, females go into
heat for 4–5 days. Once pregnant, mouse gives birth after 3 weeks, and five to eight
litters (baby mice) are produced at a time. The females can undergo mating and
reproduce up to ten times per year. At the start of the mouse life cycle, newborns are
hairless and blind but develop thin fur and gain sight and mobility after 2 weeks.
Mice reach sexual maturity in about 5 weeks and are ready to mate about 2 months
after birth. Mice can breed throughout the year if they get suitable conditions, and
17 Genetic Analysis of Development 833

Fig. 17.16 Life cycle of Mus musculus

with a short gestation period of only 21 days, they can give up to seven litters a year.
With the birth of each individual mouse and reaching sexual maturity, the total
population of a particular habitat of mice under ideal conditions can reach up to
15,000 in a year. The maximum life span of a mouse is about 3 years. Females are
capable of becoming pregnant immediately after giving birth. They can even give
birth and raise two healthy litters of normal size and weight simultaneously without
significantly changing their own food intake. However when there is scarcity of
food, the females can extend their pregnancy by over 2 weeks and give birth to
young ones of normal number and weight.
Males can ejaculate multiple times in a row and can mate with multiple females,
especially when there are several estrous females present. Males also have the
capacity to copulate at much shorter intervals than females. Dominant males have
higher efficiency of mating, and the females are more likely to use the sperm of
dominant males for fertilization. In group mating, females often switch partners and
show a clear choosing preference between an unknown male and ones with whom
they have already mated (a phenomenon called the Coolidge effect).

17.1.6.3 Mus musculus: As a Model Organism

Mice surpasses yeasts, worms, and flies in being excellent model system to studies
regarding immune, endocrine, nervous, cardiovascular, skeletal, and other complex
physiological systems that mammals share. Being a mammal, mice can easily
834 T. D. Majumdar and A. Dey

develop diseases like cancer, atherosclerosis, hypertension, diabetes, osteoporosis,

and glaucoma and therefore can act as an efficient model for their study and effective
cure. Other diseases which affect humans but not mice like cystic fibrosis and
Alzheimer’s can be induced by manipulating the mouse genome and environment.
It has relatively low cost of maintenance and has a higher multiplication rate with
reproducing once every 9 weeks. These make it advantageous in biomedical
research. Also it is nowadays finding more reliability as a robust tool in the field
of aging. Since laboratory mice have a life expectancy of only a few years, genetic
manipulation can be done on life span and aging parameters during the relatively
short period. Moreover, experiments on mice with an extended life span and those
showing premature aging, together with genetic mapping strategies, have provided
novel views toward fundamentals of aging. Again with recent advances in mapping
neural circuitry governing behavior, mouse has also evolved to be a model for
studying anesthetic action.

17.1.6.4 Threats
Mouse and other rodents can cause and spread many diseases, mostly viral. Some of
them are as follows:

1. Hantavirus—A deadly viral disease spread from rodents’ feces and urine to
humans.
2. Salmonellosis—A food poisoning spread by rodents’ feces.
3. Lymphocytic choriomeningitis—Rodent-borne viral disease which can cause
serious neurological problems.
4. Rat-bite fever—A fatal infectious disease spread by infected rodents or consump-
tion of food contaminated by them.
5. Bubonic plaque—Also known as “black death,” though almost eradicated these
days. In the Middle Ages, it was one of the most deadliest rodent-borne diseases
creating huge epidemics.

17.1.7 Zebrafish (Danio rerio) (Fig. 17.17)

Zebrafish (Danio rerio) is a teleost and a member of the minnow family that lives in
freshwater and is no bigger than 2 inches in length. In the 1970s, George Streisinger
first used it as a model organism, because of its simplicity as compared to mouse and
ease to manipulate genetically. They have various distinctive features which led to
its widespread use as a model system. These include their ease of care, fecundity,
rapid development, small size, and ease of manipulation. The brain, digestive tract,
musculature, vasculature, and innate immune system of zebrafish have physiological
and genetic similarity to humans, having genes which have functional similarity with
70% of human disease genes. They have proved to be a huge help to scientists and
had changed their way of understanding treatments for cancer, spinal cord injuries,
and potentially the regeneration of limbs in humans. It is a powerful vertebrate model
17 Genetic Analysis of Development 835

Fig. 17.17 Zebrafish (Danio rerio)

Table 17.7 Taxonomy of Domain Classification

Danio rerio
1. Kingdom Animalia
2. Phylum Chordata
3. Class Teleostei
4. Order Cypriniformes
5. Family Cyprinidae
6. Genus Danio
7. Species rerio

system for studying developmental biology and serves as a model for various
diseases and screening for novel therapeutics (Table 17.7).

17.1.7.1 Danio rerio: Structure and Components (Fig. 17.18)

The most important organs of fish are the gills and swim bladder which help in its
respiration and maintain buoyancy, respectively. The fins help in swimming. The
other important organs are the kidney, heart, spleen, pancreas, intestine, urogenital
pore, and the reproductive system.
Many of the organs of zebrafish are analogous with mammalian counterparts,
including the brain, heart, liver, spleen, pancreas, gallbladder, intestines, kidney,
testis, and ovaries.
The very first thing important for a fish’s survival is its ability to swim and
maintain its buoyancy and oxygen supply during its movement. Gills and the gas
bladder help to maintain the fish’s buoyancy. The gills play an important role in the
oxygenation of the blood. Water flows via the gills and brings in oxygen for the
blood. Blood, from the body, flows via the filament arteries and releases carbon
dioxide to the water. Besides this fish’s acid-base balance and osmoregulation are
also regulated partly by gills. They also help in excretion of waste products. The gas
bladder, or swim bladder, plays the key role in maintaining buoyancy. In the embryo
836 T. D. Majumdar and A. Dey

Fig. 17.18 Anatomy of Danio rerio

stage, it is derived from the upper digestive tract but practically has no digestive
function. In the zebrafish, the swim bladder and the esophagus are connected by a
pneumatic duct which allows the fish to fill up the swim bladder by gulping in air.
Fish’s heart is one of the simplest in the developmental hierarchy of animals. It
consists of only two chambers, i.e., one atrium and one ventricle. In zebrafish, the
heart is situated anterior of the main body cavity and ventral to the esophagus.
Deoxygenated blood from the body is carried by sinus venosus, and oxygenated
blood from the heart is distributed by the ventral aorta to the gills via the afferent
branchial arteries. The blood present in the sinus venosus subsequently passes
through the sinoatrial valve into the atrium. The atrium then contracts to force the
blood into the ventricle, via the atrioventricular valve. The ventricle first dilates to let
the blood in and then contracts to pump the blood into the bulbus arteriosus via the
ventricular-bulbar valve. From here the blood is distributed to the gills.
Zebrafish body is not distinctively divided into stomach, small intestine, and large
intestine. Instead one single long tube is present as the intestine which folds twice in
the abdominal cavity. The difference can be traced in the morphological anatomy of
the mucosa columnar epithelial cells and the number of goblet cells which depicts
that both the cells have functional differentiation between them. The intestinal
epithelium consists mainly of columnar-shaped absorptive enterocytes and secondly
of the goblet cells.
The zebrafish kidney lies on the ventral side of the vertebral column, distinctly
divided into head and trunk regions. It is further divided into nephrons with a
glomerulus, proximal tubules, distal tubules, and collecting ducts, as found in
mammals. The kidney along with the spleen filters out the foreign particles and
defective blood cells from the body. The spleen is made up of parenchyma cells,
17 Genetic Analysis of Development 837

which in turn consists mainly of erythrocytes and thrombocytes (red pulp). Bacterial
cells and other foreign bodies are trapped in splenic ellipsoids.
Though the fish possess distinct exocrine and endocrine functioning pancreatic
tissues, a discrete pancreas is absent in it. The exocrine pancreatic tissue is scattered
along the intestinal tract, while the endocrine pancreatic tissue encompasses α-cells,
β-cells, and δ-cells. The α-, β-, and δ-cells have distinctive functions of producing
glucagon-like peptide, insulin, and somatostatin, respectively.
Zebrafish testes are lateral, paired organs that comprise a series of tubules or blind
sacs. Testis contains the Sertoli cells which supports the sperm formation. Cytoplas-
mic projections of Sertoli cells lead to the formation of cysts. Spermatogenesis
occurs in these cysts which completely surrounds a single spermatogonium. Mature
spermatozoa are carried to the genital orifice by two caudally merged ducti. Ovaries
are paired, elongated structures. A short oviduct conducts the eggs to the outside.
Fish liver consists of three lobes that lie along the intestinal tract. The liver
maintains the metabolic homeostasis of the body. This includes the processing of
carbohydrates, proteins, lipids, and vitamins. It also detoxifies and synthesizes serum
proteins such as albumin, fibrinogen, complement factors, and acute phase proteins.
The gall bladder is composed of the bile ducts. The gall bladder carries the greenish
bile that reaches the intestine via the common bile duct.
Lastly, the basic components of the zebrafish brain have similarity with that of
higher animals. It is divided into five regions: the telencephalon, the diencephalon,
the mesencephalon, the metencephalon, and the myelencephalon. Senses of smell,
reproductive behavior, feeding behavior, color vision, and some other aspects of
memory are controlled by the telencephalon. The olfactory bulb connects the
olfactory (smell) organ to the telencephalon. The diencephalon is further divided
into three components: the epithalamus, the thalamus, and the hypothalamus. The
mesencephalon mainly deals with the normal sight.

17.1.7.2 Life Cycle and Reproduction (Fig. 17.19)

Zebrafish grows from egg, cleavage, gastrula, to adult. The gastrulation is seen to be
completed within the initial 16 h. The full-grown adult emerges about 4–5 days after
fertilization.
A fertile zebrafish lays approximately 200–300 embryos every 5–7 days. The
embryo develops very fast and is transparent, so the stages of development can be
viewed, which makes the study easy. The very first stage of the embryo is blastula-
tion which lasts only for 3 h; this is followed by gastrulation which ends up within
the next 5–6 h. Segmentation of the body and formation of most primary organs like
the ears, eyes, segmenting muscles, and brain are observed within the first 20–24 h.
Within the next 72 h, the embryo hatches out from the egg and, after an initial
acclimatization period, goes in search of its food by the next 48 h. In a period of just
4 days, the embryo starts developing and showing features of adulthood. The
developmental stages can be explained in brief as follows.

Zygote: It is the fertilized egg which is formed after completion of the first zygotic
cell cycle.
838 T. D. Majumdar and A. Dey

Fig. 17.19 Life cycle of Danio rerio

Cleavage: Zygote cleaves to two to six cells very rapidly in a synchronous manner.
Blastula: It is characterized by rapid and metasynchronous cell cycles, which is
followed by a lengthened and asynchronous division, the midblastula stage. This
asynchronous division is followed by the cells of one side of the blastula
spreading and surrounding the remaining cells and the yolk, in a process called
epiboly. This is the first coordinated cell movement in zebrafish embryos and
begins before gastrulation.
Gastrula: Involution, convergence, and extension from the epiblast, hypoblast, and
embryonic axis are the various morphogenetic changes which come during
gastrulation. Beginning of gastrulation also marks the end of epiboly.
Bud: Epiboly totally ends and the yolk plug gets completely covered.
Segmentation: At the end of the epiboly, the first and second somites appear which
marks the beginning of the body segmentation. Primary organogenesis and
earliest movements take place at this time. Pharyngeal arch primordia, early
neuromeres, and the tail develop.
17 Genetic Analysis of Development 839

Pharyngula: During this phase the body axis straightens from its early curvature
around the yolk sac and marks the phylotypic stage of the embryo. Metabolic
processes like circulation and pigmentation start. Fins begin to develop.
Hatching: Morphogenesis of primary organ systems completes. Cartilage develop-
ment in the head and pectoral fin occurs. With this the egg hatches
asynchronously.
Larval: Then finally comes the larval stage when swim bladder inflates. The fish
starts food-seeking with active avoidance behaviors.

After the larval stage, finally the fish reaches its sexually matured adult form.
Once reaching the adult stage, they allocate to suitable places for reproduction. The
shape of the belly is a distinguishable character between the genders as the belly of
the males is sleeker, while those of females are fuller and rounder. Zebrafish show
signs of aging when reaching 2 years of life, although they can live for 3–4 years.
The average life span of zebrafish depends on strain and rearing.

17.1.7.3 Danio rerio: As a Model Organism

Zebrafish has become much popular as a model organism in recent decades mainly
because of its certain advantages over other model organisms like ease of care,
prolific breeding, rapid external development, requirement of less space to culture,
and low maintenance cost. These features make this fish an attractive model organ-
ism for developmental, toxicological, transgenic studies, human disease, drug dis-
covery, cancer, etc. Some of the recent developments of use of the fish as a model
organism are enlisted here. Much of the preliminary work on zebra fish was on
developmental biology, in which it has done a distinctive contribution over the years.
When Streisinger and Kimmel first used it as a model system in early twentieth
century, it was for the study of developmental biology like embryonic development,
muscle development, nervous system, etc. Since then it has been used for various
similar studies, like that on development of enteric nervous system, angiogenesis,
and regeneration, mainly because it has 70% homologue in disease genes with that of
humans. It has also been used in investigating aquatic endocrine-disrupting
compounds that can cause alterations in development, physiological homeostasis,
and health of vertebrates. Complex behavior like that of learning and memory,
aggression, anxiety, and sleep had been tried to be studied on the fish. The studies
strongly suggest that conserved regulatory processes function in both zebrafish and
mammals and result in common underlying behavior between them. Apoptosis is
known to play an important role in mechanisms like embryogenesis, tissue homeo-
stasis, and immune system regulation. The zebrafish serves a powerful model
organism for studying apoptotic cell death in vertebrates, both during normal
development and under cellular stress. Zebrafish are well suited in exploring ocular
development, function, and disease as they have a well-developed eyesight and their
retina and lens show morphological similarity to other vertebrates including humans.
The most advanced use of zebrafish is in cancer research and drug testing and
targeting. It has also been extensively used in examining other human disorders
like hematopoietic disorders, kidney disorders, and cardiovascular disorders. Being
840 T. D. Majumdar and A. Dey

an aquaculture fish, Danio rerio can help in studying growth behavior of other
aquaculture fish. Research on its nutrition and growth, stress, and disease resistance
can be expected to improve husbandry and formulated feeds of aquaculture species.

17.1.7.4 Threats
It has no reported threats till date except maintaining proper hygiene while handling
the fish.

17.2 Mechanism of Gene Control in Development

17.2.1 Maternal Effect

Multicellular organisms are formed by the synchronization of the male and female
gametes, i.e., ovum and sperm. The conjunction of the gametes gives rise to a single
fertilized cell. This cell then generates more cells that will interact with one another
via highly conserved signaling pathways to build an embryo with defined axes and
organ systems. The development of the embryo continues and ultimately gives rise
to a mature adult capable of producing the cells necessary to form the next genera-
tion. On the other hand, hermaphrodites can produce both sperm and ovum. This egg
or embryo, during its development, gets a major contribution of DNA, nutrition,
mRNA, and proteins from the ovum, i.e., the mother gamete. This legacy which it
obtains from its mother is called the maternal effect. A huge number of genes
together contribute to this effect. These maternal gene products are responsible for
regulating meiosis, transitions between meiotic and mitotic cell cycles, and oocyte
development. They also contribute to the important development cycles of the
embryo including fertilization and help in activation of the embryo’s own gene
products after the preliminary utilization of mRNAs and proteins provided by the
mother, during zygotic genome activation. Besides the genetic contribution, a
mother can also pass on their behavioral traits to their offspring; for instance,
nursing, grooming, predator defense, and “decisions” on when and where to lay
eggs can all affect the offspring and its survival. Maternal effects can influence
selection of a population of offspring into a particular environment. It plays an
important role in determining various ecological and evolutionary processes like
population dynamics, phenotypic plasticity, niche construction, life history evolu-
tion, and the evolutionary response to selection. The traits expressed due to maternal
effect affect the fitness of the progeny, which in turn can play a role in selection of
the offspring in the environment and therefore govern the evolutionary dynamics of
the population. Additionally, maternal effects can also affect the change in responses
of the offspring toward adaptation with changing environments.
The maternal effect is not the same in all organisms. In some animals, only the
first few initial cleavage cycles are dependent on maternal RNAs and proteins, like
that in mice and nematodes (C. elegans), before their zygotic genes get activated.
Other organisms, such as Drosophila, Xenopus, and zebrafish, rely on maternal
RNAs and proteins for much of the developmental period before activation of the
17 Genetic Analysis of Development 841

embryonic genome. Several genes and their corresponding effect play an important
part in synchronizing the maternal effect in the above model organisms. Some of
them are discussed below.
The soil nematode C. elegans is one of the most highly studied organisms for
maternal gene effect. The fertilization and the development of the nematode require
the simultaneous action of a number of maternal genes. Some of them are mes-2,
mes-3, mes-4, mes-6, skn-1, pie-7, and mex-7. Among them mes-2, mes-3, mes-4,
and mes-6 are classified as sterile genes. It has been found that the mutant
phenotypes of these sterile genes are similar and thus were considered to have a
common origin. They are involved in a common process of encoding nuclear
proteins that are essential for germline development. MES-2 and MES-6 have
homology to members of the Polycomb group of chromatin regulators, found in
insects and vertebrates. They are homologous to enhancer of zeste and extra sex
combs. MES-3 is a novel protein, and MES-4 has function in growth control and is a
SET-domain protein. In nematodes maternal determinants are found to play a role in
the autonomous or cell-intrinsic developments of the early embryonic blastomeres.
Pharyngeal cells are produced by only the posterior blastomere P1 in C. elegans after
the first cleavage. From a study it was found that P1 can produce pharyngeal cells
with the help of the maternal gene skn-1. C. elegans embryogenesis bears a stage
called MS, which is an 8-cell blastomere. Cleavage of embryo at this stage produces
pharyngeal cells, body wall muscles, and also cell deaths. Studies have shown that
two maternal effect genes of C. elegans, pie-7 and mex-7, are involved in the
division of MS. MS can show similarities to the wild-type blastomere stage if
these two genes undergo mutations. In pie-7 mutants one additional posterior
blastomere adopts an MS-like fate, and in mex-7 mutants four additional anterior
blastomeres adopt an MS-like fate. A key molecular component of the duplication
process of centrosome/centriole during the cell cycle is a protein kinase called
ZYG-1. The paternal product of it regulates duplication and bipolar spindle assem-
bly during the first cell cycle, whereas its maternal product regulates these processes
thereafter.
In Drosophila embryo, the pattern of segmentation is controlled by both zygotic
and maternal genes. About 25 zygotically active genes and 20 maternally active
genes jointly synchronize for the process. The expression of fushi tarazu (ftz), an
active zygote segmentation gene, at the cellular blastoderm stage in progeny which
has mutations in six maternal effect genes, namely, exuperantia (a member of the
anterior class of maternal effect segmentation genes); staufen and vasa (members of
the posterior class); and torso, trunk, and fs(l)N (members of the terminal class), was
observed. The gene ftz showed disruption in its normal functioning in the mutant
varieties, depicting the importance of maternal effect on growth of Drosophila.
Staufen gene was found responsible for localization of both anterior and posterior
maternal determinants. In another study, it was found that three genes, dorsal (dl),
twist (mi), and snail (ma), undergo maternal-zygotic interactions to produce the
dorsoventral pattern of the Drosophila embryo. It was also seen that introduction of a
new maternal genetic element, named the selfish-genetic element, in wild-type
Drosophila, made them resistant to insect-borne pathogens.
842 T. D. Majumdar and A. Dey

In mouse embryo development, involvement of around 14 MEGs (maternal effect

genes) have been studied, namely, maternal antigen that embryos require (Mater),
heat shock factor 1 (Hsf1), oocyte-specific deoxyribonucleic acid (DNA)
methyltransferase-1 (Dnmt1o), DNA-cytosine-5-methyltransferase 3-like
(Dnmt3l), formin-2 (Fmn2), postmeiotic segregation increased 2 (Pms2), T-cell
leukemia/lymphoma 1a (Tcl1a), nucleophosmin/nucleoplasmin 2 (Npm2), germ
and embryonic stem cell enriched protein (Stella), zygote arrest 1 (Zar1), and
ubiquitin-conjugating enzyme.

1. The gene which transcripts Mater protein is present in the cytoplasm of growing
oocytes but not in other tissues. It is expressed till late blastocyst stages. A single
copy of gene encodes the Mater protein.
2. Stress stimuli like heat, etc. activates Hsf1, which then activates stress-inducible
genes.
3. Both the nuclei of early stage oocytes and the cytoplasm of mature oocytes
contain the Dnmt1o gene. It helps in maintaining genomic methylation patterns
in mammalian somatic cells.
4. Dnmt3l is associated with catalyzation of de novo methylation of CpG islands.
5. Fmn2 is expressed in the central nervous system, spinal cord, and brain from
development to maturation stages.
6. Pms2A is required for methyl-directed post-replication mismatch repair. It is a
DNA mismatch repair gene homologue and was identified as a MEG using null
mutation mice.
7. Tcl1a helps in the transduction of anti-apoptotic and proliferative signals in
T-cells and increases AKT kinase activity by interacting with AKT.
8. Npm2 transcripts are specified to growing oocytes. Its protein is expressed in the
nucleus of oocytes before germinal vesicle breakdown (GVBD) and in the
cytoplasm after GVBD.
9. Stella is a novel gene expressed in primordial germ cells (PGCs), cytoplasm of
oocytes and embryos, and early developmental stage pre-implantation embryos
especially in pronuclei of zygotes.
10. The transcripts of Zar1 are detected and expressed in high amount from early
oocytes to the initial zygotic embryo stages.
11. Ube2a is a ubiquitin-conjugating enzyme and associated with DNA repair. It is a
homologue to RAD-6, which is a radiation-repair gene.
12. Zfp36l2 binds to mRNAs having class II AU-rich elements and aids in its
degradation.
13. Uchl1 is a deubiquitinating enzyme which is expressed in organs like the ovary,
testis, placenta, and neuron.
14. Filia binds to Mater and is crucial for pre-implantation embryo development.

Dorsal axis in amphibians consists of notochord, somites, neural tissue, and head
structures. In a study it was found that β-catenin encoded by maternal mRNA is
needed for the development of dorsal axis of Xenopus. Xwnt-5A transcripts, a
maternal gene, are expressed throughout development of Xenopus and are well
17 Genetic Analysis of Development 843

active in the anterior and posterior regions of embryos at late stages of development.
Overexpression of Xwnt-5A in Xenopus embryos leads to complex malformations.
Xotx2, a Xenopus homeobox maternal gene, is expressed at low levels throughout
the development of the frog from unfertilized egg to late blastula, when its expres-
sion increases and has a function in the formation of the frog’s brain. Foxi1e is a
zygotic transcription factor that is essential for the expression of early ectodermal
genes. Foxi1e mRNA is maternally encoded and highly enriched in hemisphere cells
of the blastula of the frog.
Over the years, advanced researches in zebrafish have made this organism
impeccable in detailed understanding of the role of maternal factors in early verte-
brate development. A large number of maternal effect genes have been discovered in
this teleost fish. Some of them are janus (contributes in cell adhesion), yobo (has a
role in axis convergence and extension), ichabod (functions in dorsal organizer
induction), alk8 (plays a role in development of ventral cell fates), pbx4 (helps in
hindbrain segmentation and rhombomere identity), etc. A gene named mission
impossible (mis) was identified in zebrafish. The gene is a maternal gene and
plays an active part during gastrulation, contributing in activities like cell movement
and the activation of some endodermal target genes.

17.2.2 Zygotic Gene Activity

17.2.2.1 Body Segmentation (Fig. 17.20)

The genes show a coordinative and chain-wise activation of gap genes by maternal
genes, followed by activation of pair-rule genes by gap genes and then finally
activation of segment polarity genes by pair-rule genes.
Segmentation in vertebrates and arthropods is a process in which body axis is
formed by serial repetition and differentiation in patterns of similar anatomical
modules. The process involves simultaneous activation of a series of transcription
factors. The activation of these transcription factors is a chain-wise process in which
many genes are involved. These genes initiate the pathway of these transcription
factors that, in turn, induce the expression of other transcription factors, thus creating
cascades of gene expression. In this way a multistep signal pathway is initiated,
which ultimately amplifies the initial signal and results in the segmentation of the
body. In vertebrates, segmentation results in the development of the spine, head, and
limbs, whereas in insects, it results in formation of the abdomen, head, and thorax.
The segmentation is a result of the synchronous activity of a series of genes. Most
of the maternal genes taking part in segmentation are present in the eggs during the
time of fertilization. Their genetic regulation starts before the onset of the cellular
blastoderm stage. Firstly the proteins transcripted by the maternal coordinate genes,
namely, bicoid, nanos, caudal, etc. gets diffused from the anterior and posterior
poles of the embryo. The maternal transcripts also have definitive activation sites
during embryogenesis like caudal (cad) and hunchback (hb) which are ubiquitously
distributed in the fruit fly embryo, whereas transcripts bicoid (bcd) and nanos (nan)
are found only in the anterior and posterior parts of the embryo, respectively. Once
844 T. D. Majumdar and A. Dey

Fig. 17.20 Gene control in

body segmentation of
Drosophila

translated, the signals from this factors control the expressions of the zygotic genes
in the developing fruit fly. The first zygotic genes to be activated by the maternal
genes are the zygotic gap genes (the very first zygotic genes responsible for
segmentation), i.e., hunchback, Krüppel, knirps, etc. They are expressed all along
the anterior to posterior axis of the fly embryo. The gap gene domains circumscribe
the progenitors of several adjoining segments and sub-divide the embryo into
anterior, middle, and posterior regions. The gap genes regulate each other and the
next set of genes in the hierarchy, the pair-rule genes, namely, even-skipped, hairy,
fushi tarazu, etc. Once activated the pair-rule genes generate periodic gene expres-
sion events called pair-rule stripes. The strips decide the position of the boundaries
between the segments to be formed and are expressed in seven stripes of cells
corresponding to every other segment. Their formation and regulation are controlled
and governed by the pair-rule genes. Pair-rule gene expression then induces the final
set of transcription factors, i.e., the segment polarity genes, namely, wingless,
hedgehog, engrailed, etc. This set of genes is expressed in 14 segmentally repeated
stripes. The segment polarity genes, unlike the other classes of segmentation genes,
require regulatory proteins other than transcription factors (i.e., secreted signaling
molecules, receptors, kinases, etc.). These proteins mediate interactions between
17 Genetic Analysis of Development 845

cells. At the end the body is divided into identical repeated pattern of segments
expressed by the segment polarity genes. The final group of genes are the homeotic
genes which control the character (i.e., head, thorax, abdomen) of each segment.
In situ RNA and protein expression patterns of these genes gave the idea of where
the genes are first expressed:

• Maternal coordinate genes: The maternal genes which make maximum contribu-
tion to the process of segmentation are the bicoid, localized at the anterior end of
the embryo and nanos, located at the posterior end of the embryo. Both of them
are primarily expressed during oogenesis.
• Gap genes: Gap genes are first expressed at the syncytial blastoderm stage. They
are the Krüppel, hunchback, and knirps. Krüppel mRNA is found in
parasegments 4–6, whereas hunchback and knirps mRNA are found in the
anterior and posterior half of the embryo, respectively.
• Pair-rule genes: They are first expressed during syncytial blastoderm. They
decide the boundary of future segmentation around each embryo by forming
the seven stripes.
• Segment polarity genes: Primarily expressed during cellular blastoderm, segment
polarity genes play the most important part during the final segmentation process.
They form the 14 stripes of transcription around each embryo which decides the
ultimate segmentation of the body axis.

The maternal factors which are found to play an important role in segmentation in
fruit flies are studied not to be as important for other insects and neither in
vertebrates. However, the hox genes, which are said to control the ultimate fate of
each segment in the body, are found to be universal. They define what part of the
body each segment will become later in the cascades of nearly all insects and
vertebrates. Importance of hox genes in fruit flies was identified by a number of
mutation studies. Mutations in specific hox genes caused legs to grow out of a fly’s
head, wings to grow where they shouldn’t be, and a number of other physical
deformities.

17.2.2.2 Organ Formation (Fig. 17.21)

The cells during the process of embryogenesis get genetically programmed to form
the organs which make up the body. The organs are specialized tissues which
synergistically work together to maintain various functioning of the body and also
contribute to environmental adaptation (Fig. 17.21).
Flowchart depicting the cells of the embryo undergoing morphogenesis, specifi-
cation, proliferation, and differentiation to undergo organogenesis.
A number of genes work together in an organized manner to ultimately form the
mature organs. Some of the genes studied in various model organisms which
contribute in organogenesis are listed below:

1. A homeotic gene, sex combs reduced (Scr), was found to be involved in the
formation of salivary glands in Drosophila melanogaster. It was found that the
846 T. D. Majumdar and A. Dey

Fig. 17.21 Organogenesis

anterior-posterior extent of the salivary gland primordium, a placode of columnar

epithelial cells derived from parasegment 2, is established by the positive action
of the gene.
2. The development of Drosophila hindgut is a good model system for studying
establishment of epithelial cell primordium, its internalization by gastrulation,
patterning in the anteroposterior and dorsoventral axes, interaction of epithelium
17 Genetic Analysis of Development 847

with mesoderm and mesenchymal layers, cell shape change, and cell rearrange-
ment. It was found that caudal/Cdx, brachyenteron, fork head/HNF-3, and wing-
less/Wnt genes are actively involved in posterior patterning, cell rearrangement,
and gut maintenance in the fly.
3. A zygotically active gene called pha-4 is studied to have function in proper
development of the pharynx of C. elegans. The PHA-4 protein is present in
nuclei of almost all pharyngeal cells. Mutation in the gene can block the proper
formation of the pharynx.
4. A protein complex named Chromatin Assembly Factor 1 (CAF-1) aids in chro-
matin assembly during DNA replication. It was found that a mutation in CAF-1b
in zebrafish caused hindrance in cell cycle and led to defects in its progression. It
also interrupted the differentiation of several organs, including the retina, optic
tectum, pectoral fins, and head skeleton.
5. A detailed analysis of the global gene expression of mouse organogenesis from
egg to the adult was studied. Comparative analysis identified both conserved and
divergent gene regulations in mouse and human organogenesis.
6. Hox genes (regulates body segmentation) require coordination of various down-
stream target genes for its function. These genes are called the “realizator” genes.
Abdominal-b, a hox gene in Drosophila, involved in the organogenesis of the
external respiratory organ of the larva, was found to activate four intermediate
signaling molecules and transcription factors.

17.2.3 Analysis of Development in Vertebrates

17.2.3.1 Vertebrate Homologues

Homologues in vertebrates are usually referred to as genes or structures in different
organisms in different taxa, which share a common ancestry. Some of the genes in
model organisms which share vertebrate homology are discussed below:

1. A certain group of proteins are responsible for guiding the migrating cells and
axons to their targets in the nervous system of the developing embryo under
extracellular environment. One such protein family found in vertebrates is the
netrins. They are involved in guiding axons and cells to their targets by function-
ing as diffusible attractants and repellants. In C. elegans one such netrin is
UNC-5. Loss of proper action of unc-5 gene causes migration defects. Two
vertebrate homologues of UNC-5 were found in a study. It was seen that these
two homologues along with UNC-5 define a new family of immunoglobulin
superfamily. Their mRNA shows prominent expression in various classes of
differentiating neurons.
2. Polycomb group (PcG) and trithorax group (trxG) proteins are chromatin-
mediated regulators of a number of developmentally important genes including
the homeotic genes. Most of these genes are conserved from flies and humans.
Trithorax-like (Trl), a trxG member gene, is present in Drosophila. It encodes the
essential multifunctional DNA binding protein called GAGA factor (GAF). A
848 T. D. Majumdar and A. Dey

homologue of this Trl-GAF was known missing in vertebrates, until recently a

vertebrate homologue to GAF, c-krox/Th-POK, was reported on the basis of
sequence similarity and comparative structural analysis.
3. Apterous gene defines dorsal cell fate and directs the outgrowth of the wing
during Drosophila wing development. The homologue of the wings in insects is
the forelimbs of vertebrates. It was seen in a study that the function of the apterous
gene is carried out by two separate proteins in vertebrates. Lmx-1 specifies dorsal
identity and Lhx-2 regulates limb outgrowth. From the study it was found that
Lhx-2 is a more closer homologue to apterous than Lmx-1.
4. The Polycomb group (PcG) of proteins maintain expression pattern of genes
during the preliminary stages of development. They have an important role in
epigenesis and maintains the expression of a huge number of genes, most
important being the homeotic genes. Whether different vertebrates have homol-
ogy in PcG still remains much unclear. Based on the fact that all PC homologues
consist of an N-terminal chromodomain and a C-terminal Polycomb repressor
box, 100 PC homologues of different organisms were identified by their protein
and genome sequence database.
5. The nervous system and limb evolution of the vertebrates and invertebrates have
been well studied and are established to have a vast amount of gene and protein
homology.
6. Over the years the vertebrate jaw evolution has been extensively studied. The
evolution of the vertebrate jaw can be viewed as a change of developmental
program for the functioning and specification of crest cells. Various homeobox
genes like Otx2 and other Hox genes participate in the formation and homology
of the jaw.

17.2.3.2 Mouse: Insertion Mutation

Mouse has been used as a model system for various mutation studies. Various gene
mutations have been carried out in mice for the sake of studying complex disease
formation like that of cancer and tumorigenesis. Some of the gene mutations are
listed below:

1. A transgenic mice model of Alport syndrome (a syndrome of progressive loss in

kidney function) was developed. Some exons of Col4a4 and Col4a3 and the
intergenic promoter region were deleted in the mouse line (OVE250) by trans-
genic insertion. This was confirmed by genetic and molecular analysis which
identified deletion of genomic DNA at the transgenic insertion site. This insertion
led to severe progressive glomerulonephritis in the mice. It also lacked both α3
and α4 chains of collagen IV.
2. Mouse mammary tumor virus (MMTV) was used to insert mutation in both int-1
and int-2 loci of premalignant and malignant neoplasms from the GR mouse
strain. The mutations were able to determine the earliest stages of neoplastic
development as well as progression of premalignant lesions to tumors.
3. Mutations were inserted in genes like cellular proto-oncogenes or tumor suppres-
sor genes with the help of murine leukemia retroviruses (MuLVs). This led the
17 Genetic Analysis of Development 849

mice to undergo leukemia and lymphoma as seen from studies. In a study >200
viral insertion sites was sequenced, from which >35 genes was identified that
were altered by viral insertion in 4 AKXD mouse strains. It was found that the
mutations were strain specific as each AKXD strain displays a unique mutation
profile. Even some of these mutations identified genes that had no previous
reports of being involved in causing cancer.
4. Insertion of a novel transposon ETnII-β, between the genes for Dusp9 and Pnck,
causes mutation which leads to multiple malformations in polypodia mice.
Mutation due to ETn insertion includes dysregulation of nearby interval gene
expression at early stages of development.

17.2.3.3 Mouse: Gene Knockout Mutation

Many of the studies regarding mechanism behind human biological process and
diseases have been carried out in mice. Mice have many advantages for the above
purpose like they are small, have relatively short life spans, and are prolific. Most
importantly it shares 99% gene analogy to human. Target-specific gene mutation in
mice has been used recently for understanding the function of various genes respon-
sible for development, metabolism, and diseases. Gene targeting is most widely used
for designing gene knockout mice. In this a drug resistance marker replaces an
essential coding region in a genetic locus, to understand the function of the gene in
embryogenesis and in normal physiological homeostasis. Gene knockout mutations
in specific targeted genes can mold and change it into a more useful form and discern
its biological role. Homologous recombination is the most commonly used tech-
nique for knockout. It takes the advantage of the organism’s own DNA repair
machinery to replace the targeted gene with the engineered homologous genomic
sequence, in a specific locus. Only a small percentage of cells actually take part in the
process as homologous recombination takes place at a very lower rate, where only
6 to 14 kb of homology is typical for targeting constructs.
Recombination vector and Cre/loxP technology are the two most important
techniques used for knockout. Recombinant vectors are widely used and are suc-
cessful means of knockout mutations. A drug selection marker is required for its
construction and positive selection of the susceptible variety. The most common
markers are neomycin (mostly used), puromycin, and hygromycin. The marker is
placed in the genetic frame of an open reading frame. Two homologous recombina-
tion events function to insert the targeting construct containing the drug resistance
gene into a homologous genetic locus. The most widely used technique to inject the
vectors into large number of stem cells is electroporation, as it provided better
targeting ratio (1:2400) than microinjection (1:15 targeted recombinants to random
integrants).
The Cre/loxP technology works almost similar to that of the recombinant vectors.
They are also designed with a drug selection marker, flanked by two homology arms
in the construct. But unlike recombination vectors, Cre/loxP requires both positive
and negative selection to isolate cells with the properly recombined mutation, and
the entire exon is not replaced by the drug selection marker. The positive selection is
done by Cre recombinase, which is used to replace the normal coding sequence in a
850 T. D. Majumdar and A. Dey

targeted allele with the mutated version. One homology arm will carry the planned
point mutation, micro-deletion, or insertion to be introduced into the targeted gene.
To design a vector for gene targeting, the following steps are suggested:

1. Firstly, the allele of the target gene should be well studied. Its genomic structure,
exon/intron sequence information, size, chromosomal location to be targeted, and
the location of most major restriction enzyme sites for subcloning should be
researched and identified prior to designing the vector.
2. Homology arm formation requires a mouse genomic fragment which contains a
large and relatable portion of the required gene. A 129/Sv genomic clone is most
commonly used for this purpose, as most stem cells were derived from this mouse
strain.
3. Both positive and negative selection markers needed for constructing the
targeting vector, especially in the Cre/loxP method, should be relatable and
properly chosen. The most common positive and negative selection markers are
neomycin phosphotransferase (neor) gene and HSV thymidine kinase (HSV-tk),
respectively. Besides, puromycin and hygromycin are also common positive
markers. The neor gene is often inserted in the opposite orientation to transcrip-
tion in the target allele, while designing the vector.

Several reports had proved that knockouts in mice can lead to malfunctioning and
also prenatal deaths of the embryo. The death can be due to a number of disturbances
including failure of proper vascular circulation and transition from yolk sac-based to
liver-based hematopoiesis. Several other reasons like improper implantation and
formation of a yolk sac vascular circulation, defective chorioallantoic placenta
formation, etc. leads to cutoff of connections with maternal system. These can also
lead to fetal destruction. Gene knockouts can also lead to malfunctioning and
underdevelopment of a number of embryonic organ and body systems, including
the central nervous system, gut, lungs, urogenital system, and musculoskeletal
system.

17.2.3.4 Zebrafish: Gene Knockdown Mutation

Similar to mouse gene knockout, zebrafish has also been extensively used for gene
knockout studies. Some of these extensive experiments are listed below:

1. Zebrafish is a powerful model for studying forward genetics. In a study the fish
was used to study reverse genetics that produced null phenotypes in G0 zebrafish.
CRISPR/Cas9 was used to target four ribonucleoproteins in a single yolk. In this
process early embryonic and stable phenotype was produced. This helped in rapid
screening of genes which are helpful in development, physiology, and disease
models in zebrafish.
2. RNA-guided endonucleases (RGENs), in the form of Cas9 protein-guide RNA
complexes (as they were derived from the prokaryotic type II CRISPR-Cas
system), were used for the establishment of gene knockout mice and zebrafish.
To achieve this RGENs were injected into the embryo of both the species when it
17 Genetic Analysis of Development 851

was in the one-cell stage. In the process germline transmittable mutations in up to

93% of newborn mice were achieved with minimal toxicity.
3. Proviral insertions were widely used to mutagenize genes in the zebrafish
genome, using high-throughput sequencing and mapping technologies. Using
in vitro fertilization, about 0.5% of the predicted and established mutations was
understood and characterized in this study. From the results a high mutation
efficiency was predicted, and also phenotypes relevant to both developmental
processes and human genetic diseases were put forward.
4. Zinc finger-based knockout of zebrafish was also put forward by a group of
researchers, which notably enhanced the chances of the fish to be established as
an efficient experimental organism for understanding human diseases.

17.2.3.5 Mammalian Stem Cell (Fig. 17.22)

Stem cells are cells capable of growing into a specialized cell. They can develop to
various specific adult organ cells like muscle cells, intestinal cells, liver cells, blood
cells, nerve cells, cardiac cells, etc.
A stem cell is a cell that has the inherent remarkable potential of developing into a
specialized new cell. This specialized new cell can be cells of other tissues or self-
renewal to produce more of the same stem cells. A stem cell is capable of converting
into more specialized cell like muscle cell, a red blood cell, or a brain cell. They also
can internally repair damaged cells of many tissues and organs like the gut and bone
marrow, dividing in order to replace damaged cells or multiply cells in mammals. In

Fig. 17.22 Stem cells

852 T. D. Majumdar and A. Dey

certain other cases, stem cells divide only under special conditions like in the
pancreas and heart. Stem cells are distinguished from other cell types by two
important characteristics. First, they are primarily unspecialized and can renew
themselves when required, even after elongated dormant stages. Second, under
certain physiologic or experimental conditions, they can be made tissue- or organ-
specific with special functions.
Two kinds of mammalian stem cells have been proposed: embryonic stem cells
and non-embryonic “somatic” or “adult” stem cells. In the late 1990s, human stem
cells were derived and successfully grown under laboratory conditions. They were
named human embryonic stem cells (hESCs). The embryos were produced through
in vitro fertilization procedures.

1. Embryonic stem cells (ESCs)—ESCs are produced from the inner cell mass
(ICM) of the embryo. After fertilization the mammalian embryo undergoes a
series of cell divisions, without any growth in total volume. These cells called
blastomeres get progressively smaller. At a point of time, they stop dividing and
rearrange to form a hollow sphere of cells called the blastocyst, encircling a fluid-
filled cavity called the blastocoel. These cells then form the outermost epithelial
layer called the trophectoderm and the ICM. The cells of the trophectoderm or
trophoblast form the fetal part of the placenta, while the cells of the ICM give rise
to the embryo proper. Around days 5–6 after fertilization in mouse and days 8–9
in humans, the cells of the inner mass can be isolated and put in culture. In culture
only the ICM is plated and the trophectoderm is removed. ICM is plated on to a
feeder layer of mouse or human embryonic fibroblasts, which is essential for the
survival of the ICM. The ICM then develops into a close-packed colony of ESCs,
which can then be cultured and maintained to ultimately produce stable cell line.
ESCs differ from the original ICM cells, notably in their pattern of epigenetic
modifications. It was found by experimentation that the generated ESCs retain
their pluripotency and can generate all tissues in chimeric mice, including
germline tissues, when injected into the blastocyst. When the adult chimeric
mice have gametes generated from cultured ESCs, breeding of such chimeras
will produce an animal composed entirely of the progeny of cultured ESCs. This
event is called the germline transmission. But the ESCs cannot themselves
generate the entire embryo. They require the presence of feeder cells for their
survival. The feeder cells are typically mouse fibroblasts that have been treated
with mitotic inhibitors to prevent their proliferation. Human feeder cells can also
be used in conditioned medium, which presumably contains appropriate growth
factors.
2. Somatic stem cells—Many mammalian somatic cells including those of bone
marrow, skin, gut lining, blood vessels, endocrine glands, mammary gland,
prostate, lung, retina, and parts of the nervous system contain stem cell
populations that might self-renew and generate somatic cells normally as well
as proliferate and differentiate in response to wounding or disease. This multipli-
cation of the somatic stem cells is tissue specific, and therefore these stem cells
are addressed as stem cell niche. They are defined as a specialized subset of tissue
17 Genetic Analysis of Development 853

cells and extracellular matrix that produces one or more somatic stem cells and
control their self-renewal and differentiation throughout the life of an organism.
Some niches like those of CNS are said to retain multi-potency, i.e., the ability to
differentiate into major cell types appropriate to their originality.

Mainly two kinds of stem cells make up the bone marrow. They are the
hematopoietic and the non-hematopoietic stem cells. The hematopoietic stem cells
produce the blood cells in the body. The non-hematopoietic stem cells (also called
mesenchymal stem cells, or skeletal stem cells) are a class of stromal cells in the
bone marrow and generates bone, cartilage, fat, and cells that support the formation
of blood and fibrous connective tissue.
Other than bone marrow, there are also many other organ-specific stem cells. For
instance, the neural stem cells have three major categories: nerve cells (neurons),
astrocytes, and oligodendrocytes (non-neuronal cells). The epithelial layer in the
digestive tract also bears stem cells like absorptive cells, goblet cells, Paneth cells,
and enteroendocrine cells. The skin stem cells are present in the basal layer of the
epidermis and at the base of hair follicles. The follicular stem cells give rise to hair
follicle and to the epidermis, and the epidermis in turn has stem cells which give rise
to keratinocytes, which migrate to the surface of the skin and form a protective layer.
Sometimes a phenomenon known as transdifferentiation can take place. Here
some adult stem cells differentiate into organs different from their inherent cell
lineage, for example, brain stem cells that differentiate into blood cells or blood-
forming cells that differentiate into cardiac muscle cells and so forth. This process
can be used for the differentiation of one cell type into another under a well-
controlled programmed condition of genetic modification. Nowadays research on
reprogramming adult somatic cells to become like embryonic stem cells (induced
pluripotent stem cells, iPSCs) through the introduction of embryonic genes are also
being carried out.

17.2.3.5.1 Application of Stem Cells

1. Blood and immune system disorders—Blood and immune system disorders are
treated by stem cell therapy by transplantation of bone marrow containing human
somatic cells (HSCs) or purified cell fractions from bone marrow.
2. Metabolic diseases—Lysosomal storage disorders are also recently being treated
with HSC transplantation, including the mucopolysaccharidoses such as Hurler’s
syndrome.
3. Autoimmune diseases/multiple sclerosis (MS)—Multiple sclerosis, a neurologic
autoimmune disorder, can be cured by stem cell therapy. The disease affects CNS
and causes multiple impairments of motor, sensory, and cognitive functions.
Besides this, ESCs can also be a good option for treatment of demyelinating
diseases.
4. Brain tumors—Neural stem cells have a unique capacity of migrating through
tissues which can be both neural and non-neural. This property can be used
extensively to treat brain tumors especially that cannot be effectively removed
by surgery or chemotherapy.
854 T. D. Majumdar and A. Dey

17.2.3.5.2 Negative Concerns Regarding Stem Cell Therapy

1. Use of animal cells or products—According to current FDA regulations, the use
of feeder cells from other mammals to culture ESCs may limit the use of the stem
cells.
2. Tumor-forming potential of ESCs—In the undifferentiated state, ESCs have a
tendency of forming teratoma, which can lead to a substantial risk during its
implantation.

17.2.4 Analysis of Developmental Result

17.2.4.1 Sex Determination in Drosophila

The sex of individuals is the result of balance between the combined expression of
the X and Y chromosomes. But in Drosophila, sex is primarily determined by
balance of the X-A ratio, i.e., balance between female-determining factors encoded
on the X chromosome and male-determining factors encoded on the autosomes. The
process of sex determination involves the coordination of many genes. As autosomes
do not carry the sex-determining genes, the copies of the X chromosome are
primarily responsible for the purpose. However the expression of these genes is
not equal in both the sexes due to the presence of two X chromosomes in female and
only one in male. This problem is sorted by dosage compensation. It is a process by
which the gene products generated by the X-linked genes in males and females are
equalized. This is accomplished in the flies by adjusting the levels of X-linked gene
products by doubling expression from the X chromosome in males. In flies sex
determination begins immediately at fertilization with no specific time period. The
transcription for the male and female gender specificity is triggered on sensing the
definite X-A ratio and does not require the involvement of any hormones. The
number of X chromosomes with respect to the autosomes plays an important part
in defining the gender type. When one X chromosome is present in a diploid cell
(1X-2A), the fly is male, whereas two X chromosomes in a diploid cell (2X-2A)
leads to female progeny. The XO variety is sterile male. Drosophila can generate
gynandromorphs, i.e., animals in which certain regions of the body are male and
other regions are female. Gynandromorphs are formed when an X chromosome is
lost from one embryonic nucleus and the cells which are formed from such cell are
XO male instead of XX female. Sex hormones are absent in insects and such traits
are controlled by body itself.
The sex determination in Drosophila is governed by the integrated action of a
number of genes like Sex-lethal (Sxl), transformer (tra), transformer-2 (tra2), inter-
sex (ix), and doublesex (dsx) genes. XX progeny can convert into males when these
genes undergo mutations, whereas XY males remain unaffected. Under homozygous
condition the intersex (ix) gene leads the XX flies to develop an intersex phenotype
having portions of male and female tissue in the same organ, whereas absence of the
doublesex (dsx) gene leads both XX and XY flies to turn into intersexes.
17 Genetic Analysis of Development 855

1. Sex-lethal (Sxl)—Present in both male and female, sxl is a switch gene for female
progeny. It is active in XX female from early stages of development. Its activation
requires high X-A ratio, a series of transcriptional and posttranscriptional factors,
and a number of regulatory proteins. It becomes active in XX females from the
initial 2 h of fertilization, and once activated it remains in this state as its protein
product is able to bind to and activate its own promoter. Immediately after
activation the gene transcribes Sxl mRNA (an embryonic mRNA) that is found
for only about 2 h more. The resulting SXL female-specific RNA-binding protein
modulates the expression of a set of downstream genes, ultimately leading to
sexually dimorphic structures and behaviors. In contrary in XY cells, Sxl remains
inactive during the early stages of development. A certain class of proteins called
the numerator (encoded by the X chromosome) is said to be responsible behind
this female-specific activation of Sxl. The other group of proteins called the
denominator block the binding or activity of the numerator proteins. These are
autosomally encoded proteins such as Deadpan and Extramacrochaetae.
2. The transformer genes—Transformer (tra) is one of the somatic genes that take
an active part in sex determination. It is also a switch gene and can be both female
and male specific. It regulates sexual dimorphism based on RNA splicing in many
insects. The female transcript encodes a functional TRA protein, and the male
transcript encodes a nonfunctional truncated TRA protein. The gene activates
sex-specific splicing of doublesex (dsx) pre-mRNA, along with
TRANSFORMER-2 by binding to dsx repeat elements. Dsx is the final gene in
the genetic cascade of sex determination and promotes female sexual develop-
ment. The activation of the tra gene is also responsible for the activation of Sxl
gene, and when Sxl is switched “off,” a nonfunctional TRA protein is formed.
This results a switch to male specificity of dsx, which then generates the male
DSX-M protein.
3. Double sex gene (dsx)—The final and most important gene in the sex determina-
tion gene cascade is the doublesex (dsx) gene. It is the final gene to be activated in
the series and needs the consecutive activation of both srl and tra genes for its
functionalization. It is active in both males and females, but its primary transcript
is processed in a sex-specific manner. When the X-A ratio equals to 1, the sxl gene
leads to activation of tra gene in a female-specific manner. A female-specific
splicing factor gets initiated which causes the splicing of the tra gene transcript.
This tra gene product interacts with the Tra2 splicing factor to cause the
doublesex pre-mRNA to be spliced in a female-specific manner. When tran-
scribed in this manner, the dsx transcript promotes female development and
inhibits male development. If the doublesex transcript is not acted on in this
way, it will be processed in a manner that will be male specific.

17.2.4.2 Dosage Compensation in Drosophila

The presence of Y chromosome in Drosophila is necessary just for the specification
of male gender and lacks any gene related to phenotypic development or sexual
maturity. Instead the sex-related genes and proteins are associated with the X
chromosome and its number. As males consist of only one X chromosome, it is
856 T. D. Majumdar and A. Dey

necessary to equalize the products of male X chromosome with that of female. This
is done by a process called dosage compensation. In this animals equalize the
amount of gene products released by the X-linked genes in both male and female.
This phenomenon was first observed in the fly by H.J. Muller in the early 1930s who
also coined the name “dosage compensation.”
The X chromosomes in Drosophila are identical in shape and genetic content and
are active in all somatic cells. They carry many housekeeping genes and other genes
which take active part in developmental pathways. Males have one X and a Y
chromosome. The Y chromosome differs from the X in morphology and genetic
information.
The X-autosome (A) ratio mainly controls both sex and dosage compensation and
not the X-Y ratio. Y chromosome is only required for male fertility. For proper
maintenance of dosage compensation, it is very necessary to have a control on the
number and function of X chromosomes, failure to which can be lethal. The first
gene which gets activated during initiation of dosage compensation is a critical
binary switch gene called sex lethal (Sxl). It is present on the X chromosome and is
regulated by transcription factors encoded by the chromosome. XX chromosomes
are able to initiate Sxl expression from promoter, Pe, whereas embryos with XY fail
to express Sxl from Pe. In flies, dosage compensation is mediated by the dosage
compensation complex (DCC) also known as male-specific lethal complex (MSL),
as loss in proper function of the complex can lead to lethality of the male phenotype.
The Drosophila MSL is a ribonucleoprotein complex and is composed of at least five
proteins, namely, MSL-1 (male-specific lethal 1, scaffolding protein), MSL-2 (male-
specific lethal 2, RING finger protein), MSL-3 (male-specific lethal
3, chromodomain protein), MOF (males absent on the first, histone
acetyltransferase), and MLE (maleless, RNA helicase). The SXL protein, present
only in females, works to suppress the activity of MSL complex by repressing the
translation of msl2 mRNA by binding in both the 50 and 30 untranslated regions
(UTRs) of the mRNA. If SXL is absent in females, dosage compensation is aber-
rantly turned on, and these females die. Conversely, if SXL is expressed in males,
dosage compensation is turned off and males die.
The five components of the DCC complex have their specific functions and
combinedly lead to the activity of the complex. The MLE protein binds single-
stranded RNA or DNA and is an ATP-dependent RNA-DNA helicase. MSL2 takes
part in ubiquitination of itself and the other MSLs and targets them for proteolysis
when required. It also binds DNA through its CXC domain—a stretch of 37 amino
acids rich in cysteine. MSL3 contains a chromodomain that targets the MSLs to
active X-chromosome genes in association with nucleosomes which contain histone
H3 methylated at lysine 36 (H3K36me). MSL3 like MSL2 can also bind to DNA and
methylated histone H4 at lysine 20. All the MSL proteins are male-specific and
absent in females. It was found from various studies that the presence of MSL1 or
MSL2 was compulsory for all the other proteins in the complex to bind to X
chromosome. These sites are therefore considered nucleation sites for MSL targeting
and spreading. DNA sequence motifs and histone acetylation are largely responsible
for the targeting of MSL to the X chromosome and dosage compensation. Gene
17 Genetic Analysis of Development 857

activation for dosage compensation involves the MSL-associated MOF acetyl trans-
ferase activity on H4K16 (histone H4 lysine 16), which represents a hallmark of the
male X chromosome.
Two non-coding RNAs (ncRNAs), called RNA on X (roX), are responsible for
targeting the MSL complex to the male X chromosome in Drosophila. They lack a
significant open reading frame, are dissimilar in size, and are not colocalized with the
MSL complex along the length of the X. roX RNA function was understood by
mutation of X chromosome carrying both roX1 and roX2. It was found from the
studies that MSL complex becomes mislocalized when both the RNAs underwent
mutation, and the males showed an unknown phenotype when any one of them faced
mutation. Partial purification of the complex suggests the presence of a tight core
consisting of MSL1, MSL2, MSL3, and MOF proteins, with roX RNA and the MLE
helicase lost except under very low salt concentrations. It was seen from studies that
MSL complex can bind with acetylate histone H4 on lysine within nucleosomes
in vitro, even if it lacks roX RNAs. From this it was put forward that the MSL
complex possessed every components essential for dosage compensation and only
need the RNAs to stimulate assembly and spreading. Besides this overexpression of
MSL proteins can partially overcome the lack of roX RNAs.
MSL1 and MSL2 play the primary role in the MSL complex, as their interaction
marks the initiation of the complex. Two subunits of MSL2 interact with an MSL1
dimer for the initiation. The interaction occurs near the RING finger of MSL2
(a C3HC4 zinc-binding domain) and an amino-terminal coiled coil domain in
MSL. MSL1 further associates with MSL3 and MOF. It leads to chromatin binding
and scaffold formation for interaction with MSL3 and MOF via adjacent conserved
carboxy-terminal domains. MSL2 also functions in ubiquitination of itself and other
members of the MSL complex components, including MSL1, MSL3, and MOF, but
not MLE in vitro. The MSL3 has an active chromodomain-bearing histone acetyl
transferases. It uses this domain in locating target genes for the MSL complex by
interacting with active chromatin marks such as H3K36me3. The next member of the
MSL complex is the MOF, which is the most important part in the MSL complex as
it is the principal component which mainly controls the gene regulation by the MSL.
It is a part of the MYST subfamily of HATs and is characterized by the presence of a
chromodomain and enzymes that specifically acetylate lysine 16 in vivo. The vital
role of the rest of the complex is to support MOF and to localize it to its targets on the
X chromosome. MOF recruitment is particularly important as MOF also participates
in the nonspecific lethal (NSL) or MBD-R2 complex in both sexes. The NSL
complex is necessary for mortality in both the sexes. It is found at 50 ends of most
active genes. The last member of the complex, MLE, shows RNA/DNA helicase,
adenosine triphosphatase (ATPase), and single-stranded RNA/single-stranded DNA
binding activities in vitro. The function of MLE denotes that RNA has a potential
role in MSL function. MLE most probably interacts with RNA or alters its structure,
particularly the roX RNAs. Other than male-specific factors, many general factors
are also responsible for dosage compensation. They are involved in chromatin
organization and transcription in both sexes. For example, JIL-1, a tandem kinase,
is found along all chromosomes in both males and females but is more highly
858 T. D. Majumdar and A. Dey

concentrated on the male X chromosome. JIL-1 functions in modifying

nucleosomes, specifically on the male X chromosome.

17.2.4.3 Sex Determination in Mammals

In placental mammals, the presence or absence of a Y chromosome determines the
sex of the individual as the X chromosome is common in both male and female
(XX in females and XY in males). Even in cases of aneuploidy, the sex determina-
tion depends upon presence of the Y chromosome.
Painter, in 1923, showed that the presence of X and Y chromosomes in the
karyotype is characteristic of the male sex of humans. Sex of an individual is
determined by the presence or absence of the Y chromosome. Sex chromosome
aneuploids are males if they retain the Y chromosome and females if they lack it,
irrespective of the number of the X chromosomes in the chromosome set. Different
systems of chromosomal sex determination were put forward which studied the
various combinations of sex chromosomes which ultimately determined the sex:
females with XX and males with XY in mammals and females with ZW and males
with ZZ in birds and snakes. The most ancient part of the X chromosome was found
in marsupial and eutherian species. In various studies it was found that genes in the
ancient conserved region of humans and also in the recently discovered regions have
similarities with chromosomal arrangement of chickens and other birds. The sex
chromosomal arrangement of humans has also resemblance with that of turtles, fish,
marsupials, and eutherians.
In mammals gonads develop within the urogenital system and are formed from
the intermediate mesoderm. The urogenital system is divided into three regions:
pronephros, mesonephros, and metanephros, which develop anterior to posterior
along the nephric or Wolffian duct. Gonads arise from the ventrolateral surface of
each mesonephros, which serve as primitive kidney during embryogenesis. The
pronephros is vestigial. The genital ridges are composed of somatic cells derived
from the mesonephros and primordial germ cells.
The process is carried out by a series of genetic regulation (Fig. 17.23).

Fig. 17.23 Location of the SRY gene on Y chromosome

17 Genetic Analysis of Development 859

The SRY gene (blue) dictates the sex determination in mammals. It is located on
the Y chromosome:

1. Sex-determining region Y (SRY) gene—The SRY is the most important gene in

sex determination in mammals. It is located on the Y chromosome and functions
in development of testis, the male-specific organ which defines the preliminary
stage in sex determination and gender differentiation. Initially the embryo has the
capacity of differentiating into both testis and ovary. It possesses two ducts,
namely, Wolffian and Müllerian ducts, which develops into the male and female
reproductive tracts, respectively. At about seventh week of embryo development,
the SRY gene activates and encodes a unique transcription factor that activates a
testis-forming pathway. Once testis is formed, it produces testosterone and anti-
Müllerian (AMH) hormone. Testosterone and one of its derivatives, dihydrotes-
tosterone, induce formation of other organs in the male reproductive system,
while AMH causes the degeneration of the Müllerian duct. SRY protein is
however absent in females, and a different set of proteins activate the ovary-
forming pathway. Developed ovary then produces estrogen, which triggers
development of the uterus, oviducts, and cervix from the Müllerian duct.
Müllerian duct plays important role in females and is not degenerated as in males.
2. Steroidogenic factor 1 (SF1)—SF1 functions along with SRY and is a member of
the orphan receptors, nuclear receptors which have no known activating ligand.
This transcription factor has two highly conserved regions which are specific to
mammals, a DNA-binding domain composed of two zinc fingers and a carboxyl
terminal domain in the zinc fingers. It is studied that SF1 is involved in steroid
biosynthesis and was found to be associated with endocrine function such as
gonads, adrenals, pituitary, and hypothalamus.
3. Lim 1 and Pax 2—These genes help in the differentiation of the intermediate
mesoderm. They take part in early gonad development and the final formation of
the urogenital system. They also help in kidney development during the early
stages of the organ.
4. Wilms’ tumor 1 (WT1)-associated gene—This also takes an active part in early
gonad and kidney development. Mutations in this gene are found to be associated
with three different but related syndromes in humans. The first are childhood
tumors in the kidney which can develop when the gene undergoes heterozygous
deletions. Second is Denys-Drash syndrome in which urogenital malformations
are caused when WT1 undergoes heterozygous missense mutations in the zinc
finger DNA-binding domain. Third is Frasier syndrome, specific to females
where they display urogenital malformations.
5. M33 gene—It was discovered in mouse. It also takes part in the early develop-
ment of the gonads but in a different way than the above genes.

Three different cell lineages altogether make up the gonads and germ cells. They
are the supporting cell lineage, steroidogenic cell lineage, and connective cell
lineage. The supporting cell lineage gives rise to Sertoli cells in the testis and follicle
cells in the ovary. The Sertoli and follicle cells provide protection and the required
860 T. D. Majumdar and A. Dey

growth environment to the germ cells. The steroidogenic cell lineage gives rise to the
Leydig and the theca cells in male and female, respectively. These cells are respon-
sible for producing the sexual hormones during the maturity and development of the
gonads. They contribute to the development of the secondary sexual characteristics
of the embryo. The connective cell lineage leads to the formation of the gonads and
germ cells as a whole. During the early stages of development of testis, Sertoli and
germ cells are produced in the testicular cords, whereas Leydig cells are excluded to
the interstitium. Basal lamina in testis is collectively formed by the cord and Sertoli
cells. The action of SRY gene (remains active for a day and a half during early gonad
development) triggers differentiation of the Sertoli cell lineage in the testis. After
activation the Sertoli cells direct the differentiation of the rest of the cell types in the
testis. Ovary is less structured than testis, and its development and organization take
place later than that of the testis. The connective tissue lineage, in the case of ovary,
gives rise to stromal cells with no myoid cell equivalent.

17.2.4.4 Dosage Compensation in Mammals

As most of the sexual development related genetic loci is present in the X chromo-
some, it is necessary that the output of the genes is equal in both male and female.
Disproportionation in the level of expression of sex chromosomes can lead to
different copy numbers of X-linked genes in males and females. Due to this there
can be dissimilarity in the level of gene products in terms of RNA and proteins,
which would, in turn, require differences in metabolic control and other cellular
processes. In order to maintain the equality in genetic output of both the sex
chromosomes X and Y, dosage compensation is carried out in the genes. It balances
the level of X-linked gene products between the sexes. Dosage compensation is
carried out in three ways in mammals: by twofold upregulation or twofold
downregulation in the expression of X-linked genes in males and females, respec-
tively, and by the complete inactivation of one of the two X chromosomes in
females.
Many genes are involved in the sex determination process, and consecutive
activation and inactivation of these genes control the sexual differentiation in
mammals. The product of these genes then initiates the activation of another cascade
of genes and the consecutive regulatory cycles that mediate the progression of the
various pathways which ultimately leads to sex identification. In humans, it is the
protein product of the SRY gene on the Y chromosome that directs the embryo
toward the male pathway. In the absence of dosage compensation, chromosome
degeneration takes place which has two major problems: first, a high amount of
chromosomal difference generates between the male and female progeny and con-
sequently among the members of the same species, and second, the heterogametic
sex is monosomic for a large chromosome and thus monoallelic for a large number
of genes.
Males of both mammals and drosophila have one copy of X and females have two
copies. The Y chromosome only functions in sex determination in both the
organisms. It has a relatively poor number of genes and is largely heterochromatic.
But the X is much larger and has much more number of genes. This difference in the
17 Genetic Analysis of Development 861

number of X chromosomes (2:1) can cause a twofold difference in many gene

products between the sexes. XY males are monoallelic for the great majority of
X-linked genes. This problem faced by males was addressed by Susumu Ohno about
40 years ago, who hypothesized that this problem could be solved if the X in males
produces twofold products and matched the female. This led to the studies in the fruit
fly Drosophila melanogaster using high-throughput gene expressions, which con-
firmed the occurrence of dosage compensation through upregulation of X-linked
genes in XY males.
The evolutionary development of both the sex chromosomes X and Y are
co-related. They differ significantly in their genetic content where the human X
chromosome contains about 1100 genes, whereas the Y chromosome contains only
100 genes. Over the evolutionary period, the Y chromosome went through many
changes in function and genetic composition to maintain the difference between the
male and female progeny, including controlling the activity of SRY gene to develop
the testis and mark the difference between the genders. The X chromosome, on the
other hand, acquired the maximum percentage of sexual related genes. Genes that
enhance male sexual reproduction and spermatogonia also accumulate on the X
chromosome. But genes of later spermatogenesis are not expressed on X.
The other important mechanism of dosage compensation is X inactivation.
During the early period of mammalian development, the differentiation in both the
X chromosomes is governed and controlled by the Xic gene. Only the chromosomes
which have the Xic gene can induce X-chromosome inactivation (XCI), as they
contain a region of about 1 Mb occupying the Xic gene.
There are several genes responsible for dosage compensation. Some of them are
sdc-1, sdc-2, sdc-3, dpy-21, dpy-26, dpy-27, dpy-28, and dpy-30. Mutations in them
reduce the viability of XX but not XO animals. The mutations cause stoppage of
dosage compensation, and the genes in XX individuals undergo twofold
upregulation in their expression, though it does not affect the XO individuals in
any way.
DPY-26, DPY-27, and DPY-28 proteins showed similarity to 13S condensin, a
complex essential for control and proper functioning of mitotic and meiotic
chromosomes from yeast to man and hence is believed to have function in the
above mechanisms.
SDC-2 functions in chromosome specificity and hermaphrodite specificity during
dosage compensation. It is the switch gene that activates dosage compensation in
hermaphrodites.
The hermaphrodite formation is controlled by the activation of xol-1. Under
active conditions xol-1 leads to male development in XO embryos, whereas it
leads to hermaphrodite formation in XX embryos when in inactive state. Also
when inactive in XX it leads to activation of dosage compensation. The ratio of
the X chromosomes to sets of autosomes (X-A signal) regulates the activation of the
xol-1. The twofold differentiation in expression in X-chromosome dose between
males and hermaphrodites is dominated by the on/off state of xol-1.
862 T. D. Majumdar and A. Dey

Dorsal

Anterior Posterior

Ventral
9 nuclear divisions Pole
(syncytial blastoderm) cells

Nuclei migrate
Zygote to periphery
nucleus (2N)
Fertilized egg

Adult
Head

Cellular
blastoderm

Protein gradients
establish segmentation
Thoracic Abdominal
segments segments Segments
Embryo at 10 hours

Fig. 17.24 Developmental stages in the life cycle of Drosophila

17.2.4.5 Developmental Stages of Drosophila (Fig. 17.24)

The Drosophila life cycle starts from fertilized egg to the syncytial blastoderm,
cellular blastoderm, and finally the embryo. The adult fly hatches out from the
embryo and undergoes various developmental stages to finally result the segmented
body.
The Drosophila life cycle consists of a number of stages: embryogenesis, three
larval stages, a pupal stage, and the adult stage.
The nuclear division (mitosis) starts soon after fertilization. Cytokinesis (division
of cytoplasm) does not take place in the early stages of embryogenesis; instead a
multinucleate cluster of cells is formed called syncytial blastoderm. By the tenth
nuclear division, the nuclei formed during syncytial blastoderm migrates toward the
periphery of embryo and gets partitioned into separate cells (cellular blastoderm) at
the 13th division. The major body axes and segment boundaries start developing and
are characterized by formation of morphogen gradients, which play a key role in
pattern formation. Subsequent development results in an embryo with morphologi-
cally distinct segments.
Early embryogenesis—The Drosophila egg is the shape of a sausage. The sperm
enters through a micropyle which is present at the anterior end. After fertilization,
mitotic division occurs within the first 90 min. No cleavage is formed which results
in a common cytoplasm and many nuclei. This is called a syncytium. After about
17 Genetic Analysis of Development 863

nine to ten divisions, nuclei move to the periphery to form the syncytial blastoderm
(2 h).
Embryogenesis—By the 13th mitosis division, individual cells begin to be
formed when the membranes start surrounding the nuclei. This stage is called the
cellular blastoderm. After this about 15 cells move to the posterior as pole cells and
ultimately become the germline. After blastoderm formation, gastrulation starts at
about 3 h.
Gastrulation—Cells do not divide during gastrulation, instead they separate from
one another and move to internal locations under the ectoderm. Formation of
mesodermal tube and nerve cord takes place at this stage. Mesodermal tube forms
from ventral tissue and ultimately becomes muscle and connective tissues, whereas
nerve cord occupies the ventral region. Neuroblasts lie between mesoderm and outer
ectoderm. Both posterior and anterior midgut fuse and ectoderm becomes epidermis.
Segmentation—The ventral blastoderm or the germ band develops into the trunk
region. It further pushes the posterior end of the body over the dorsal side and marks
the beginning of segmentation. The very initial segmentation grooves begin to
appear from the posterior of one parasegment and the anterior of the next. There
are 14 parasegments: 3 mouth, 3 thorax, and 8 abdominal.
Larvae—After 24 h of fertilization, the larva comes out. It gets divided into
anterior (acron) and posterior (telson) ends. It is segmentally divided into the head,
three thoracic segments, and eight abdominal segments. The ventral side of the
larvae possesses denticle belts, alternating patches of denticle hairs, and cuticle on
each segment, used for locomotion.
Metamorphosis—Once the egg moves into larval stage, the cycle of the fly goes
through three instar stages, separated by molts. Pupae come out from the third instar
larvae and undergo metamorphosis. The metamorphosis includes all the develop-
mental processes which take place after the pupal stage to the adult body structure,
including the final segmentation. Small sheets of epidermis called the imaginal discs
develop into the adult tissues. The discs grow throughout life cycle. In addition to
imaginal discs, tissues are also formed from histoblasts, especially the abdominal
segments. The adult fly ultimately develops six legs, two wings, two halteres, and
two eye antennas, plus genital, head discs, and about ten histoblasts.
Maternal effect genes play an important part during zygotic development. They
take an active part in oogenesis as well as fertilization. Two such genes are bicoid
and nanos whose protein product plays important function during embryo develop-
ment and are present in the egg at fertilization. Besides these genes also have a
significant role in the process of segmentation in the fly. They activate the transcrip-
tion of the zygotic genes, namely, gap genes, pair-rule genes, and segment polarity
genes, which control the segment patterning in the fly. The gap genes roughly
subdivide the embryo along the anterior/posterior axis, the pair-rule genes divide
the embryo into pairs of segments, and the segment polarity genes set the anterior/
posterior axis of each segment. The segmentation takes place in a synchronized
manner by the consecutive activation of these three genes. Firstly, the maternal genes
encode transcription factors that regulate the expression of the gap genes, and the
gap genes then encode the transcription factors of the pair-rule genes, which in turn
864 T. D. Majumdar and A. Dey

encode the transcription factors of the segment polarity genes. After the division of
the body into segments, another set of genes called the homeotic genes gets
activated. These genes then control the formation of anatomical structures like
legs, wings, and antennae on the segments. The homeotic genes include a 180 nucle-
otide sequence called the homeobox, which is translated into a 60 amino acid
domain, called the homeodomain.
Besides the maternal and zygotic genes, other growth factors also take part in the
development of Drosophila. For instance, Torpedo (an epidermal growth factor
(EGF) receptor homolog) is expressed in the dorsal follicle cells and takes part in
the dorsoventral patterning of the embryo. Besides this another transforming growth
factor (TGF) alpha homologue called gurken is also required for follicle cells to
adopt dorsally.
Anterior-posterior axis specification in Drosophila is controlled during oogenesis
by localization of bicoid and oskar mRNA, more precisely, localization of bicoid
mRNA at the anterior pole and oskar mRNA at the posterior pole. This transfer of the
mRNAs depends on microtubules. Initially, oocyte is present at one end of the nurse
cells (cells that provide food, helps and provides stability to other cells). The
adjacent follicle cells then move to the posterior side. Microtubules then aid toward
the posterior side and help the necessary nutrients to flow from nurse cells to
posterior. The microtubules are then directed in an opposite direction by a signal
from the follicle cells, and the oocyte nucleus moves anteriorly and to one corner.

17.2.4.6 Microarray Study of Drosophila Development

The developmental pathway of Drosophila needs the conjoint activity of thousands
of genes, starting from embryogenesis to adult and metamorphosis. In order to know
the mechanism behind the growth cycles of the fly, it is necessary to understand,
identify, and analyze the function and expression of these genes. In recent times,
advanced approaches like microarray are used to understand the expression of
various genes. A microarray basically consists of an array of microscopic slides,
each containing a particular kind of gene which acts as probes to detect the
expression of the respective genes. This technique is used now for studying the
functioning of the genes of Drosophila. Many genes have been discovered over the
years. Among them some take part in developmental process and are active during
metamorphosis, whereas some others are not directly associated with metamorpho-
sis. Additionally, many genes of unknown function were identified that may be
involved in the control and execution of metamorphosis.
DNA microarray and mRNA in situ hybridization are two widely used techniques
for epigenetic and other genomic studies. Time-related assessment of differences in
gene expressions from mRNA extracted at various stages of embryonic development
can be efficiently done by microarray studies. Integrative analysis of in situ expres-
sion patterns and microarray gene expression data is a very efficient way to find out
the roles that genes play during development and to identify sets of genes that
contribute somehow to developmental processes.
Advantages of microarray (expression screens)—These experiments are fre-
quently performed in order to screen for molecules regulating a specific biological
17 Genetic Analysis of Development 865

Fig. 17.25 The insertion and function of ovo+ gene in germline sex determination. (a) Molecular
map depicting the ovo locus with the consecutive insertion and deletion sites of the ovo and svb
genes. The bold arrows represent the deletions, whereas the arrowheads represent the insertions
(filled in (insertion of ovo and svb) and unfilled (insertion of ovo)) sites. These are the sites
where multiple insertions and deletions were carried out in order to understand the function of both
the genes. Below the domains the wild-type fragments are shown in which the mutation studies have
been carried out. At the bottom the three reporter gene fragments of ovo gene, used for building the
gene constructs for mutation, are indicated. The transcription proceeds from left to right. (b) Map
showing that the activity of ovo gene is not required in XY female germ cells. Females carrying both
ovo+ and ovo genes were crossed with males to obtain the XY female germline. The siblings were
then scored for the activity of the ovo gene, and no significant difference was found

process. There are many genes whose regulations were studied by microarray. Some
of them are as follows:

1. Target genes induced by b-catenin, a key component of the WNT signaling

pathway—A human renal carcinoma cell line was used for the purpose. The
cells did not express catenins and were stably transfected with a b-catenin
construct. From microarray studies it was found that LEF/TCF binding sites
were necessary for transcription of b-catenin by Nr-CAM promoter and their
proper functioning.
2. Similar screens have been performed in order to identify the targets of other WNT
signaling pathways, GDNF signaling, Mitf-dependent SCF/Kit signaling, and the
targets of the transcription factors TBX2, SIX5, PAX3, WHN, HOXA11, and
HOXA13. While studying HOXA11 transcription, one gene, Integrin a8, was
upregulated 20-fold after induction of Hoxa11 expression.

Technical issues: The microarray till date has many technical limitations. In order
to perform genome-wide expression data in detail, it is necessary to have the genome
information. The complete genomic data of various important model organisms like
frog and chicken is missing. This hinders the proper utilization of the technique.
866 T. D. Majumdar and A. Dey

Another disadvantage and perhaps one of the most important ones is the limiting
amount of RNA available from standard embryonic dissections. Lastly in the case of
mammals, the main limitation is their complexity both at transcriptional and cellular
level that limits the efficient use of microarray in the study of mammalian genomics.
Imaging gene expression in four dimensions: Microarray in the near future can be
used to study the gene expression patterns and their activity in four dimensions. It
can lead to excellent studies of cell differentiation, communication, death, migration,
and division in time and three- and four-dimensional space.

Box 17.1: Scientific Concept: Function of Drosophila ovo + in Germline

Sex Determination Depends on X-Chromosome Number (Brian Oliver
et al.) (Fig. 17.25)
The differentiation of the gonads and the development of the germline are
mainly characterized by the process of spermatogenesis or oogenesis. It is
known that in drosophila after fertilization and formation of embryo, the sex of
the individual is not determined by the X-Y ratio but by the X-A ratio. One of
the principal steps in this process is counting the number of X-chromosomes.
Some studies have shown in somatic cells that this function is carried out and
controlled by the Sxl+ gene, but nothing much is known about how the process
is carried out in germ cells. It is often thought that both the germ and somatic
cells follow the same pathway, which is studied not to be true. Though both the
cell types have the functioning of the Sxl+ gene in common, the germline sex
determination also needs the association of some other X-chromosome genes.
These are the ovo+, otu+, and snf+ genes. These genes act in association with
the Sxl+ gene and helps in its transcription and activation in a female-specific
manner. It is studied that Snf+ is an RNA-binding protein and may therefore act
directly on Sxl + pre-mRNA. But the function of ovo + and otu + was not
known for a long time. In this work, Oliver et al. first estimated a function of
ovo+ in the germline sex determination and that it is highly active in the
females. They made ovo reporter genes and found that they show high activity
in the germline of females and low activity in the germline of males. They saw
that when XY flies are transformed into somatic females, they do not show
high levels of reporter activity, but when XX flies were transformed into
somatic males, they showed high level of reporter activity. This shows that
high-level ovo+ expression depends on the number of X chromosomes, not on
the X-A ratio. It was found that the ovo+ functioning is only required for XX
flies which showed that the gene played an important role in sensing the
number of X chromosomes. Mutations in ovo have no effect on XY males,
X0 males, or XY females but have pronounced effects on germ cell viability in
XX females, XX females with sex transformed germlines, and XX males
indicating that ovo+ gene products are required for events occurring only in
flies with two X chromosomes.
17 Genetic Analysis of Development 867

17.3 Summary

• “Genetics is about how information is stored and transmitted between

generations,” John Maynard Smith.
• A gene is a functional unit of heredity. These genes control the growth and
development of an organism throughout its life cycle. The study of function
and mechanism of genes in the evolution of a living being is called developmental
genetics or genetic analysis of development.
• When life begins from a single cell, it involves three processes: cell division
(production of cells), cell differentiation (diverging of cells to different cell
types), and morphogenesis (forming structure of organism). This whole phenom-
enon of conversion of a single cell to an organism is due to the orchestrated
synchronization of genes which forms the basis of development.
• During the onset of life from an egg to an embryo, two groups of genes come to
play. They are the maternal genes (in development of egg to embryo) and zygotic
genes (in further embryonic development). These two sets of genes are among the
earliest, governing the onset of life from egg to embryo to infant. Maternal genes
are genes that are present in the ovum during fertilization and have an immense
contribution in the initial development of the zygote in terms of DNA, nutrition,
mRNA, and proteins. Zygotic genes play an important role in segmentation and
differentiation of the body in organisms and also in various organ formations.
Many model organisms are used as important tools to study various developmen-
tal processes and the role of genes in it.
• A model organism is a species that has been widely studied, because it is easy to
maintain and breed under laboratory setting and has many experimental
advantages. It is used to enlighten many biochemical, genetic, and signaling
pathways and serves as disease models, like that of cancer and neurological
disorders, etc. Saccharomyces cerevisiae, Drosophila melanogaster,
Caenorhabditis elegans, Xenopus tropicalis, Mus musculus, and Danio rerio
are the most widely used model organisms. These organisms also serve as
vertebrate homologues for analyzing the various developmental stages in
vertebrates.
• Studying the phases of development is very important for the diagnosis of
diseases and studying various signaling and biochemical pathways. These
homologues have a much simpler life cycle and genetic framework than higher
vertebrates, which helps researchers in giving an idea of the activity and impor-
tance of any gene. For this many gene knockout and gene mutation studies have
also been carried out in organisms like mouse and zebrafish. Drosophila
melanogaster or the fruit fly is one of the most studied model organisms. It has
many gene homologues in humans. So, developmental stages of the fruit fly and
the genes associated with it give an idea of both insects and vertebrate
developments. Many robust and advanced techniques are also available to
study the interaction of these genes like microarray.
• Besides, other important areas of research in developmental genetics are studying
the process of sex determination of any individual. The interdependency and the
868 T. D. Majumdar and A. Dey

cumulative functioning of the sex chromosomes are major factors of research in

developmental biology and are well studied from the last century.
• Another field which is gaining immense importance in present-day research is
mammalian stem cells. Stem cells denote certain primary cells which have the
pluripotent ability to give rise to a cell lineage and ultimately a tissue. They are
finding wide applications in various immune disorders, hematopoiesis, metabolic
disorders, tumors, etc.

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Molecular Genetics of Cancer
18
Bhawna Chuphal

18.1 Cancer: Overview (Fig. 18.1)

Coined by Hippocrates in the fifth century BC, cancer has proven to be a leading
cause of death in Western countries mostly. The tale of cancer goes back to 1761
when Giovanni Morgagni did autopsies for the first time to relating after death
pathogenic findings to illness of patients and laid the foundation for study of cancer
scientifically known as oncology, the study of cancer. Over a million people are
diagnosed each year in the USA (Table 18.1) with at least half of them died and the
treatment being often costs a fortune as well. Looking at the statistics provided by a
study published in the journal of American Cancer Society, it is quite evident that the
USA reported approximately 1.7 lakhs new cancer cases and more than 6 lakhs death
by cancer toward the end of 2019. When it comes to cancer medical expenses, it has
been estimated by the Agency for Healthcare Research and Quality (AHRQ) that the
USA alone has spent up to 80.2 billion dollars in the year 2015. Patients suffering
from cancer feel as if their bodies have been invaded by an extraterrestrial force;
however, the malignancies arise from the self. Cancer is considered a group of
disorders in which the normal regulation of cell cycle is lost. In fact, a series of
genetic mutations are well established to be the leading cause of cancer arising from
a single cell (Cavenee & White 1995). A healthy cell comprises a portion of an
ordered array of other cells around it and undergoes cell division only when the
stimulatory and inhibitory signals from external environment balance out and favor
the event. This cell is replaced by new ones if worn out or damaged. However, with
replication or growth comes an inevitable hazard of genetic mutations impairing the
regulatory circuits inside a cell, occasionally leading to unscheduled cell division
(Fig. 18.1). The growth of cancer cells is a consistent process, producing new cells,
thereby crowding out normal cells and creating ruckus at the onset site of cancer.

B. Chuphal (*)
University of Delhi, Delhi, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 871
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_18
872 B. Chuphal

Fig. 18.1 SEM of two dividing prostate cancer cells. Two cancer cells shown here are undergoing
cytokinesis

Cells having cancer are well known to possess the ability of spreading via a
process known as metastasis (meh-TAS-tuh-sis) and reaching to various other body
parts. For instance, lung cancer cells can spread to the bones and divide there, but it
is still called lung cancer unless it started in the bones. No matter where a cancer
originates, the cell loses its native shape and boundary, ceases responding to inhibi-
tory signals, and goes haywire in case of division. The resulting mass of cells, in
turn, can crowd out and rob healthy tissue of nutrients. The worst scenario is when it
can invade the barriers separating organs and metastasize to distant sites.

18.2 Cancer Types

There exist several different cancer types, and it may start in several tissues including
the lungs, the skin, the eye, or even in the blood (Table 18.2). Although alike in some
criteria, cancers differ in manner of growth and invasion. With loss in normal
functioning, cancer cells lose their regular shape forming a distinct mass known as
tumor. Tumor is generally of two types: benign, where tumor is localized, and
malignant in which the cells invade other tissues via metastasis.

18.2.1 Gonadal Tumors

There are various classes of tumors under this category that may arise from totipotent
cells giving rise to a variety of tissue-type tumors, often within “germ cell” tumors
and mainly consist of endodermal sinus tumor, choriocarcinoma, seminoma/
dysgerminoma, teratocarcinoma, and embryonal carcinoma. Although common in
the gonads (ovaries/testes), these germ cell tumors might sometimes occur at sites
18 Molecular Genetics of Cancer 873

Table 18.1 Top 10 cancer types along with reported new cases and deaths in males and females in
the USA, 2016 (Siegel et al. 2016)
In males Estimated new cases Estimated deaths
Cancer sites Percentage of Number of Percentage of Number of
cases cases cases cases
Brain and other – – 3% 9440
nervous system 8% 70,820 8% 26,020
Colon and rectum – – 4% 12,720
Esophagus 5% 39,650 – –
Kidney and renal 4% 34,090 4% 14,130
pelvis 3% 28,410 6% 18,280
Leukemia 14% 117,920 27% 85,920
Liver and intrahepatic 6% 46,870 – –
bile duct 5% 40,170 4% 11,520
Lung and bronchus 4% 34,780 – –
Melanoma of the skin 21% 180,890 8% 26,120
Non-Hodgkin – – 7% 21,450
lymphoma 7% 58,950 4% 11,820
Oral cavity and
pharynx
Prostrate
Pancreas
Urinary bladder
Total 100% 841,390 100% 314,290
In females Estimated new cases Estimated deaths
Cancer sites Number of Percentage of Number of Percentage of
cases cases cases cases
Brain and other – – 6610 2%
nervous system 246,680 29% 40,450 14%
Breast 63,670 8% 23,170 8%
Colon and rectum 23,050 3% – –
Kidney and renal 26,050 3% 10,270 4%
pelvis – – 8890 3%
Leukemia 106,470 13% 72,160 26%
Liver and intrahepatic 29,510 4% – –
bile duct 32,410 3% 8630 3%
Lung and bronchus – – 14,240 5%
Melanoma of skin 25,400 3% 20,330 7%
Non-Hodgkin 49,350 6% – –
lymphoma 60,050 7% 10,470 4%
Ovary
Pancreas
Thyroid
Uterine corpus
Total 662,640 100% 281,400 100%

which are extragonadal. There exists another type of gonadal tumors which may
arise from stroma of connective tissue, for instance, tumors of granulosa-theca cell,
hilar cell, and lipid cell in females and Sertoli-Leydig cell tumors in males,
depending upon the nature and function of stromal cells.
874 B. Chuphal

Table 18.2 The different kinds of tumors associated with tissue type
Tissue Malignant tumors Benign tumors
Adult fibrous tissue Fibrosarcoma Fibroma
Bile duct Cholangiocarcinoma Bile duct adenoma
Blood vessels Angiosarcoma, hemangiosarcoma Hemangiopericytoma,
hemangioma
Bone Osteosarcoma Osteoma
Breast Cystosarcoma phyllodes Fibroadenoma
Fat Liposarcoma Lipoma
Glandular Adenocarcinoma Adenoma
epithelium
Hematopoietic cells Aleukemic leukemia, Leukemia (various Myeloproliferative
types) disorders, preleukemias
Kidney Hypernephroma; renal cell carcinoma Renal tubular adenoma
Liver Hepatocellular carcinoma Hepatic adenoma
Lymph vessels Lymphangiosarcoma Lymphangioma
Lymphoid tissue Hodgkin lymphoma and Non-Hodgkin Plasmacytosis
lymphoma, multiple myeloma,
plasmacytoma
Placenta Choriocarcinoma Hydatidiform mole
Smooth muscle Leiomyosarcoma Leiomyoma
Stratified squamous Malignant skin adnexal tumors, Skin adnexal tumors,
epithelium epidermoid carcinoma and squamous cell seborrheic keratosis, and
carcinoma papilloma
Nerve cells Medulloblastoma, neuroblastoma Ganglioneuroma
Nerve sheath Malignant schwannoma Neurilemmoma,
Neurofibrosarcoma neurofibroma,
Malignant meningioma schwannoma
APUD system
• Adrenal medulla Islet cell carcinoma Islet cell adenoma,
• Pancreas Malignant carcinoid gastrinoma
• Pituitary Malignant carcinoid Insulinoma
• Parathyroid Parathyroid carcinoma Chemodectoma
• Thyroid (C cells) Medullary carcinoma Paraganglioma
• Stomach and Malignant Pheochromocytoma Basophilic adenoma
intestines Chromophobe adenoma
• Carotid body and Eosinophilic adenoma
chemo-receptor Parathyroid adenoma
system C cell hyperplasia
Pheochromocytoma

18.2.2 Classification of Cancer

Cancers are broadly classified based on tissue type in which they originate and
primary site where they develop (Table 18.3). Tissue type also known as histological
type as established by the International Classification of Diseases for Oncology
includes hundreds of different cancer types which have been primarily grouped
18 Molecular Genetics of Cancer 875

Table 18.3 Some common types of cancers

Types
1. Anal cancer, adrenal cancer, acute myeloid leukemia (AML), acute lymphocytic leukemia
(ALL)
2. Basal and squamous cell skin cancer, bile duct cancer, bladder cancer
Bone cancer, brain and spinal cord tumors, breast cancer
3. Colorectal cancer, Castleman disease, chronic myeloid leukemia (CML), cervical cancer,
chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL)
4. Ewing family of tumors, eye cancer (ocular melanoma), esophagus cancer, endometrial
cancer
5. Gestational trophoblastic disease, gastrointestinal neuroendocrine (carcinoid) tumors,
gallbladder cancer, gastrointestinal stromal tumor (GIST)
6. Hodgkin lymphoma
7. Kidney cancer, hypopharyngeal cancer, Kaposi sarcoma, laryngeal cancer
8. Lung cancer, lymphoma, liver cancer, lung carcinoid tumor, leukemia
9. Myelodysplastic syndromes, Merkel cell skin cancer, multiple myeloma, melanoma skin
cancer, malignant mesothelioma
10. Non-small cell lung cancer, neuroblastoma, non-Hodgkin lymphoma, nasopharyngeal
cancer, cavity and paranasal sinuses cancer
11. Ovarian cancer, osteosarcoma, oropharyngeal cancer, oral cavity
12. Prostate cancer, pituitary tumors, penile cancer, pancreatic neuroendocrine tumor (NET),
pancreatic cancer
13. Rhabdomyosarcoma, retinoblastoma
14. Stomach cancer, soft tissue sarcoma, small intestine cancer, small cell lung cancer, skin
cancer, salivary gland cancer
15. Thyroid cancer, thymus cancer, testicular cancer
16. Uterine sarcoma
17. Vulvar cancer, vaginal cancer
18. Wilms’ tumor, Waldenstrom macroglobulinemia

into six major categories, namely, carcinoma, leukemia, lymphoma, myeloma,

sarcoma, and mixed types. Here four of them are dealt with in detail.

18.2.3 Carcinoma

Carcinoma often refers to cancer related to body lining (internal or external) or

epithelial malignant neoplasm and accounts for majority of the cancer cases
(80–90%) since the involved tissue type is found throughout the body ranging
from the skin to internal passages such as alimentary canal tract or gastrointestinal
tract and exterior or interior lining of organs. Carcinomas are again classified into
two major subtypes: adenocarcinoma and squamous cell carcinoma (SSC).
Adenocarcinomas develop inside a gland or organ and are often seen as thickened
mucosa at first. It generally occurs in mucus membranes and easily spreads through-
out the soft tissue at their place of origin. SCCs mainly originate from squamous
epithelium, chiefly found in lining of body cavities, lung, and skin. It has been long
876 B. Chuphal

Fig. 18.2 A schematic view of TGFβ signaling pathway. Ligand TGF-β binds to its type II and
type I receptor (a) leading to complex formation and type I receptor phosphorylation which
subsequently phosphorylates Smad2 or 3 (b), binding with Smad4 and translocating to the nucleus
shown in c. The complex associates with enhancers in target genes (d) inside the nucleus. TNF
upregulates Smad7 inhibiting the signaling pathway (e) and CSE might also interfere with the Smad
pathway (f)

established that SCC caused by mutation increases transforming growth factor-beta

(TGFβ) secretion levels in the surrounding microenvironment. Briefly, after binding
with ligand, TGFβ receptor complex takes part in phosphorylation of Smad2 and
Smad3 resulting in complex formation with Smad4 translocating into nucleus,
binding to protein binding elements specific to Smad, and regulating various down-
stream transcriptional pathways. The activated complex in turn also activates a
number of non-canonical signaling pathways which are independent of Smad
proteins (Fig. 18.2).

18.2.4 Immunotherapy

TGFβ promotes immune suppression in a number of ways and hence provides a

target for cancer immunotherapy. In addition, it also inhibits growth of tumor cells at
early stages of cancer, thereby rendering them unresponsive to its underlying
signaling. In the recent past, various TGFβ receptor inhibitors have undergone
development, for instance, galunisertib (LY2157299), with focus on their impact
on patients with heart diseases. The current range of TGFβ inhibitors has been
proven effective in combination with other agents as well as a single agent. For
instance, in a preclinical trial, TGFβ has been demonstrated to drive away response
18 Molecular Genetics of Cancer 877

related to immune suppression in mice with prior EGFR inhibitor treatment. Perhaps
in combination with immune checkpoint blockade, inhibition therapy targeting
TGFβ will have the most potential as the signaling is closely related to immune
checkpoint signaling. Moreover, in murine SCC, it has been shown that addition of
TGFβ depleting antibody abrogates the elevated TGFβ signaling and Treg expansion
induced by anti-PD-1 treatment. Reports including bifunctional antibodies such as
TGFβ/CTLA-4 and TGFβ/PD-1 which have shown antitumor response in certain
breast cancer lines and melanoma in both clinical and preclinical trials show that
bifunctional antibodies which work by combining antibodies for immune
checkpoints blockade with ligand binding domain are also well effective. Another
such example is M7824, antibody against PD-L1 and bifunctional antibody of
TGFβRII ligand trap, which has shown positive results in colon and breast cancer
in the murine model system by activation of T cell and NK cell inside the tumors.

18.2.5 Sarcoma

The words sarco and oma have Greek origin meaning fleshy and tumor, respec-
tively. Although relatively rare and often malignant, sarcomas are derived from
embryonic mesodermal layers of mesenchymal tissues (Table 18.4). They constitute
less than 10% of all cancer types and have been shown to have high morbidity as
well as mortality rate among children and young adults rather than older adults.
Associated with sarcoma, there are certain environmental exposures and genetic
predisposition syndromes even though the majority of these cancer types are consid-
ered sporadic with unknown etiology.
Most of the sarcomas are reported to have alterations in either retinoblastoma
(RB) pathway or p53 pathway; hence hereditary retinoblastoma patients are often at
higher risk of developing sarcomas. Since these two pathways have members which

Table 18.4 Examples of sarcomas

S. no. Name Origin
1. Osteosarcoma or osteogenic sarcoma Bone
2. Chondrosarcoma Cartilage
3. Leiomyosarcoma Smooth muscle
4. Rhabdomyosarcoma Skeletal muscle
5. Mesothelial sarcoma or mesothelioma Membranous lining of body cavities
6. Liposarcoma Adipose tissue
7. Hemangioendothelioma or Blood vessels
angiosarcoma
8. Fibrosarcoma Fibrous tissue
9. Glioma or astrocytoma Neurogenic connective tissue found in the
brain
10. Myxosarcoma Primitive connective tissue
11. Mesenchymous or mixed mesodermal Mixed connective tissue
tumor
878 B. Chuphal

are viewed as either brakes or accelerators of cell cycle, usually in tumors, loss of
proteins acting as brakes takes place, whereas those acting as accelerators get
amplified. For instance, when protein such as INK4A is lost, it results in amplified
phosphorylation of RB protein through cyclin D1/CDK4 inhibitory loss, thereby
preventing cells from undergoing cell division by disarming RB protein. On the
other hand, in cases of sarcomas, increased expression of CDK4 or cyclin D1 has
often been demonstrated and leads to same results as during loss of INK4A protein.
In addition, with sarcomas, RB protein loss has been associated, rendering its
inability to block the process of cell division. Another example is ARF protein
loss which leads to HDM2 inhibitory loss in turn enhancing its ability to block
normal functioning of p53 protein. Further, amplified HDM2 in case of sarcomas
enhances its ability to inhibit normal functioning of p53 protein. It has been well
documented that p53 loss of function in sarcomas impairs the ability of affected cells
with already damaged DNA to undergo apoptosis (Helman & Meltzer 2003).

18.2.6 Therapeutic Implications

At the present, treatment strategy is based largely on histological grade of tumor,

ease of resecting, along with absence or presence of metastases. This grading is
entirely dependent on features often determined medically such as degree of nuclear
atypia, mitoses, necrosis, and others. The main purpose behind histological grading
is to be able to predict most likely biological behavior of a tumor under consider-
ation, which specifically includes spread due to metastasis or recurrence of the tumor
locally after its surgical removal. Low-grade tumors have high possibility of getting
cured with complete surgical resection though they do occasionally metastasize,
whereas high-grade ones are likely to recur and spread distantly following local
therapy. However, it has been suggested that further expression of genes along with
proteomic analysis in case of such tumors may in turn improve our ability to predict
future behavior of various sarcomas. Interestingly, there have been documented
successes in recent development of therapies targeting specific genetic alteration
involved in these tumors. Gastrointestinal stromal tumors (GISTs) represent such
examples involving improved diagnosis as well as treatment strategy depending on
mechanisms driving the process of tumorigenesis. Before this improved diagnosis
and treatment, these stromal tumors were thought to be tumors of smooth-muscle
cells, i.e., leiomyosarcomas of the GI tract (gastrointestinal tract) and hence never
expected to give positive response to chemotherapeutic agents. Subsequently, after
careful histological grading, these tumors were considered to be derived instead from
Cajal’s interstitial cells and thereafter majorly characterized by activation in tyrosine
kinase of c-KIT receptor through point mutations. Luckily, imatinib, a drug well
known to inhibit the activity of c-KIT tyrosine kinase, led to pronounced effects
when tested in GIST patients, leading to major regression in tumor activity in
previously thought unresponsive patients. However, in the recent past, GISTs having
lack of KIT mutation were found to be associated with an activating mutation in
PDGFα receptor (PDGFRA) in many cases. The interesting part discovered was that
18 Molecular Genetics of Cancer 879

mutations in either of these receptors led to a similar downstream signaling pathway

which included activation of MAPK (mitogen-activated protein kinase) as well as
AKT pathway. By far, mutated tumor-specific target blockage such as c-KIT in case
of GISTs has been most successful at molecularly targeting therapeutic
interventions, but further therapeutic advances are required as many more kinases
are involved in sarcomas which seem to be activated and not yet mutated. Although
potential kinase inhibitors being developed in cancer therapeutics have been recently
listed, it still remains to be largely determined as to whether any of the drugs/
inhibitors will have any pronounced effect in sarcomas having non-mutated activa-
tion of kinase signaling pathways.

18.2.7 Leukemia

Increased numbers of leucocytes in our blood and/or bone marrow (the production
site for blood cells) lead to development of several malignant disorders commonly
called as leukemia. Leukemia often renders patients prone to infection as it
associated with the overproduction of inefficiently functioning immature white
blood cells. However, there are cases as seen in chronic lymphocytic leukemia
(CLL) where either leukemia cells present in dominance have been reported to be
matured, or like in the acute leukemias where precursor cells of various lineage are
found, or as in chronic myeloid leukemia (CML) where both precursor and mature
cells have been reported. While leukemias may be found in all age groups, each type
is reported to have specific age distributions, with acute myeloid leukemia (AML)
progressively common in people with older ages, whereas acute lymphoblastic
leukemia (ALL) is most commonly seen in early childhood. CML on the other
hand is reported to be very rare among young children, and being most common in
the West, CLL is considered almost exclusive to people above 40 years. Few
examples of different leukemia are granulocytic, lymphatic, lymphoblastic, and
polycythemia vera.
Studies in AML have characterized several genes with recurring mutations with
both prognostic and biologic implications, especially within normal functioning
karyotype and/or cytogenetic subset having intermediate risk involved of the host
cells. The genes involved with leukemia via mutations include CEBPA, FLT3, MLL,
NPM, and NRAS. Two major approaches based on cytarabine and anthracycline are
currently being followed for AML treatment, but the associated outcomes still
remain poor and unsatisfactory, especially for high-risk patients or patients of
older age. Hence one promising treatment strategy would be study and development
of novel agents having diverse mechanisms to target AML by targeted therapeutics
including oligonucleotide constructs and certain kinase inhibitors along with histone
deacetylase inhibitors, leading to arrest of cell growth and thus apoptosis via
conformational changes and histone acetylation. Furthermore, new class of therapies
are more concerned with targeting repair of DNA, replication of DNA, along with
cell cycle and underlying signaling. While a few of this class of therapies are under
early study and development phases, others have given away promising results at
880 B. Chuphal

preclinical and clinical trials. For instance, immunohistochemical investigation of

p53 protein expression in patients with leukemia has shown positive expression of
said protein in 7 out of 24 AML cases, in 3 out of 15 CLL cases, while in 1 of
11 CML cases and in 4 out of 8 ALL cases. From the study, it was concluded that
abnormalities in p53 protein expression may have a vital role in leukemogenesis as
well as development of chronic leukemia’s more malignant clones because in all,
leukemia cases with p53 protein expression was 26%.

18.2.8 Lymphoma

Lymphomas are defined as clonal neoplasms arising from subsets of innate and
adaptive immune cells such as natural killer (NK) cell, T cell, and B cell, respec-
tively, at their different maturational stages. With almost 4% of all new reported
malignancies, lymphomas are the fifth most common cancer type with highest
mortality rate in Western countries with B-cell deprivation representing more than
80% of mortality cases. In addition, major pathogenetic mechanisms have been
reported for B-cell lymphomas opposed to derived from other two cell types, and
till date, the genome level characterization has been done for mostly a huge subclass
of B- and T-cell lymphomas (TCLs). These studies unraveled into genetic
mechanism’s novel insights leading to immune escape along with various cellular
mechanisms such as activation of B-cell receptor, epigenetic and spliceosome
alterations, GTPase families, non-coding sequences, TCR signaling, certain
regulators and their dependencies, unregulated proteolysis, and altered metabolism
of tumor cell. Immunotherapies related to cancer have further highlighted that
immune escape depends on genetic mechanisms (Table 18.5). For lymphomas,
excellent platforms are provided by regulatory checkpoints leading to circumvention
of antitumor response for exploitation of mechanisms involved in initiation, pro-
gression, and therapy resistance (Elenitoba-Johnson & Lim 2018).

Table 18.5 Immune evasion and contributing genetic mechanisms in lymphoma

S. no. Mechanism Genes involved Lymphoma
1. 30 Untranslated-region PD-L1 Adult T-cell leukemia/lymphoma,
(UTR) disruption diffuse large B-cell lymphoma
2. Transcriptional STAT3 regulation Anaplastic lymphoma kinase-positive
regulation of PD-L1 anaplastic large cell lymphoma
MYC regulation of Burkitt lymphoma
PD-L1, CD47
MEK/ERK Anaplastic lymphoma kinase-positive
regulation of anaplastic large cell lymphoma
PD-L1
3. Mutations Β2M, CD58 Hodgkin lymphoma, diffuse large
B-cell lymphoma
4. Structural X/PD-L1, Primary mediastinal large B-cell
rearrangements X/PDCD1LG1 lymphoma, Hodgkin lymphoma
18 Molecular Genetics of Cancer 881

18.2.9 Therapeutic Targets

Therapy development for lymphoma has employed the E3 ubiquitin ligase’s

substrates along with insights into the regulatory mechanism. For example, cereblon
encoded by CRBN gene, one of the E3 ubiquitin ligase (CRL4) substrate receptors, is
directly targeted by thalidomide and IMiDs which is a collective of all related
immunomodulatory drugs. After binding, these drugs alter the specificity of cereblon
to CRBN-CRL4 complex, leading to lymphoid transcription factor’s degradation,
thereby suggesting that drug collective, IMiDs, may prove effective in cases of
multiple myeloma and primary effusion lymphomas which are types of B-cell
tumors. Comparable applications of these drugs have further been proposed for
both T-cell and B-cell Hodgkin lymphoma as well as non-Hodgkin lymphomas. In
addition, studies have been done in a B-cell neoplasm, Waldenstrom macroglobuli-
nemia, which is dependent for sustenance of BCR-driven cell growth on aberrant
proteasome functioning, activation of MYD88, and BCR signaling. These studies
used a combinatorial targeting approach in clinical trials wherein BTK/MYD88 node
was targeted using ibrutinib and 20S proteasome was inhibited using bortezomib.
Further extending the same approach, various inhibitors with small molecular weight
were used for targeting ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5)
and ubiquitin-specific protease 14 (USP14). Both being deubiquitinating enzymes,
the inhibitors resulted in tumor-specific apoptosis of otherwise resistant lymphoma
cells. Further, exposure to an inhibitor of both UCHL5 and USP14, known as
VLX1570 residing in the cap of 19S proteasome, resulted in downregulation of
proteins associated with BCR signaling such as BTK, CXCR4, MYD88, NFAT, and
NF-κB suggesting its potential to inhibit components of the proteasome for clinical
translation.
In spite of the fact that histological type classifies the cancer in a more efficient
way, it is the cancer name based on primary sites that the general public is more
familiar to. The common sites at which development of cancer takes place include
the following.

18.2.10 Lungs

Lung cancer (Fig. 18.3), considered to be the world’s most common cancer type, can
be of various types, namely, lung carcinoid tumor, small cell lung cancer (SCLC),
and non-small cell lung cancer (NSCL) with NSCL being common with prevalence
rate of over 85%. SCLC averages about 10–15% of lung cancers and tends to
quickly spread, whereas less than 5% of cancers are lung carcinoid cancer which
rarely spread due to slow growth. Till recently, translocation of anaplastic lymphoma
kinase and mutation in epidermal growth factor receptor are among numerous
molecular events having been identified offering hopes to patients with metastatic
lung cancer.
There have been many oncogenes related to lung cancer and include RAS, MYC,
and HER-2/NEU dominant oncogenes which act by overtaking normal cell growth
882 B. Chuphal

Fig. 18.3 Pictograph showing lung cancer. The enlarged view depicts the metastatic form of lung
cancer

and functions and RB, p15, p16, and p53 among various tumor-suppressor genes
(TSGs) that act in controlling further cellular growth. For cancer development and
progression, molecular alterations in either proto-oncogene or TSG are involved.
Lung cancers have been shown to exhibit multiple genetic lesions involving
mutations leading to dominant cellular proto-oncogene activation or activation of
those involved in inactivation of TSGs (Singh & Kathiresan 2014). These alterations
prime to cellular capabilities often acquired and can be grouped further based on
function in sets of six: self-sufficiency in growth signals, antiproliferative signal
insensitivity, apoptosis evasion by anti-apoptotic molecule upregulation or
pro-apoptotic molecule downregulation, boundless replicative potential as a result
of telomerase activation, sustained angiogenesis, and metastasis.

18.2.11 Therapeutics

Cancer treatment generally includes procedures such as surgery, chemical therapy,

and radiation therapy or therapy including drug targeting. Chemotherapy often uses
a combination of drugs which can be given orally or intravenously to kill the cancer-
affected cells and is generally a pursued treatment after the surgery for lung cancer.
Application of chemotherapy along with cisplatin and etoposide is reported to have
pronounced positive results in case localization of the tumor is within the irradiation
field, while for a longer time, the standard treatment is mentioned to be chemother-
apy along with etoposide and cisplatin. For advanced lung cancer treatment, current
trend includes efficacy of several treatments often combining antineoplastic and
platinum-based antineoplastic agents which have been recently developed. In addi-
tion, the marine flora has displayed a plethora of pharmacological properties
18 Molecular Genetics of Cancer 883

including antioxidant, immunostimulatory, and antitumor activities and is often

considered a rich source of medicinal polyphenols and sulfated polysaccharides.
The mechanism of action of these marine florae includes prevention of oxidative
damage to DNA by the phytochemicals released, induction of apoptosis, and
activation of macrophages, thereby possibly controlling the process of carcinogene-
sis. Another example of marine flora exhibiting anticancer properties are the man-
grove resources which are enriched in anticancer lead compounds. Associated
species and around 45 mangroves have been reported to possess anti-cancerous
properties, yet very scarce studies have been done on cancer types such as lung
cancer.

18.2.12 Female Breasts

Breast cancers (Fig. 18.4) mostly begin either in the ducts carrying milk, also known
as ductal cancer, or in the glands producing the breast milk, called lobular cancer.
Breast cancer may be detected even before it causes lump formation or develops
the symptoms. The important part is that many of the lumps developing in the breasts
are benign. Non-cancerous breast tumors are often abnormal growths but do not
metastasize and hence are not life threatening.
Accounting for ~10% of all breast cancers, hereditary breast cancer is caused by
deletion of a tumor suppressor gene rather than mutation leading to an oncogene
gain. A plethora of mutations in a variety of genes are known for susceptibility to
breast cancer, the most significant being BRCA2 and BRCA1 (Deng & Scott 2000;
Osborne et al. 2004) genes typically responsible for about 80–90% of high-risk type,

Fig. 18.4 Breast cancer. Breast cancer either begins in the ducts or the lobules. The figure shows
breast anatomy and development of breast cancer. (Image #WebMD, https://www.
emedicinehealth.com/breast_cancer/article_em.htm)
884 B. Chuphal

genetically determined tumors. These genes have been reported to be sporadically

seldom mutated, highlighting their role in pathogenetic promotion of carcinogenesis.
Many of these genes are reported in breast cancer in case of males. For instance, men
carrying germline mutations in either BRCA2 or BRCA1 gene are usually at a higher
risk of carcinoma development than the rest of the general population not carrying
the same mutations. Additional genes supposedly involved in the breast cancer
genesis include TSGs, namely, p53 and PTEN. Studies have shown that mutation
in the p53 coding region is the most common factor contributing to the inactivation
of p53 signaling pathway in human cancers as it has a vital role as a transcription
factor in regulating DNA repair and cell cycle. The level of protein is reported to be
increased inside cells during cellular stress periods, and hence the cell cycle arrest
gives time for DNA damage repair or for cellular death. In case of mutation, one or
all of these mechanisms fail leading to an increase in abnormal protein production,
thereby allowing cells with DNA damage to proceed with replication. Li-Fraumeni
syndrome often associated with various tumors such as sarcomas, lymphomas,
melanomas, carcinomas, leukemia, etc. is also caused by p53 germline mutations.
Another tumor suppressor gene involved in cell division, proliferation, and death,
located at chromosome 10q23, is phosphatase and tensin homologue (PTEN), and
PTEN with germline mutations is associated with syndromes such as Cowden
syndrome (includes aberrant protein expression and increased breast cancer fre-
quency), rare Mendelian syndrome, certain high-risk cancer types such as thyroid
and endometrial cancers, and benign manifestations. PTEN acts in tumor suppres-
sion which regulates chromosomal disruption, and its integrity leads to extensive
breakage at centromere breakage as well as chromosomal translocations. Other
pathways often associated with cancer including breast cancer are PI3K/AkT and
Ras/Raf/MEK/ERK signal transduction pathways generally involved in regulation
of apoptosis and cell cycle process in various cellular types.

18.2.13 Therapeutic Strategies

18.2.13.1 Chemotherapy
Cyclophosphamide. It is a derivative of nitrogen mustard, an alkylating agent,
initially synthesized and used for improving the nitrogenated mustard selectivity.
Over time it has been a cytotoxic agent, clinically implemented and proven effective
on a wide range of tumors, including breast cancer. Most of the tumoral cells have
enzymatic systems, phosphamidasis, and phosphatases, responsible for the activa-
tion of cyclophosphamide. The conversion of cyclophosphamide to
4-hydroxycyclophosphamide begins in the liver, a tautomerization process that
yields aldophosphamide. One of the byproducts of this cleavage process is N,
N-bis-2-(2 chloroethyl)phosphorodiamidate, which is a bifunctional alkylating
agent, an active product of cyclophosphamide reported to act as an alkylating
agent on DNA with N7 position of particularly susceptible guanine. The ability of
this drug to disrupt mitosis and cell differentiation in rapidly proliferating cells takes
the main focus. It has been used as an adjuvant therapy and in numerous combination
18 Molecular Genetics of Cancer 885

therapies along with fluorouracil and methotrexate (MTX) for treating patients with
high risk for relapse.
Methotrexate (MTX). Belonging to antimetabolites and folic acid analog class
of drugs, MTX is known to prevent cell division as it gets embedded in prerequisite
material for nuclear neosynthesis, or because it combines with the life necessary
enzymes in irreversible manner, hence preventing normal cellular division. Treat-
ment with this drug results in blocking the synthesis of thymidine 50 -monophosphate
(TMP) by prevention of N5,N10-methylenetetrahydrofolate synthesis required for
DNA synthesis.
5-Fluorouracil (5-FU). This drug also belongs to antimetabolite class and is
known to successfully prevent biosynthesis of nucleotide pyrimidines. 5-FU is
considered inactive in normal as well as tumor cells and acquires its cytotoxic
activity only after the cell’s bioregulation is disrupted. The mechanism of action of
5-FU is similar to that of MTX and prevents DNA synthesis leading to arrest of cell
cycle.
Anthracyclines. Anthracyclines, belonging to cytotoxic antibiotics, are antican-
cer agents whose antineoplastic action is mainly due to interaction with genetic
material eventually triggering cell death. Although very effective, these antibiotics
are also known to be toxic as they act as intercalating agents and often fail to
discriminate between malignant and healthy cells. The mechanism of actions leading
to tumor cell death are (a) p53-independent and/or p53-dependent DNA damage,
(b) DNA topoisomerase II inhibition, (c) apoptosis induction mediated through
cytochrome c, (d) proteasome interactions, and (e) free radicals’ generation which
results in oxidative damage. Doxorubicin (Adriamycin) and epirubicin are the major
anthracyclines used in the treatment of breast cancer.
Taxanes. Taxanes consist a group of drugs which include docetaxel and pacli-
taxel, known by their trade names Taxotere and Taxol, respectively. These drugs are
known to treat various types of malignancies, including lymphomas and leukemias,
and many solid tumor types, such as breast, brain, lung, neck, prostate, and ovarian
cancer, and are progressively being used at early stages of the disease. They function
by disrupting microtubules’ structure which play vital role in several important
cellular functions. During the growth of normal cell, microtubules are responsible
for division of cell, and once a cell stops dividing, these microtubules disintegrate.
Taxanes, however, stop the disintegration of these microtubules, and as a result,
cancer cells cannot divide and grow because these are clogged with the intact
microtubules. Epothilones are one such example which possess anti-tubulin activity
hence inducing tubulin polymerization and microtubule stability causing the cell
cycle arrest at the G2/M transition (Ligresti et al. 2008).

18.2.13.2 Hormone Therapy

Aromatase inhibitors. The hormone estrogen is well known to promote the growth
in many breast cancers. After menopause, the main source of estrogen acquirement is
the conversion of androgens from adrenal cortex into estrogen via an enzyme called
aromatase cytochrome P450 (CYP19) mainly found in the adipose-filled tissues in
the body. Aromatase inhibitors work via prevention of cell proliferation in breast
886 B. Chuphal

cancer by blocking CYP19 and thereby estrogen synthesis. Two major classes of
these inhibitors exist which differ in chemical composition and mechanism of
function, namely, Type 2 and Type 1 inhibitors. Type 1 inhibitors are steroids like
exemestane which bind to the aromatase enzyme in irreversible manner, whereas in
nature, Type 2 inhibitors are non-steroidal such as letrozole and anastrozole and bind
to enzyme in a reversible manner. These inhibitors, especially anastrozole, provide
an alternative to tamoxifen (TAM) due to their ability to significantly increase
thymidine 50 -triphosphate (TTP) and lower rate of incidences such as vaginal
bleeding and thromboembolic events.
Tamoxifen. Hormonal intervention has been implemented for breast cancer
treatment as early as the 1800s, when Beatson treated it by performing an ovariec-
tomy. In years that follow, the underlying mechanisms to this treatment were
demonstrated to be related to hormonal responsiveness of these cancers and their
dependence on estrogen receptor (ER) signaling. The antiestrogen with
non-steroidal, TAM, is now being used for breast cancer treatment in postmeno-
pausal women given its attribute to both the cell growth arrest and cell death
induction. In spite of the belief that the primary mechanism of action of TAM is
related to the ER signaling inhibition, studies in the recent past have indicated the
presence of non-ER-mediated mechanisms which seem to include signaling
proteins, such as c-myc, transforming growth factor-ß (TGF-ß), calmodulin, and
protein kinase C (PKC). Research has also showed vital roles played by caspases and
MAPK, including p38 and c-Jun N-terminal kinase, in its apoptotic signaling. TAM
has significantly been able to decrease the mortality rate in case of breast cancer
having ER-positive cells in both postmenopausal and premenopausal women.

18.2.13.3 Therapy Based on Antibody

The term “targeted therapy” came into existence due to a newly discovered genera-
tion of cancer drugs which were specifically designed to interfere with targeted
molecules and were alleged to play a crucial role in either tumor progression or
growth. Antibodies are considered as another type of targeted therapy by many
scientists. They are often used when conjugated with cytotoxic cellular poisons,
drugs, or radioisotopes, killing the malignant cells with target antigen. The role and
inherited nature of genetic polymorphisms in prolonging the effect of drugs and their
disposition is being rapidly elucidated by pharmacogenomic studies. These studies
thus greatly enhance the process of drug discovery, thereby providing a stronger
scientific basis for optimizing drug therapy on the genetic constitution basis respec-
tive of the patient.
Trastuzumab. Being the pioneer approach concerning neoplastic cell growth,
trastuzumab (Herceptin®, Genentech Inc., San Francisco, CA) is a recombinant
human monoclonal antibody which targets the human epidermal growth factor
receptor 2 (HER2 or ErbB-2) protein extracellular ligand binding domain, generally
expressed in all epithelial cell types and belongs to the human epidermal growth
factor receptor (HER) family. The receptor gene seems to be overexpressed in 30%
of breast cancers, compromising an unfavorable prognosis and the tumor’s aggres-
sive behavior. The most significant predictor of relapse time and overall survival in
18 Molecular Genetics of Cancer 887

breast cancer patients is HER-2/neu gene amplification which promises to distin-

guish between patients who will not show any response and those who may have
registered a significant response to the antibody. Nonetheless, this high expression of
HER2 receptor protein is suitable as antibody therapy target using trastuzumab.
Even though most patients usually tolerate trastuzumab, however, its adverse effects
include chills and/or acute fever and are responsible for the cardiac dysfunction
(CD) among patients having anthracycline prior exposure as well as death and
anaphylaxis in patients receiving anthracycline and cyclophosphamide treatment
combined with trastuzumab, and therefore treatment of patients with trastuzumab
previously are at a higher risk of cardiomyopathy development, suggestive of the
cardiomyopathy associated with anthracyclines. Till today, the molecular
mechanisms associated with trastuzumab-associated cardiomyopathy are vague,
and the risk can be substantially lowered by using trastuzumab with concurrent
paclitaxel and trastuzumab or as a single agent.

18.2.13.4 Colon and Rectum

Colorectal cancer as the name suggests starts as a growth on the inner lining of either
the colon or the rectum. These growths are termed polyps. Only some polyps can
turn cancerous over time depending upon the type. There are mainly two types of
polyp:

1. Adenomatous polyps (adenomas): Adenomas are often called a precancerous

condition as there are chances that these polyps may cause cancer.
2. Hyperplastic polyps and inflammatory polyps: Being very common, this type is
not precancerous.

If development of polyp takes place, it can further penetrate and grow into the
colon or rectum wall eventually. Colorectal cancer usually develops in the innermost
layer (the mucosa) and further grows outward via the other layers.
Once inside the colorectal wall, cancer cells can metastasize into blood or lymph
vessels and may grow into nearby lymph nodes or spread further to distant parts of
the body. The stage of the cancer depends on the extent of how much it has
metastasized outside and growth into the wall of the colon or rectum. Owning
majority of colorectal cancers (96%), adenocarcinomas tend to produce the mucus
for lubrication of inside of colon and rectal wall.

18.3 Cancer and Apoptosis

Lying under the idiopathy and complexity of each cancer type is a restricted set of
“mission-critical” events which propel the tumor and the associated progeny cells
into unrestrained division and invasion. One of the key events is known to be
unregulated cell proliferation, which, along with compensatory suppression of
apoptosis required for support, gives a minimal “stage” inevitable for the support
of neoplastic advancement. Inside our body, many surplus and unwanted cells are
888 B. Chuphal

usually disposed of via a mechanism that is already programmed into them.

Programmed cell death, which is also known as apoptosis (Greek roots meaning
falling off), is vital among animals as its absence might impair the proper functioning
of organs. Apoptosis is also equally important for prevention of tumor formation
because if a cancer cell is killed, it cannot multiply to form a dangerous mass of cells.
Hence, apoptosis keeps a proper check against the mutated cells which would
otherwise divide uncontrollably leading to the formation of HULK! A detailed
analysis of apoptosis mechanism is important in a way that it is essential in many
of the disease pathologies. In some cases, too much apoptosis is associated with
some major problems, such as the case of degenerative diseases, whereas in other
cases, the culprit is too little apoptosis, cancer being the perfect model. Kerr et al.
described apoptosis in the late 1900s for its morphological characteristics, including
chromatin condensation, membrane blebbing, cell shrinkage, and nuclear fragmen-
tation. Our understanding of development biology has had insightful implications
through the discovery of the fact that apoptosis is a gene-directed program, which
implies that factors distressing cell proliferation and survival and differentiation of
cells can regulate cell numbers.
Moreover, any mutations in the genes controlling apoptosis lead to the disruption
in the process. This process is the most investigated one in biological research.
During apoptosis, a family of proteases called caspases play an inevitable role. These
proteases cleave peptide bonds, thereby inactivating the target proteins. The target
consists of a plethora of proteins including components of cytoskeleton, and lamins,
which are the building blocks of the inner lining of the nucleus. The proteolytic
cleavage results in chromatin fragmentation, formation of cytoplasmic blebs at cell
surfaces, and shrinkage of cells themselves. Cells with these integrated defects are
usually engulfed by host phagocytes, one of the key players of the innate immunity,
and are processed for destruction. If the machinery of programmed cell death is
impaired or inactivated, a defective cell would have the potential to survive and
divide uncontrollably and hence become cancerous.

18.3.1 Morphological Changes in Apoptosis

Across all the cell types and species, these changes remain quite the same and alarm
the cytoplasm as well as nucleus. For nucleus, the hallmarks of death by apoptosis
are nuclear fragmentation and chromatin condensation often with cell rounding up;
reduced cellular volume, i.e., pyknosis; and pseudopod retraction. During the con-
densation of genetic material, i.e., chromatin, periphery of the nuclear membranes
starts to form ring-like or crescent structure. Further, the chromatin condenses till it
breaks up inside the cell with intact plasma membrane throughout the process. The
morphological features at the later stages of apoptosis include membrane blebbing, a
loss of membrane integrity, and ultrastructural cytoplasmic organelle modifications.
Under normal circumstances, the phagocytic cells engulf cells undergoing
apoptosis (Elmore 2007).
18 Molecular Genetics of Cancer 889

18.3.2 Biochemical Changes in Apoptosis

The main biological changes observed during apoptosis: (a) caspase activation,
(b) DNA and protein breakdown, and (c) phagocytic cells recognize the cell mem-
brane changes. The expression of phosphatidylserine (PS) during early apoptosis is
flipped out to the outer cell membrane from the inner membrane, which eventually
allows macrophages for early recognition of the dead cell, followed by phagocytosis
without pro-inflammatory cellular component release. For the detection of apoptosis,
a recombinant phosphatidylserine-binding protein, Annexin V, has been developed,
which has higher affinity for the phosphatidylserine amino acid residues, thus used
as recognition signal for apoptosis. Another protein, Calreticulin, has the ability to
bind with the LDL receptor-related proteins, thereby indicating the engulfing cells.
In the microvascular endothelial cells, expression of an adhesive glycoprotein,
thrombospondin-1 and CD36, has been observed. Then DNA breaks into 50–300
kilo bp pieces and eventual inter-nucleosomal cleavage by endonucleases into
oligonucleosomes into 180–200 bp fragments. An essential step of apoptosis
includes caspase (c-caspase, caspase-aspartic acid, a group of cysteine proteases)
activation which has signature cleaving site after aspartic acid residues. Once
caspases are activated, they break down the cytoskeleton and nuclear scaffold,
cleave vital cellular proteins, and activate DNAse, hence degrading nuclear DNA.

18.3.3 Mechanism of Apoptosis

Two initiation pathways, (a) mitochondrial (intrinsic) and (b) death receptor (extrin-
sic) pathways (Fig. 18.5), exist eventually leading to execution phase of apoptosis
known as common pathway. A third pathway is the perforin/granzyme pathway;
however a fourth lesser known intrinsic endoplamic reticulum pathway is also
present for apoptosis. The extrinsic death receptor pathway starts when an appropri-
ate ligand, Fas (fatty acid synthetase) ligand (FasL) and TNF-α (tumor necrosis
factor alpha), is bound to the receptor, Fas (CD95) and type 1 TNF receptor
(TNFR1), respectively. The TNFR has an extracellular domain rich in cysteine
amino acids and a cytoplasmic domain consisting of 80 amino acid residues
(known as death domain). These receptors are well known for having cytosolic
death domain that plays a critical role in recruitment of proteins such as
Fas-associated death domain (FADD), pro-caspase-8, and TNF receptor-associated
death domain (TRADD). The death effector domain (DED) at the amino-terminal of
pro-caspase-1 is seen to be involved in the interaction with DED of FADD.
The complex so formed by ligand-receptor-adaptor known as death-inducing
signaling complex (DISC) leads to autocatalysis of pro-caspase-8 and hence activa-
tion of caspase-8. In addition to the FasL/FasR and TNF-α/TNFR ligand/receptor
combination, other lesser known ligand/receptor combinations have also been dis-
covered such as Apo2L/DR4, Apo2L/DR5, and Apo3L/DR3 (Apo3 ligand/death
receptor 3).
890 B. Chuphal

Fig. 18.5 Schematic representation of the apoptotic intrinsic and extrinsic pathways

Several internal stimuli such as high cytosolic Ca2+ concentration, permanent

genetic damage, and severe stress by oxidative species are known to trigger the
intrinsic mitochondrial pathway. There are basically two types of stimuli, negative
and positive, that control the intrinsic mitochondrial pathway. Absence of cytokines,
hormones, and growth factors leads to apoptosis suppression, which acts as negative
stimuli, thereby leading to apoptosis. The positive stimuli include hypoxia, radiation,
viral infection, hyperthermia, and reactive oxygen species which directly trigger
apoptosis events. Irrespective of the stimuli, increase in mitochondrial permeability
and pro-apoptotic molecule release in the cytoplasm are the ultimate result of this
pathway, which is under Bcl-2 family protein regulation. There are two main groups
of the B-cell lymphoma protein 2 (Bcl-2) proteins: the first one involves
pro-apoptotic proteins (Bad, Bcl-Xs, Bak, Bax, Bid, Bim, and Hrk), and the second
one consists of anti-apoptotic proteins (Bcl-2, Bcl-W, Bcl-XL, Bfl-1, and Mcl-1).
Interaction of caspase recruitment domain (CARD) with Apaf-1 (apoptotic protease
activating factor) is essential for the formation of apoptosome from pro-caspase-9,
Apaf-1, and cytochrome and leads to the caspase-3 activation. On the other hand,
DIABLO (direct IAP binding protein with low PI), Omi/HtrA2 (high-temperature
18 Molecular Genetics of Cancer 891

requirement), or Smac (second mitochondrial activator of caspases) disrupts the

interaction of caspase-3 or caspase-9 with the IAPs by binding to inhibitor of
apoptosis proteins (IAPs) and results into caspase activation. When the cell has
committed for apoptosis, AIF (apoptosis induce factor), CAD (caspase-activated
DNase), and endonuclease G released from the mitochondria at a later stage promote
the apoptotic events. All these pro-apoptotic signal molecules, CAD,
endonuclease G, and AIF translocate to the nucleus and result in the DNA fragmen-
tation and peripheral nuclear chromatin condensation.
The perforin/granzyme pathway involved the release of granzyme enzyme
(Granzyme A and Granzyme B) and perforin from the cells, which are specifically
linked to the cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells. The
release of perforin from CTL and NK cells results in formation of pores in the
membrane of targeted cells, through which influx of cytoplasmic granules occurs.
Granzyme A and Granzyme B are serine proteases and have the ability to cleave at
the aspartate residues. By interaction with the mannose-6-phosphate receptors
(IGFR2), Granzyme B enters the target cells and also through the pores formed by
the perforin enzyme. By cleaving the ICAD (inhibitor of caspase-activated DNase),
Granzyme B has potential to activate the pro-caspase-10. Granzyme B cleaves the
anti-apoptotic signal and leads to mitochondrial cytochrome c release, subsequently
inducing apoptosis. In addition, Granzyme B possesses the capacity of direct
activation of caspase-3 and leads to the execution pathway of apoptosis. Since
Granzyme B activates caspase-dependent pathway, Granzyme A leads to apoptosis
in caspase-independent pathway. Through the activation of DNase NM23-H1, a
product of tumor suppressor gene, Granzyme A, leads to DNA nicking. This DNase
has the ability to prevent cancer in the CTL, thereby essential in the immune
surveillance. The HM23-H1 gene is under the control of a nucleosome assembly
protein, SET. The SET protein complex comprises of SET, Ape1, pp32, and HMG2
proteins which protects the structure of DNA and chromatin. Granzyme A abolishes
the functions of SET complex by cleaving the SET proteins and induces apoptosis in
the target cell. However, a nuclear pathway has also been proposed which depends
on the Pml oncogenic domains (PODs) or nuclear bodies. It has been reported that
mice with defective pml gene develop resistance against apoptosis through unknown
mechanisms. The PODS includes Daax, Zip kinase, and Par4 proteins, which has a
role in apoptosis induction, and defects in these have been reported in cancers.
Caspase-3 acts as a converging point for the intrinsic and extrinsic pathways and
is responsible for cleaving the inhibitor of the caspase-activated deoxyribonuclease
(ICAD), thereby activating CAD, known to have indispensable role in nuclear
apoptosis. In addition, downstream caspases (caspase-6 and caspase-7) cleave cyto-
skeletal proteins, protein kinases, inhibitory subunits of endonuclease family, and
DNA repair proteins. These processes result in the abovementioned morphological
changes involved in apoptosis. Caspase-3 cleaves the gelsolin protein, which is
involved in the polymerization of actin protein and serves as the binding site for
phosphatidylinositol phosphate, resulting in the disruption of the cytoskeleton and
intracellular signaling. Due to evasion of cell death, cancer is often seen as an
892 B. Chuphal

outcome of a series of genetic changes which involve transformation of normal cell

into malignant one.
Generally reduced caspase function or impaired death receptor signaling or
disruption of pro- and anti-apoptotic protein balance due to an overexpression or
an underexpression or both leads to cell death evasion. The caspases are placed in
two groups: (1) those related to caspase-1 (e.g., caspase-1, caspase-4, caspase-5,
caspase-13, and caspase-14) and are involved in inflammation and cytokine
processing and (2) those involved in apoptosis (e.g., caspase-2, caspase-3,
caspase-6, caspase-7, caspase-8, caspase-9, and caspase-10). The other group has
been divided into (a) effector caspases (caspase-3, caspase-6, and caspase-7) essen-
tial in cleavage of cellular components and (b) initiator caspases (e.g., caspase-2,
caspase-8, caspase-9, and caspase-10) involved in initiation of apoptotic pathway.
Thus, decrease in apoptosis and carcinogenesis is the result of low concentration or
impairment in caspases. However, few reports suggest that caspase disruption is only
responsible for cell death delay. On the other hand, protease loss leads to pathologi-
cal increases in cell numbers. The cellular FADD-like interleukin-1 beta-converting
enzyme inhibitory protein, i.e., c-FLIP, acts as an inhibitor of extrinsic death
receptor pathway by binding to caspase-8 and FADD. Another inhibitor of the
death receptor mediated apoptosis is Toso which regulates T-cell apoptosis by
inhibiting the activation of caspase-8. Endometrial, colon and prostate cancer,
non-small cell lung carcinoma (NSCLC), hepatocellular carcinoma, and melanoma
reported higher expression of c-FLIP protein, whereas in some cancers (ovarian,
prostate, endometrial, and colon cancer) increased expression of c-FLIP reported
poor prognosis and increased progression of cancer. To date, only few reports are
available which show the caspase mutation involvement in cancers. Noonan’s
syndrome is a result of disruption in Apaf1, autoimmune lymphoproliferative syn-
drome type II is caused by mutations in caspase-10, and mutations in caspase-5
including frameshift mutations are reported to occur in endometrial tumors, gastro-
intestinal tumors, and hereditary non-polyposis colorectal cancers. Abnormalities in
death receptor and ligand signaling pathway lead to dodging of apoptosis extrinsic
pathway as they play a crucial role in the same. Regardless of the type or mechanism
of defects, these abnormalities may include receptor function impairment or
downregulation of receptor expression and reduction of the levels of death signals
eventually contributing to reduction of apoptosis. CD95 reduced expression is
associated with neuroblastoma cells or treatment-resistant leukemia. Reesink-Peters
et al. (2004) reported that the factors responsible for carcinogenesis in cervical
cancer include loss of Fas and dysregulation of DR5, DR4, FasL, and TRAIL
(tumor necrosis factor-related apoptosis-inducing ligand) in the cervical cancer
sequence of cervical intraepithelial neoplasia (CIN). TRAIL (also known as
Apo2L) is essential for the tumor suppression through the NK-mediated
apoptosis (Delbridge et al. 2012), and FasL is critical for killing the cancerous
cells through the CTL. However, with progression of tumor cells, these cells develop
resistance against the FasL and TRAIL either through expression of nonfunctional or
downregulation of Fas receptor, thereby escaping the immune attack and preventing
resistance against apoptosis. The c-FLIP and RIP are crucial in the activation of
18 Molecular Genetics of Cancer 893

NF-κB and ERK pathway in the TRAIL-resistant NSCLC. The constitutive expres-
sion of NF-κB and upregulation of c-FLIP by the NF-κB lead to various human
cancers. The apoptosis-inducing proteins (IAPs) are essential for caspase activation.
IAPs consist of at least one copy of BIR (baculovirus IAP repeat) and one to three
copies of zinc-bonding fold which are essential for the anti-apoptotic activity of
IAPs. The IAP family includes cIAP1, cIAP2, and XIAP (X-linked mammalian
inhibitor of apoptosis protein), member that binds via BIR domain and results in
suppression of the caspase-3, caspase-7, and caspase-9 activity, thereby evasion of
cell from the apoptosis. Survivin and livin (ML-IAP) are other members of IAP
family involved in the inhibition of caspase-9 only. In several cancers,
overexpression of IAPs has been documented, and IAPs are also associated with
the resistance of cancer against chemotherapy and maintains the survival and growth
of cancer cells. These IAP molecules suggest a potential target to fight against
cancer. The development of synthetic peptides of IAP antagonists which mimics
the natural IAP antagonists (Smac and DIABLO) present in the mitochondria may
help to curb the cancer development. The upregulation of anti-apoptotic proteins
such as Bcl-2 and downregulation of pro-apoptotic proteins such as Bax are
observed in several cancers that leads to inhibition of intrinsic apoptotic pathway.
The tumor suppressor protein, p53, regulates Bcl-2 and Bax expression, and p53
mutation accounts for 50% of human cancers. Upon sensing the DNA damage,
ataxia telangiectasia-mutated gene (ATM) activates the p53 pathway of apoptosis,
and ATM gene mutation is reported in the several cancers. In addition, several
signaling pathways lead to tumor development. For the development of cancer,
phosphatidylinositol 3-kinase/AKT pathway activation without the requirement of
ligand (cell survival signals) is responsible. Several researches are ongoing to
develop the specific molecular target therapy against the cancer. For the treatment
of cancer, the pro-apoptotic and anti-apoptotic proteins, p53 protein, caspases, and
several signaling components present potential molecular targets. These molecular
target therapies are less toxic with less side effects compared to the chemotherapy.

18.3.4 Apoptosis During Cancer

The question now appears as to what triggers apoptosis during tumor development.
Extracellular triggers include loss of cell-matrix interactions, radiation, hypoxia, and
depletion of growth/survival factor. Disruption in proliferative signals produced by
oncogenic mutations, malfunction of telomeres, and DNA damage are among the
various factors misbalancing the internal system and triggering apoptosis. Although
very unlikely, in some cases, apoptotic “trigger” leads to the alleviation of an anti-
apoptotic signal. For instance, IGF-1 promotes cell survival by activating the PI-3
pathway; however, “death by default” can be triggered by survival factors such as
IGF-1. In contrast, p53 activation under stress and stimuli forms pro-apoptotic
factors promotes apoptosis through the involvement of pro-apoptotic molecules
such as Bax.
Apoptotic trigger identification may provide valuable insights into the tumor
evolution. Excessive exposure of skin to UV radiation leads to apoptosis induction,
894 B. Chuphal

Fig. 18.6 Evasion of apoptosis and carcinogenesis: the contributing mechanism

and p53 function loss leads to damaged cell survival, thereby initiating tumor
development (Fig. 18.6). The developing tumors encounter hypoxia as they outgrow
the blood supply which then activates p53 eventually promoting apoptosis. Cells
which have underlying apoptotic defects can survive the hypoxic stress, which leads
to clonal expansion. For telomere malfunction-induced apoptosis, not only hypoxia
but p53 is also required. Thus, cells with p53 mutation survive and are unstable
genomically where loss of p53 and telomerase stimulates the development of tumor.
Every target of cancer treatment arose from every abnormality or defect along the
apoptotic pathways like a double-edged sword. Treatment strategies and drugs
represent the potential approach in eliminating cancer cells and have the ability for
restoring normal apoptotic signaling pathways because cancer cells in a way depend
on these defects for thriving (Hassan et al. 2014; Lowe & Lin 2000; Wong 2011).
Potential classes of anticancer drugs opened by recent advancement and important
discoveries are summarized in Table 18.6.

18.4 Telomere Shortening, Telomerase, and Cancer

Present toward the end of chromosomes and considered as protective structures,

telomeres contain repetitive nucleotide sequences (TTAGGG in case of vertebrates)
along with linked proteins, commonly termed as shelterin consisting of six proteins:
TRF1, TRF2 (telomere double-stranded DNA-binding proteins), RAP1, TIN2,
TPP1, and single-stranded DNA-binding protein (POT1) (Table 18.7).
By preventing nucleolytic degradation, irregular recombination, and chromo-
somal end-to-end fusion, telomeres bring about genomic stability and chromosome
integrity. At a rate of 50–200 bp per cell replication, the telomeric DNA shortens and
has an average length of 10–15 kb in humans (Okamoto & Seimiya 2019).
18 Molecular Genetics of Cancer 895

Table 18.6 List of potential new anticancer drugs

S. no. Strategies of treatment Remarks
1. Target the proteins of Bcl-2
family
(a) Bcl-2 family proteins In combined action with the conventional anticancer
targeting agents drugs, chemosensitizing effects have been shown in
i. Oblimersen sodium chronic myeloid leukemia patients and improved
ii. Bcl-2 family protein their survival
inhibitors Sodium butyrate, depsipeptide, flavopiridol, and
iii. BH3 mimetics fenretinide affect gene and protein expression.
(b) Silencing the Bcl family Molecules like HA14–1, gossypol, ABT-737,
anti-apoptotic proteins/genes GX15–070, and ABT-263 act on the proteins
ABT-737 inhibits anti-apoptotic proteins and
exhibits cytotoxicity in SCLC cell line, lymphoma,
and primary patient-derived cells
ATF4, NOXA, and ATF3 bind and inhibit Mcl-1
Bcl-2 specific siRNA inhibits the target gene
expression with antiproliferative effect and
promotes pro-apoptotic effect in pancreatic
carcinoma cells under both in vivo and in vitro
conditions
Bmi-1 silencing in MCF breast cancer cells
downregulates Bcl-2 and pAkt expression and
increases apoptosis in vivo and in vitro along with
increased sensitivity of these cells to doxorubicin
2. Targeting the p53
(a) Gene therapy based on Retroviral vectors containing normal p53 gene were
p53 injected into tumor cells derived from NSCLC
(b) Drug therapy based on patients, and this therapy is feasible
p53 Wild-type p53 introduction sensitize the colorectal
i. Small molecules and prostate and neck and head cancers and glioma
ii. Other agents: Nutilins, to the ionizing radiation
MI-219, and Tenovins ONYX-015, a genetically engineered oncolytic
(c). Immunotherapy based on adenovirus, selectively replicates in and lyses tumor
p53 cells, which are p53 deficient
Phikan083 binds and restores mutant p53
CP-31398 destabilizes the DNA-p53 core domain
complex and intercalates with DNA, thereby
restoring unstable p53 mutant
Nutilins inhibit MSM2-p53 interaction and stabilize
p53, resulting in induction of cancer cell senescence
selectively
MI-219 disrupts the MDM2-p53 interaction,
thereby inducing selective apoptosis in tumor cells
and inhibits tumor growth and proliferation
Tenovins under in vivo condition decrease tumor
growth
A recombinant replication-defective adenoviral
vector containing vaccine with normal p53 gene
given to advanced-stage cancer patients and
reported stable disease
(continued)
896 B. Chuphal

Table 18.6 (continued)

S. no. Strategies of treatment Remarks
3. Targeting IAPs
(a) Targeting XIAP In vivo improvement in tumor control by the
i. Antisense approach radiotherapy
ii. siRNA approach Antisense oligonucleotide use with chemotherapy
(b) Targeting Survivin in vivo and in vitro exhibits enhanced activity of
i. Antisense approach chemotherapeutic in lung cancer cells
ii. siRNA approach Using this method increased the sensitivity of
(c) Other IAP antagonists human cancer cells toward radiation
i. Small molecule antagonists The sensitized hepatoma cells lead to death receptor
and chemotherapeutic agent-induced cell death by
siRNAs
Spontaneous apoptosis is induced in LOX and
YUSAC-2 malignant melanoma cells when
transfected with anti-sense Survivin
It sensitizes squamous cells of the neck and head to
chemotherapy and induces apoptosis
Inhibits medullary thyroid carcinoma cell
proliferation and growth
Diminishes pancreatic cancer cell’s radioresistance
through downregulation of Survivin
Induces apoptosis in human lung adenocarcinoma
cells (SH77 and SPCA1) and inhibits proliferation
Enhances apoptosis, inhibits cell proliferation, and
suppresses expression in ovarian cancer cells
(SKOV3/DDP)
Enhances the sensitivity of NSCLC cells
Gene therapy and CDK and Hsp90 inhibitors are
attempted in targeting survivin
Cyclopeptidic Smac mimetics 2 and 3 bind to XIAP
and restore the caspase-9/caspase-3/caspase-7
activity, which is inhibited by XIAP
SM-164 enhances activity of TRAIL by targeting
XIAP and IAP1
4. Targeting the caspases
(a) Drug therapy based on Apoptin induces apoptosis in malignant cells
caspases selectively
(b) Gene therapy based on Small molecule caspase activators lower the caspase
caspases threshold activation and increase the cancer cells
sensitivity toward drugs
In addition with etoposide, human caspase-3 gene
therapy induces apoptosis and reduces volume of
tumor in AH130 liver tumor model
Constitutively active caspase-3 gene transfer into
HuH7 human hepatoma cells results in selective
apoptosis
A recombinant adenovirus with immunocaspase-3
promotes in vivo and in vitro anticancer effect in
hepatocellular carcinoma
(continued)
18 Molecular Genetics of Cancer 897

Table 18.6 (continued)

S. no. Strategies of treatment Remarks
5. Targeting signal transduction
pathways
(a) Targeting NF-κB In response to TNF and chemotherapeutic agents,
(b) Targeting PI-3/Akt NF-κB signaling is inhibited, resulting in inhibition
pathways of cell growth
(c) Targeting Ras-GAP I-κB or proteasome inhibitor inhibits NF-κB activity
and induces tumor cell death in association with
radiation and chemotherapy
Small molecule inhibition of enzymes involved in
PI-3/Akt pathways leads to tumor cell apoptosis
Farnesyltransferase is responsible for active Ras.
Farnesyltransferase inhibitor, as an antitumor agent,
leads to massive apoptosis and mammary carcinoma
regression in ras transgenic mice

Table 18.7 List of shelterin proteins and their function

Shelterin proteins
Function involved
End protection from ATM- and ATR-dependent DNA damage responses TRF2, POT1
End protection from DNA repair pathways: non-homologous end joining
and homologous recombination
Higher-order telomere loop structure called t-loop TRF2
DNA bending activity, telomere replication at the S phase of the cell TRF1
cycle
Telomere localization TPP1
Negative regulation of telomerase TIN2
NB: The t-loop is formed with the help of TRF1 DNA bending ability which prevents the
telomerase and machinery involved in response to DNA damage to be recognized by the telomere
ends resulting in a single-stranded G-overhang (G-tail, 30 -overhang) invasion at the ends to double-
stranded telomeric DNA

Inside normal somatic cells of human, the semi-conservative mode of replication

leads to the gradual shortening of telomeric ends, and cells with awfully short
telomeres undergo apoptosis. However, there are cells with self-renewal powers,
which surprisingly maintain telomeres to overpower the programmed cell death
caused by short telomeres and become immortal such as embryonic stem cells and
almost all cancer cells. Generally, a critically short length of telomere is the trigger
for a cell entering subsequent replicative senescence. The ultimate fate of the
triggered cell is cell death; alternatively, on the other hand, if death doesn’t take
place, cells continue to divide, often resulting in genomic instability and chromo-
somal abnormality.
The literature available up to now suggests that telomere maintenance occurs
through two important pathways: activation of telomerase-independent mechanism
employing homologous recombination DNA repair pathway also known as
898 B. Chuphal

Fig. 18.7 Telomerase assembly, maturation, and recruitment to telomere. The synthesis of hTERT
takes place in the cytoplasm. The hTR and hTERT assembly into functionally active telomerase
(“?” subcellular location still unknown) is assisted by reptin and pontin (AAA+ ATPases). The
recruitment of telomerase to telomeres takes place via interaction of TPP1 with TEN domain of
hTERT in the S phase of cell cycle

alternative lengthening of telomeres (ALT) and telomerase transcriptional activation,

telomerase reverse transcriptase (TERT). Out of all cancer cells, 85–95% express
telomerase; the rest undergo ALT pathway activation. Telomerase in general
consists of two main components, TERT and telomerase RNA (TERC/TR), along
with some proteins such as pontin/reptin, NOP10, DKC1, TCAB1, and NHP2.
The recruitment of telomerase to telomeric single-stranded DNA takes place via
interaction with TPP1, a telomerase-localizing protein. Using TERC as a template,
telomeric sequences are synthesized by TERT. The mechanism taking place is
shown in Fig. 18.7. Studies have reported that TERC is constitutively expressed in
human somatic cells under normal condition, whereas TERT expression is silenced
epigenetically. Interestingly, most cancer cells re-express the limiting factor, TERT,
and hence acquire telomerase activity.
Many groups of scientists have studied the mechanism regulating TERT tran-
scription over the past years. In 1999, in order to isolate the TERT gene 50 promoter
region, three independent groups were successful. The core promoter region essen-
tial for the transcription of the gene was demonstrated to fall upstream of the
transcription start site by 260 bp. Further, the E-box having the signature sequence –
CACGTG- is reported to bind with C-MYC, SP1, and other transcription factors at
165 bp and + 44 bp along with five GC boxes with conserved sequence –
GGGCGG-, respectively, resulting in the induction of TERT mRNA expression.
The promoter region also possesses binding sites for other transcription factors such
as AP-1, E2F, and ERE (estrogen response element) which are essential for TERT
18 Molecular Genetics of Cancer 899

mRNA expression. Another important factor which creates loops in the chromatin
and functions as insulator across the genome is CTCF (CCCTC binding factor). It is
also involved in positive and negative regulation of gene expression by either
promoting the promoter-enhancer association or blocking it in a manner dependent
on the position, respectively. In addition, phosphatidylinositol-3 kinase (PI3K)/AKT
kinase pathway enhances TERT activity via phosphorylation of TERT at the post-
translational level. Hence, the regulation of expression of TERT takes place at
multiple levels through several factors. TRF1 promotes telomere replication at the
S phase and in contrast negatively regulates telomerase through recruitment of TIN2.
Telomerase is actively also regulated by TPP1-POT1 wherein TPP1 interacts with
telomerase and promotes telomerase processivity, whereas POT1 limits the access of
G-overhangs to telomerase by binding to single-stranded DNA. In addition, for
TPP1-TERT interaction, phosphorylation of TPP1 is required, which is dependent
on cell cycle.

18.4.1 TERT Expression: Epigenetic Regulation

Studies have reported that TERT expression levels correlate with DNA methylation
levels at promoter region of the gene, which is in contrast to the role of methylation
in gene silencing. The levels of methylation in TERT are reformed between explicit
positions in the promoter region.
For instance, the highly methylated regions lie between 600 and  200 bp,
and  200 to +150 bp region corresponds to relatively low methylation rate. The
200 to 100 bp area has the presence of GC box and E-box, collectively called
core promoter region known to activate TERT mRNA expression by binding to SP1
and C-MYC as mentioned before. Hence, the methylation levels are reduced as
compared to +1 to +100 bp though both the areas fall under low methylation rate.
Also,
the positive correlation between TERT expression and methylation level in the
promoter region has been demonstrated by whole genome sequencing which also
shows that the expression negatively correlates to methylation level of gene body.
To further support this, Sterna and group have shown that 600 bp methylation level
inversely correlates to TERT transcription. In addition, when compared to
monoallelic mutant cancer cells, wild-type allele had higher level of DNA
methylation.

18.4.2 Regulation of Gene Expression by Telomeres

The length of telomere affects the expression of genes located near the region, and
this phenomenon is known as telomere position effect (TPE). This phenomenon was
first discovered by Gottschling and group in Saccharomyces cerevisiae and reported
that the expression of RNA polymerase II was suppressed when inserted next to
telomere locus. The reason behind TPE was suggested to be silent chromatin
900 B. Chuphal

conformation in the telomeric region. An example includes ISG15 which shows

higher expression in the aged cells. ISG15 is located at 1 Mb from the telomeric end
and on the chromosome number 1p36.33. Interestingly, Robin and group
demonstrated that not only adjacent genes but those far from telomeric region
(upto 10 Mb) are also affected by TPE by a process known as long-range loop
formation. This phenomenon involving atypical TPE is known as TPE-OLD (telo-
mere position effect over long distances). TRF2 is a component of shelterin and falls
under TPE-OLD. This protein tethers interstitial telomere repeats to telomere and
generates the so-called long-range loop of chromatin. These repeats are present
100 kb downstream of TERT gene and suppress its expression by forming chromatin
loop in a TRF2-dependent manner. This suppression is aided by LDS1 (lysine-
specific demethylase 1) and REST (RE-1 silencing transcription factor) which bind
to promoter region bound to TRF2 and add silencing histone marks. The regulation
of gene expression in telomere length-dependent manner might involve TERRA,
telomeric non-coding RNA associated with telomere length regulation, telomere end
protection, and maintenance of chromatin structure. TERT overexpression leads to
increase in number of telomeric tracts and TERRA signal enhancement, the reason
being telomere elongation. Therefore, in telomere-elongated cancer cells, decreased
expression of ISGs is associated with TERRA signal enhancement. This is further
validated by the fact that ISG expression in cells having short telomeres is repressed
by oligonucleotides which mimic TERRA. All these observations point toward the
possibility that downregulation of ISG expression is dependent on upregulated
TERRA signals in cancer cells having longer telomeres (Zhu et al. 2016).

18.4.3 TERT Promoter Mutations in Cancer

In sporadic melanoma, point mutation in the TERT promoter at 146 base pairs
(C > T) and  124 (C > T) from TSS (transcription start site) was discovered by
Horn’s group and Huang’s group. Furthermore, in familial melanoma, Horn and
group reported point mutation at 57 bp (T > G) from TERT transcription start site.
These mutations lead to upregulation of TERT mRNA expression by providing E-
twenty-six (ETS) transcription factor (GGAA, reverse complement) novel consensus
binding motifs in the promoter region. These mutations being common type in
noncoding somatic cells are present in different types of cancers (Table 18.8).
Except in myxoid liposarcoma, there seems to be a low percentage of mutation in
TERT promoter as activation of ALT and not telomerase occurs in about 60% of
sarcoma. In light of these observations, TERT promoter mutations are likely to be
mutually exclusive with death domain-associated protein (DAXX), the
α-thalassemia/mental retardation syndrome X-linked (ATRX), and other ALT
pathway-associated chromatin modeling proteins. In case of mutations in TERT
promoter in the case of ovarian carcinoma, the telomerase activity is regulated by
ARID1A and PIK3CA.
18 Molecular Genetics of Cancer 901

Table 18.8 TERT promoter mutations involved in cancer types

Type of cancer % Occurrence of TERT promoter mutation
Melanoma 67
Glioma 51.1
Primary glioblastoma 83.3
Myxoid liposarcoma 79
Bladder cancer 59
Urothelial carcinoma 50.8
Hepatocellular carcinoma 44
Medulloblastomas 21
Ovarian clear cell carcinoma 15.9
Squamous cell carcinoma 14.4
Thyroid cancer 10
Osteosarcoma 4.3

18.4.4 Telomere Shortening in Cancer

In contrast to the acquired ability such as elongation of telomere involved in cancer

having activated telomerase, the length of telomere appears to be shorter in cancers
such as prostate cancer. The fact was validated through whole genome/exome
sequencing data from TCGA (The Cancer Genome Atlas) wherein Barthel and
group examined 31 cancer cohort types for length of telomere. They demonstrated
that while 30% of cohorts were suspected to be regulated by ALT, a majority (70%)
exhibit shorter telomeres compared with normal samples.
Nevus and melanoma section staining using fluorescence in situ hybridization
provided further validation. Chiba and group suspected and examined that telomere
maintenance through cell division in human embryonic stem (ES) cells involved the
TERT point mutants. Upon inactivating checkpoint proteins along with TERT
promoter mutation, immortalization phenotype was achieved. Interestingly, in
approximately 70 cell cycles, telomere length was shortened despite TERT mutation,
followed by emergence of TERT expression and telomerase activity. As compared
to non-cancerous tissues, cancerous ones show high expression of shelterin genes
such as POT1, TRF (1–2), and TIN2. Interestingly, telomere length is reported to be
inversely correlated with elevated expression of these genes/TERT/telomerase activ-
ity (Fig. 18.8). In normal telomerase silent cells, hTERT or its ectopic introduction
activates telomerase activity and bypasses senescence leading to cell immortaliza-
tion, demonstrating that for both crisis and senescence, the telomeres are mechanis-
tically important (Shay & Wright 2011).

18.4.5 Telomerase Therapeutics

In general, immunotherapy, gene therapy, and small molecule inhibitors are among
three classes of agents that have been developed for targeting telomerase molecular
902 B. Chuphal

Fig. 18.8 Implication of telomere shortening in cancer development. Telomere length inversely
correlates with TERT or even telomerase activity (b). In normal telomerase silent cells, hTERT or
its ectopic introduction (c) activates telomerase activity and bypasses senescence leading to cell
immortalization (a)

biology. Among the more advanced approaches, immunotherapy and small mole-
cule inhibitor therapy are prevalent. Gene therapy targets a suicide vector by using
either hTR (template RNA component) or hTERT promoter specific for oncolytic
virus. Immunotherapy is currently being employed for advanced cases of pancreatic
cancer and is undergoing phase III clinical trials, whereas small molecule therapy is
being pursued for non-small cell lung cancer and breast cancer currently in phase II
trials. In the case of lung cancer (non-small cell), use of small molecule therapy
under controlled manner involving telomerase inhibitor, namely, imetelstat, is prev-
alent for prolonging the remissions after chemotherapy. Under this treatment
method, the patients of lung cancer are randomized such that few of them receive
bevacizumab, an angiogenic inhibitor, some receive imetelstat, while others receive
imetelstat along with the angiogenic inhibitor. In case of multiple myeloma, small
molecule therapy involving imetelstat is being verified as a cancer biomarker for
depletion of stem cell.

18.5 Carcinogens

Carcinogens are the substances that have the capacity to induce cancer. Carcinogens,
genotoxic agents, have the potential to directly bind the DNA and result in DNA
damage, impairment of DNA repair machinery, and alteration in the proto-oncogene
18 Molecular Genetics of Cancer 903

and tumor suppressor gene and hence tumor development (Moschel 2001; Sugimura
2000). In addition, carcinogen alters expression of genes through epigenetic effects.
In the 1950s an idea was put forward that a carcinogen causes cancer by causing
some mutations, but a proof was not available initially. The first proof was put
forward by Bruce Ames. The name of the test which he introduced is Ames test
which assesses the mutagenic capability of a chemical. A special strain of bacteria
which could not synthesize histidine was taken, which is an essential amino acid, and
since these bacteria were made to grow in a medium lacking histidine, the cells
couldn’t survive. They added a chemical whose mutagenic capability was to be
tested to the media; that caused mutations in the bacterial cells: some of them were
back mutations, and in some bacterial cells, the capability of synthesizing histidine
was restored, and those bacterial cells started dividing.
Carcinogen has been classified under six groups by the International Agency of
Research on Cancer:

1. Biological agents.
2. Arsenic, fibers, metals, and dust.
3. Pharmaceuticals.
4. Chemical agents and related occupation.
5. Radiation.
6. Personal habits and indoor combustions.

Biological carcinogens include several viruses (HBV, HTLV, KSHV, EBV, and
HPV) (Butel 2000), bacteria (Chlamydia trachomatis, Helicobacter pylori), and
animals (Schistosoma haematobium, Opisthorchis viverrini, Clonorchis sinensis).
The ultraviolet radiations cause cataract and skin cancer. Several pharmaceuticals
including anticancer drugs, analgesics, and estrogen are documented as carcinogens.
Chemical carcinogen directly acting on DNA includes alkyl and aryl epoxides,
nitrosoureas, sulfonate, sulfate, and nitrosamides, while indirectly acting chemical
carcinogens include hydrocarbons with aromatic rings and amines, alkyl
nitrosamines, or aflatoxin B1. The effluents released from the industries (vinyl
chloride, benzene, chromium compounds, and aromatic amines) and through com-
bustion of fossil fuels (polycyclic aromatic hydrocarbons) are causing various
cancers. The chemicals used as pesticides, fungicides, and pesticides are potential
carcinogens. Chemical compounds dysregulate the cytochrome P-450 cellular
enzyme in the liver and induce cancer development. Because of cigarette smoking
habits, nicotine chemicals cause lung, esophagus, mouth, pharynx, bladder, pan-
creas, and larynx cancer. Several assays are discovered to check carcinogenic
potential of the particular substance which includes Ames test for the early detection
of carcinogen, single-cell gel assay for DNA strand break, FISH, DNA adducts
assay, and Syrian hamster embryo cell transformation assay. Ames test can detect the
mutagen agent but does not confirm its carcinogenic capacity. To detect the carcino-
gen, differential genetic expression assay, cDNA hybridization method, and
checking protein synthesis pattern are used.
904 B. Chuphal

18.6 Oncogenes

The oncogenes are initially reported as tumor-inducing genes encoded by retrovirus

in rodents and birds. Retroviruses picked up these genes from the host genes (proto-
oncogenes) as the dominated mutated forms of gene (Lee & Muller 2010). The
signals usually regulating cell division are molecules which either stimulate the cell
division process or inhibit it. Oncogenes cause cancer development when present,
and they can be either introduced via viruses into the host cells that cause cancer or
some genes in the host genome itself can be mutated to form oncogenes. These genes
can code for proteins which result in stimulation of excessive proliferation of cells
and can lead to enhanced cell survival. The activation of oncogene is the result of
epigenetic modifications, mutation, gene rearrangement or gene amplification, and
subsequently tumor development (Botezatu et al. 2016). The early stages of tumor
are associated with the gene mutation and translocation, while gene amplification is
associated with the late stage of tumor. The proteins encoded by oncogenes discov-
ered till date are classified among four major classes: growth factors, GFs (v-sis), GF
receptors (v-erbB, v-fms, v-kit), GF response transducers (v-src, r-raf, v-ras), and
transcription factors; TFs which mediates gene expression of growth factors induced
genes (v-jun, v-fos). The identification and sequencing of the simian sarcoma virus
resulted in the discovery v-sis oncogene. The cellular homologue of PDGFB protein
encoded by v-sis gene is PDGF (platelet-derived growth factor), which initially
thought to be responsible for the proliferation of arterial smooth muscle cells.
PDGF binds to the PDGFR (PDGF receptor) and Src family of tyrosine kinases
(SFKs) and activates PI3K-Akt pathway, therefore, involved in regulation of prolif-
eration and cellular growth (Cantley et al. 1991; Shih & Holland 2006). The
overexpression of PDGF and PDGFR is documented in the cancer of the central
nervous system, sarcoma, glial cell tumor, germ-cell tumor, and gastrointestinal
sarcoma. The GF receptor tyrosine kinase, epidermal growth factor receptor
(EGFR), overexpression is reported in the tumor growth progression and involved
in NSCLC, esophageal, ovary, cervix, breast, endometrium, and bladder
cancer (Nicholson et al. 2001). EGFR family includes HER4, HER3, and HER2,
of which overexpression of HER2 is associated with numerous cancers. The v-kit
oncogene has a cellular homologue, c-kit, that encodes a KIT protein, belonging to
the family of receptor tyrosine kinase. Mutation of the c-kit gene has been reported in
the exon numbers 11, 13, and 17, and valine (V) to an alanine (A) amino acid
substitution at 599 position (V599A) in the KIT is the most common mutation found.
These mutations are observed in the acral, mucosal, conjunctival and cutaneous
melanoma, and gastrointestinal stromal cancer. Another oncoprotein belonging to
tyrosine kinase family, c-ErbB, has been discovered in humans, along with identifi-
cation of v-ErbB protein in avian erythroblastosis virus, which is encoded by
oncogene, v-erbB. The c-erbB gene amplification is found in gliomas and many
other carcinomas, while point mutation in the c-erbB gene results in lung
carcinomas. With the discovery of McDonough feline sarcoma virus, v-fms onco-
gene has been identified, which is analogous to the colony-stimulating growth factor
receptor and associated with various cancers. In Rous sarcoma virus, v-src is derived
18 Molecular Genetics of Cancer 905

from c-src of the host genome, thereby responsible for the transformation of
fibroblasts. Oncogene ras of Kristen and Harvey sarcoma virus encodes for
oncoprotein Kristen-ras (Ki-ras) and Harvey-ras (Ha-ras) which results into the
transformation of the fibroblasts, hematopoietic cells, and epithelial cells into
cancerous cells. The cellular homologue of v-ras is c-ras, and mutation in the
c-ras leads to thyroid, colon, lung, and pancreatic carcinoma and acute myeloid
and lymphocytic leukemia. The cellular homologue in humans of v-jun oncogene
from the avian sarcoma virus (ASV17) is c-jun gene, which encodes for the c-Jun
protein with 39 kDa molecular weight, consisting of leucine zipper DNA-binding
domain at the C-terminus with basic in nature and transcription activator domain
present at the N-terminal region. Under the stress, c-Jun promotes apoptosis through
JNK signaling. Mutation in the c-Jun leads to the transformation of fibroblasts and
tumor development in mice. Detailed analyses of viral oncogenes, proto-oncogenes,
and tumor suppressor genes are discussed below.

18.7 Viral Oncogenes

In 1909, Dr. Francis Peyton Rous provided the first evidence of an infectious
etiologic agent that causes cancer, and the RNA virus inducing chicken sarcoma
was named Rous sarcoma virus in honor of Dr. Rous. He injected the cells, extracted
from the hen breast tumor, into other hens who developed sarcoma later. For this
discovery, he was awarded with Nobel Prize in 1966. Now approximately 20–25%
human cancers in the world have a viral etiology (Table 18.9). The first

Table 18.9 Human DNA and RNA virus

Taxonomic
grouping Examples Oncogenes Tumor types
1. DNA viruses
Adenoviridae Adenovirus types 12, 18, E1A, E2B Various solid tumors only in
Hepadnaviridae 31 HBx rodents
Herpesviridae HBV LMP-1, Hepatocellular carcinoma
Papovaviridae EBV BARF-1 Burkitt lymphoma, B-cell
Papillomaviridae KSHV vGPCR lymphoma, NPC
Merkel cell polyomavirus T antigens Kaposi sarcoma, primary
BK virus, JC virus E6, E7 effusion lymphoma
HPV 16, 18, 31, 45 Merkel cell carcinoma
Solid tumors in rodents and
primates
Cervical and anal cancer, oral
cancer
2. RNA viruses
Flaviviridae Hepatitis C virus – Hepatocellular carcinoma
Hepacivirus Human T-cell leukemia Tax Adult T-cell leukemia/
Retroviridae virus type I lymphoma
HTLV
906 B. Chuphal

demonstration of human retrovirus was human T-cell leukemia virus type 1 (HTLV-
1), which causes adult T-cell leukemia. Another group of scientists were identifying
the involvement of human DNA viruses in the transformation and development of
cancer. This led to the discovery of herpes simplex virus type 2 (HSV2) and
association of human papillomaviruses (HPVs) in cervical cancer development.
Some viruses possess an oncogene which is derived from cellular proto-oncogene
of host cell, known as viral oncogene. These viral oncogenes are integrated into
cells, while a particular virus is infecting cells, resulting into cancer
development (Bishop 1985).

18.7.1 Human DNA Tumor Viruses

18.7.1.1 Human Papillomavirus (HPVs)

The integration of viral genome into the genome of cancer cell represents the most
common characteristics of infection by these HPVs. Being a DNA virus, the genome
size is 8 kb and is known to cause tumors, both benign and malignant in animals
including humans. The two main viral oncoproteins of HPVs, E7 and E6, are
involved in cervical cancer induction, which destabilize p53 and pRb, the two
most important tumor suppressors of the cell. Sexual contact is the primary mode
of transmission of these viruses. In view of this, Gardasil, the first cancer vaccine,
was approved by the US Food and Drug Administration in 2006. Females in age
group 9–26 years use this vaccine for cervical cancer, genital warts, and precancer-
ous genital lesion prevention, which are caused by HPVs (HPV18, HPV16, HPV11,
and HPV6).
HPVs consist of high-risk (oncogenic) HPVs and low-risk (non-oncogenic)
HPVs. High-risk HPVs are considered to be oncogenic, which include the HPV16
and HPV18 and cause various cancers including cervical cancer. The HPV
oncoproteins, E6 and E7, from high-risk HPV have the ability to transform the
normal keratinocytes and epithelial cells into carcinogenic cells. Only the high-risk
E6 and E7 and not the low-risk E6 and E7 have the capacity to interact with the p53
and pRb, tumor suppressor proteins. In order to comprehend the molecular mecha-
nism behind E6 and E7 oncogene interaction with the tumor suppressor genes, first
E6 and E7 structure needs to be known (Fig. 18.9). The E6 oncoprotein contains
approximately 150 amino acids, having molecular weight of ~18 kDa, and is a
nuclear protein with basic nature. Because of high-risk HPV16, 16E6 oncoprotein
encoded by the HPV16 has been analyzed in detail. Structurally, 16E6 contains three
nuclear localization signals (NLS), a PDZ domain-binding motif at the C-terminal
along with four zinc-binding motifs (Cys-X-X-Cys), which results into formation of
two Cys/Cys fingers leading to direct zinc binding. The active form, E6AP
(E6-associated protein), interacts with the p53 and leads to the ubiquitination
mediated p53 proteasomal degradation. 16E6 has higher binding affinity for p53
and efficiently causes the degradation, compared to the 18E6 (encoded by the
HPV18), whereas 11E6 oncoprotein, encoded by the low-risk HPV11, has the
least binding affinity for p53 and weakly influences the p53 degradation. The
18 Molecular Genetics of Cancer 907

Fig. 18.9 Schematic representation of HPV16 oncoproteins E6 (a) and E7 (b)

amino acid present at 47 position (F47) in the E6 oncoprotein is essential for

interaction with p53. Any mutation in the E6 F47 amino acid leads to the abrupt
ubiquitination process and p53 degradation. The E6 from high-risk HPVs has shown
to inactivate the CYLD (cyclindromatosis tumor suppressor protein) tumor suppres-
sor, under hypoxia condition, by interacting with CYLD deubiquitinase and causing
unstoppable NF-κB activation. 16E6 is known to regulate the gene transcription by
interacting with various transcription factors and coactivators, such as p300/CBP
(EP300/cAMP response element-binding protein-binding protein), IRF-3 (interferon
regulatory factor 3), and c-Myc. 16E6 oncoprotein interacts with the c-Myc and
leads to immortal cells by inducing the transcription of h-TERT. With the help of
nuclear localization signal present near C-terminal, 16E6 is known to regulate the
E6E7 bicistronic RNAs splicing by interacting with the RNA. In addition to above
functions, 16E6 is reported to interact with the protein phosphatase H1, TNFR1
(tumor necrosis factor receptor 1), and PDZ proteins, thereby regulating the signal
transduction (Zheng 2010).
Similar to the E6, high-risk E7 has a higher affinity for interaction with the pRb
compared to the low-risk E7. E7 oncoprotein has approximately 100 amino acids
and is a nuclear protein like E6. The detailed analysis of 16E7 of HPV16 leads to the
findings that 16E7 contains nuclear localization signal and casein kinase II phos-
phorylation sites in the N-terminal domain and two zinc-binding motifs near the
C-terminal domain. The N-terminus of 16E7 has a sequence similarity with the
complete CR1 (conserved region 1) and CR2 (conserved region 2) of the adenovirus
E1A oncoprotein and also related sequences found in T antigen of SV40. In the CR2
region, LXCXE motif is present, which upon interaction with the LXCXE-binding
cleft of the p107, p130, and pRb results into the degradation of these proteins
908 B. Chuphal

through interaction with the ubiquitin ligase complex, cullin-2. E7 not only degrades
the pRb but is also essential for the viral life cycle and HPV-infected cells for various
cellular functions. E7 has been reported to upregulate the expression of p21 and p16
(cyclin-dependent kinase inhibitor), thereby resulting in dysregulation of cell cycle.
E7 oncoprotein interacts with the regulator of centromere, γ-tubulin, and inhibits its
recruitment at the centromere and thereby leads to chromosomal abnormalities,
resulting in mitotic defects and aneuploidy. In addition to above functions, E7 is
reported to interact with various other proteins also, such as steroid receptor
coactivator 1, p300, p600, and PCAF (P300/CBP-associated factor). These
interactions of E7 and E6 oncoprotein with the tumor suppressor proteins and cell
cycle regulator result in induction of the malignancies.

18.7.1.2 Epstein-Barr Virus (EBV)

The main target of this virus is B lymphocytes; the virus transforms B lymphocytes
into lymphoblasts, instead of replicating within them, resulting in immortality of
cells. The EBV contains oncogene, LMP1 (latent membrane protein-1 or BNLF1),
which is mainly expressed in lymphoma associated with the EBV and is crucial for
B-cell transformation as well as for signal transduction disruption inside a cell. EBV
is also involved in nasopharyngeal carcinoma and some types of Hodgkin disease. In
nasopharyngeal carcinoma (NPC), early gene BARF1 (BamHI-A reading frame-1) is
essential for carcinogenesis, which is reported to express as latent gene in some NPC
samples.
Depending on the latency forms, different expressions of EBNA (EBV nuclear
antigen) and LMP1 are observed in B cells. EBNA-2 and EBNA-5 are crucial for the
expression of LMP1 gene. Expression of only EBNA1 and LMPs is observed in
NPC, T and NK lymphoma, and Hodgkin lymphoma with type II latency. With type
I latency in Burkitt lymphoma, expression of only EBNA1 is observed. It has been
reported that cytokines (IL-10 and IL-21) are involved in the expression of
the LMPs.
The molecular weight of EBV integral membrane oncoprotein LMP1 is 62 kDa.
LMP1 contains 386 amino acid residues with 24 amino acids present at the cyto-
plasmic N-terminal, 186 amino acids are responsible for six transmembrane
domains, and 200 amino acids present at the cytoplasmic C-terminal. The full-
length LMP1 with 386 amino acids is required for B cell transformation into
cancerous cells. LMP1 interacts with the TRAF (TNFR-associated factor) and
JAK3 (Janus kinase 3) through the C-terminal and induces the signaling molecules
activation in B cells, resulting in the proliferation of B cells. The levels of LMP1
oncoprotein in the clonal population of B cell vary largely, and this leads to different
functions of oncoprotein in clonal population such as promotion of cell proliferation
by NF-κB in cells with intermediate expression of LMP1. However, with high
expression of LMP1, protein synthesis is inhibited by activation of PERK (protein
kinase R-like endoplasmic reticulum kinase) kinase which prevent phosphorylation
of eIF2α, leading to higher expression of activating transcription factor 4 (ATF4)
and, thereby, transactivation of its own promoter. In addition, LMP1 leads to the
upregulation of anti-apoptotic genes, prevention of p53-mediated apoptosis through
the inhibition of pro-apoptotic proteins, and activation of A20 gene.
18 Molecular Genetics of Cancer 909

The BARF1 gene which is involved in NPC is located at the BamHI-A region and
encodes a 31 kDa protein. The 54 amino acid regions at the N-terminus of BARF1
protein are responsible for the expression of anti-apoptotic proteins, resulting in the
transformation of rodent’s fibroblasts and B cells. It has been reported that, in B cells
of Burkitt lymphoma with EBV-negative, BARF1 has potential to induce tumor
growth in these cells. BARF1 expressing B cells reported to have higher expression
of c-myc, CD23, and CD2. The secreted form of BARF1 acts as receptor for the CSF
(colony-stimulating factor) and is also involved in the inhibition of IF-α (interferon-
α) from the mononuclear cells. The secreted form of both BARF1 and LMP1 is
detected in NPC patient’s serum and acts as mitogenic factor.

18.7.1.3 Kaposi Sarcoma-Associated Herpesvirus (KSHV)

KSHV contains 165–170 kb genome size with double-stranded DNA. Several viral
oncoproteins encoded by the viral oncogenes are involved in the tumorigenesis
through the inhibition of apoptosis, immune modification, cell cycle regulation,
and regulation of signal transduction pathway. KSHV is responsible for KS (Kaposi
sarcoma), PEL (primary effusion lymphoma), and MCD (multicentric Castleman
disease). Virus encodes for various oncoproteins including latency-associated
nuclear antigen (LANA); v-FLIP (viral-FLIP) also known as K13; Kaposin C, B,
and A; K1 also as named VIP (variable ITAM-containing protein); viral interleukin-
6 (vIL6); viral interferon regulatory factor-1 (vIRF-1); and viral G protein-coupled
receptor (vGPCR). The LANA is responsible for the inhibition of p53 and pRb
proteins and interaction with the bromodomain of RING3/Brd2 protein complex,
thereby progresses the cell from G1 to S transition. LANA protein is essential for the
upregulation of hTERT, cooperates with H-ras oncogene, and leads to the expression
of Myc gene through β-catenin by preventing its degradation from the glycogen
synthase kinase-3β (GSK-3β), resulting in cancer development. The K13
oncoprotein interacts with the FADD and caspase-8, thereby inhibiting the death
receptor-mediated apoptosis. The K13 also activates the NF-κB, responsible for the
viral latency and Kaposi sarcoma. Through maintaining the levels of IL-6 and
GM-CSF, Kaposin protein is reported to be involved in PEL, and high levels of
Kaposin protein are found in PEL cancer cell lines. K1 is a 46 kDa glycoprotein
which contains an immunoreceptor tyrosine-based activation motif (ITAM). K1
leads to the constitutive expression of NF-κB and PI-3 K/Akt pathway, thereafter
resulting in immortalization of the cells. Abundant levels of K1 have been reported
in the KS, PEL, and MCD. The vGPCR encoded by KSHV is involved in human
endothelial cell immortalization and angio-proliferative tumors. Through
JAK/STAT activation and MAPK pathway, vIL-6 is involved in the pathogenesis
of the KS and MCD. The vIRF-1 inhibits the host system antiviral response by
blocking the IRF3/CBP/p300 complex formation and suppressing the interferon
response. vIRF-1 promotes cancer development through inhibiting the TNF-α- and
p53-mediated apoptosis.
910 B. Chuphal

18.7.1.4 Human Polyomaviruses

Simian virus 40 (SV40) from the rhesus monkeys is the most important member of
this family. Merkel cell polyomavirus (MCV) is a newly discovered human poly-
omavirus which causes approximately 80% of Merkel cell carcinoma (MCC). The
other polyomaviruses, Polyomavirus-6 (HPyV6) and HPyV7, along with the MCV
inhabit the human skin.
During the early viral infection, the mRNA of human polyomavirus undergoes
alternative splicing mechanism, resulting in the translation of two viral nonstructural
oncoproteins: large T (LT) antigen and small t (st) antigen. LT antigen interacts with
p53 and pRb, tumor suppressor proteins. In addition to the role of st antigen in
interaction with the protein phosphatase 2 (PP2A), it also promotes the transforming
capacity of LT. To understand the molecular mechanism behind the interactions of
LT antigen with the p53 and pRb, LT antigen structure has been analyzed in detail.
LT antigen protein contains two conserved regions (CR1 and CR2) in the J domain
of the N-terminal, DNA origin-binding domain (OBD) at the middle, and ATPase/
DNA helicase region in the C-terminal domain (Fig. 18.10). The J domain of LT
N-terminal contains Hsc70 chaperone binding sites and is essential for the proper
protein folding. The LXCXE motif is also present near the N-terminal of LT antigen,
which interacts with the LXCXE-binding motif of the pRb protein, thereby
regulating the pRb activity. The ATPase/DNA helicase region at C-terminus of LT
antigen is crucial for its interaction with the p53 binding region and regulating the
p53 activity. Further, malignant cell growth requires activation of insulin-like
growth factor I (IGF-I), which is done by the LT-p53 complex. For viral DNA
replication, DNA origin-binding domain of LT antigen is required. Similar to LT
antigen, the J domain is also present in the st antigen because of common derivation
from the exon 1 region of mRNA, while other parts are different from st antigen.
With the presence of a unique PP2A-binding site, st antigen interacts with the PP2A,
which is required for various signaling pathways and leads to inhibition of its
phosphatase activity.

Fig. 18.10 Schematic representation of the SV40 large T antigen

18 Molecular Genetics of Cancer 911

18.7.1.5 Human Adenoviruses

The human adenoviruses are the DNA viruses mainly responsible for the infections
of the respiratory system. The two main oncogenes of the adenoviruses are E1A and
E1B, which are responsible for tumor development and help to understand the
cellular processes of the tumor developing cells.
Till now, no reports are present in which adenovirus caused the tumor in humans,
but many adenovirus serotypes (adenovirus types 2, 5, 12, 18, and 31) are known to
cause tumors in rodents and hamsters. The main oncoproteins encoded by the
adenovirus include E1A and E1B. The E1A protein (289R) is a 289 amino acid
residue nuclear protein, which comprises of four conserved regions: CR1 located in
the N-terminal domain, CR2 and CR3 located on the middle, and CR4 present at the
C-terminal domain (Fig. 18.11). The E1A protein is responsible for the
transactivation of various early genes including E1B genes. Because of the alterna-
tive splicing in the E1A mRNA, 243 amino acid residue protein (243R) has been
encoded, which lacks the CR3 region. During the early viral infection, levels of both
289R and 243R E1A proteins are high, which are involved in the stimulation of
DNA synthesis in cells and transformation, inactivation of the pRb, and induction of
apoptosis through the interaction with various proteins. The CR2 region of E1A is
known to interact with higher affinity to the pRb, p107, and p130 proteins, and along
with CR1 region (which has low binding affinity for pRb and its related proteins)
E1A inhibits the binding of pRb with the E2F target genes, thereby continuous
expression of E2F transcriptional factors, which is responsible for expression of
various cell cycle genes. The residues of the CR1 and N-terminus domain of E1A are
required for transformation and interact with p300/CBP protein and lead to reduction
in the acetylation of the H3 histone protein at lysine 18, thereby regulating the
chromatin structure. For the acetylation of H3K18, the CBP and p300 are required.
By interacting with the promoter of various genes temporarily and in time-dependent
manner, E1A regulates the genes essential for cell cycle and growth, development,
differentiation, and antiviral activities.

Fig. 18.11 Schematic representation of the adenovirus E1A-289R protein

912 B. Chuphal

Fig. 18.12 Schematic representation of the adenovirus E1B-496R protein

The pre-mRNA of E1B under alternative splicing and encodes two main proteins
of 176 amino acid residues (176R/19 K) and 496 amino acid residues (496R/55 K).
There is no sequence homology between the 176R and 496R, but both have the
capacity to upregulate the cell growth and transformation. The E1B-496R protein
contains nuclear export signal (NES) near the N-terminal domain, ribonucleoprotein
motif (RNP) in the middle and casein kinase I/II phosphorylating sites, nuclear
localization signal (NLS), and zinc-binding motif near the C-terminal domain
(Fig. 18.12). During viral infection, prevention of Bcl-2 family proteins oligomeri-
zation results in inhibition of caspase-mediated apoptosis of host cells, and transfor-
mation of rodent cells is promoted by the E1B-19 K and E1B-55 K. Both E1B-19 K
and E1B-55 K along with the E1A protein is required for cancer induction in the
lungs of transgenic mice, which is expressing both E1A and E1B transgenes. With
the presence of nuclear export signal, transport of viral late mRNA is carried out by
the E1B-55 K protein. E1B-55 K protein is also essential for the interaction of p53
and causing its degradation, thereby hampering p53 functions.

18.7.1.6 Hepatitis B Virus (HBV)

HBV contains DNA genome and is of only 3 kb size. This virus can cause cell
transformation by the presence of a gene called gene X, which codes for transcrip-
tional regulator HBx protein, through which it can cause hepatocellular carcinoma.
However, HBx itself cannot cause tumors as evident from its inability to induce
cancer in X gene transgenic mouse strains. HBV is mainly found in Southeast Asia
and sub-Saharan Africa. HBx gene has the ability to disrupt the signal transduction,
thereby affecting the cell growth; thus it can be a viral oncogene. The first human
cancer vaccine to be developed, with high immunogenic effect and lasting protective
efficacy, is HBV vaccine. Currently, more than 170 countries include the HBV
vaccine under their vaccination program, which is cheap and safe to use.
18 Molecular Genetics of Cancer 913

18.7.2 Human RNA Tumor Viruses

18.7.2.1 HTLV-1
HTLV-1 causes T-cell lymphoma, mainly affecting the CD4+ lymphocytes, and this
can be transmitted through blood transfusion, breastfeeding, or sexual contact. Only
1% of people carrying this virus develop leukemia after decades. This virus is
endemic in the Caribbean Basin, South Africa, and Japan. This retrovirus does not
integrate into host genome and does not contain the viral homologue of the cellular
proto-oncogenes. Tax oncoprotein of this virus is involved in the progression of cell
cycle in the T cells and also sets up the continuous proliferating system.
Tax is also known as p40tax/Tax1 nuclear phosphoprotein, which comprises
353 amino acids with 40 kDa molecular weight. Tax1 acts as transcriptional activa-
tor of the virus promoter region and transformation of the T-cells, but not involved in
maintaining T-cell transformation. Transgenic mice with Tax1 expression are prone
to develop various type of tumors, making the concept of Tax1 as oncogenic clear.
Recent reports indicate that Tax1 is essential for the transformation, but for
maintaining the T-cell leukemia, another viral protein HBZ (HTLV1 basic leucine
zipper factor) or expression of microRNA is required. In T-cell leukemia samples,
detectable amounts of Tax1 are not observed.
The structure of tax1 protein consists of zinc finger motif and nuclear localization
signal in the N-terminal, leucine zipper-like motif and nuclear export signal at the
middle, and PDZ binding domain, activation domain, Golgi localization motif, and
secretion motif at the C-terminal domain. The N-terminal domain of Tax1 acts as
transcriptional factor for various cellular genes through interaction with the CREB
and is also crucial for the transport of various RNAs and proteins within the cell. The
leucine zipper motif is essential for the interaction with PP2A and NF-κB, thereby
regulating the expression of various genes. In addition, Tax1 is essential for the
regulation of various cell cycle genes, chromatin remodeling proteins, as well as
activation of viral and cellular transcription. Another protein, Tax2/p37tax, encoded
by the HPLV-2, shows similar properties to the Tax1 such as lymphocyte
transformation.

18.7.2.2 Xenotropic Murine Leukemia Virus-Related Virus (XMRV)

XMRV is first identified from the patients with prostate cancer. This virus is also
involved with chronic fatigue syndrome. The receptor, XPR1 (xenotropic and
polytropic retrovirus receptor 1), is essential for the infection of XMRV. However,
recent studies are controversial related to the XMRV roles in chronic fatigue
syndrome and in prostate cancer.

18.7.2.3 Hepatitis C Virus (HCV)

The Flavivirus family contains HCV, having RNA genome which carries a long
open reading frame involved in encoding a 3010 amino acid precursor polypeptide,
which results in ten different proteins, seven nonstructural proteins, and three
structural proteins. However, HCV’s role in oncogenesis is not properly understood;
liver cancer associated with HCV requires persistent infection. No oncogenes are
914 B. Chuphal

discovered in the viral genome, but for the HCV replication, liver-specific miR-122
is involved. Till now, there is no vaccine available for the HCV, but various
researches are ongoing to develop a safe vaccine with long-lasting effects against
HCV infection.

18.7.2.4 Rous Sarcoma Virus (RSV)

Rous sarcoma virus is an avian sarcoma virus, involved in the transformation of
fibroblasts. Using the RNA fingerprinting technique of RSV genome, identification
of gag, pol, env (genes required for the virus reproduction), and src gene (required
for the oncogenic activity of virus) can be carried out. The src gene comprises 20%
of the virus genome and is present in the C-terminal of the virus RNA. The first
oncogene to be discovered is src oncogene and encodes v-src protein with 60 kDa
molecular weight which belongs to the tyrosine kinase protein family. The src gene
is essential for the viral oncogenic activity but not responsible for the virus replica-
tion and reproduction. The sequencing of src gene leads to the finding of its source of
origin, as it is a cellular gene, not a viral gene. The src DNA sequence of the host is
incorporated into the virus DNA during viral infection, since loss-of-function or
gain-of-function mutation in certain genes converts them to oncogenes. The viral src
gene has deletion at carboxy-terminal, which includes essential phosphorylation
sites and also several point mutations in comparison to the cellular SRC gene. The
SRC protein kinase activity is under the regulation of a polyproline-binding SH3
domain and a phosphotyrosine-binding SH2 domain, two modular domains of SRC
protein, and disruption in the intramolecular interactions in these SH2 and SH3
domains is essential for the activation of catalytic domain of SRC protein. Because
of these regulations, SRC protein has lower kinase activity and no oncogenic activity
in comparison to the viral src protein, since, because of point mutations in the SH3
domain and deletion in the C-terminal, these regulations are absent in viral src
protein, resulting in the constitutive expression of viral src protein with higher kinase
activity and oncogenic activity.

18.7.2.5 Avian Myelocytomatosis Virus MC29

Avian myelocytomatosis virus MC29 is the cause for the development of Burkitt
lymphoma, B-cell lymphoma, neuroblastoma, and medulloblastoma because of the
presence of myc gene in its genome. Basically, myc gene is responsible for several
human cancers. With the development of RNA fingerprinting technique and DNA
sequencing techniques, it has been reported that myc is not a viral gene but
incorporated into viral RNA as a contiguous stretch. The myc is the second oncogene
to be discovered. The avian retroviruses CMII, OK10, and MH2 also contain myc
gene. MYC gene is also reported in the human genome. Presence of three MYC genes
in the human genome has been discovered: c-MYC (or MYC), MYCN, and MYCL1.
The homologous gene of viral myc gene is cellular MYC gene. With the discovery of
MYC protein location in the cell nucleus, it is represented as a transcription regula-
tor. But MYC protein alone has lower gene-binding affinity and is not able to
regulate the gene expression, but in association with the MAX protein,
MYC-MAX heterodimer has higher gene-binding affinity and has potential to
18 Molecular Genetics of Cancer 915

regulate gene expression. With regard to c-MYC gene chromosomal translocation at

the immunoglobulin gene region and under the immunoglobulin enhancer,
overexpression of c-MYC protein was observed in Burkitt lymphoma (Boxer &
Dang 2001). The MYCL1 and MYCN genes are responsible for several human
cancers. The amplification and expression of MYCN gene was observed in the
neuroblastoma. The c-MYC gene is not only essential for the cancer induction but
also involved in providing resistance against inhibitors of PI3K and inhibition of
RAS-induced tumors in mice.

18.7.2.6 Harvey Sarcoma Virus and Kirsten Sarcoma Virus

During the murine leukemia virus infection in rats, recombination between the host
genome and viral genome leads to the origin of two new retroviruses: Harvey
sarcoma virus and Kirsten sarcoma virus. Both the retroviruses contain ras gene in
their genome and encode 21 kDa protein, Ras. The Kristen-ras (Ki-ras) from the
Kristen sarcoma virus and Harvey-ras (Ha-ras) from the Harvey sarcoma virus have
similar properties. These oncogenes are responsible for the transformation of the
fibroblasts, hematopoietic cells, and epithelial cells into cancerous cells. The ras
gene is also involved in pancreatic cancer and its homologue is also found in the
human genome. The Ras protein is a GTPase, bound to the GTP in its active form,
and located at the cytoplasmic side of plasma membrane and interacts with lipid
membrane through isoprenylation post-translational modification of the protein. The
GTPase-activating protein (GAP) stimulates the GTPase activity of Ras. Mutations
in the 12 and 61 amino acid residues of RAS protein affect its binding with the GAP
protein, and GTP-bound Ras protein level increases with the decrease in GTP
hydrolysis rate, thereby stimulating the oncogenic activity of Ras. The Ras protein
interacts with various signaling components such as with the PI3K catalytic subunit
and affects the MAPK pathway through binding with the RAF protein.

18.7.2.7 Abelson Murine Leukemia Virus

Abelson murine leukemia virus contains an oncogene, v-abl, which is responsible
for the pre-B-cell transformation into the cancerous cells by the two-step method.
Initially, during viral infection in the bone marrow, progressive proliferation of the
pre-B cells is observed which marked the first step. The second step includes a
period of apoptosis and abrupt growth marked as crisis phase. Some pre-B cells
escape this crisis period and give rise to fully malignant cells. The v-abl oncoprotein
negatively regulates the p53 tumor suppressor protein and p19Arf protein expres-
sion. The p19Arf protein is essential for the activation of p53 through interaction
with the Mdm2 protein. The v-abl oncoprotein interacts with the c-myc promoter
region, resulting in cellular Myc protein expression and is also involved in the
cellular Ras expression, which are essential for virus stimulated transformation
and growth. The v-abl activates the PI-3 K and JAK pathway which promote the
growth and survival of cancerous cells. The Akt pathway is also under the regulation
of v-abl oncoprotein, which interacts with the Mdm2 protein, thereby suppressing
the p53 activity. Through regulating various signaling proteins and tumor suppressor
protein p53, v-abl leads to oncogenesis of pre-B cells.
916 B. Chuphal

18.7.2.8 Avian Erythroblastosis Virus

The avian erythroblastosis virus causes leukemia of erythrocytes, known as erythro-
blastosis because of the presence of oncogene erbB. In the virus-infected cells,
presence of 74 kDa ErbB protein has been detected which is encoded from the
fusion of cellular ErbB mRNA and viral erbB mRNA. The ErbB viral protein is
phosphorylated and glycosylated and belongs to the tyrosine kinase family which
functions as integral plasma membrane protein. The sequence analysis shows the
close similarity of ErbB protein with the cellular EGFR. The viral ErbB protein has
large deletion in the extracellular domain compared to EGFR, modification at
N-terminal domain, and mutation at the C-terminal that comprises the kinase
domain. Because of the mutation at the kinase domain of ErbB protein, it works in
ligand-independent manner with constitutive expression. These mutations lead to the
disruption in the signaling component of the host system and result in oncogenesis.
Another viral oncogene from the avian erythroblastosis virus has been sequenced,
named erbA (Table 18.10), and encodes an ErbA protein, which belongs to the
hormone receptor family and shows close similarity with the cellular THRA.

18.7.2.9 Mode of Action of Viral Oncogenes

Presently there are only three strategies known through which viral oncogene
products might act.

1. Phosphorylation: The primary activity occurs on the plasma membrane surface.

The transforming protein which acts as potential substrate strikes the surface,
ultimately spanning the whole plasma membrane or residing at the inner surface.
2. DNA synthesis: Cancer cells exhibit unrestrained cell division by involving some
transforming proteins eliciting the property to initiate DNA synthesis.
3. Transcription regulation: Many transforming proteins may interact with
enhancers or promoters to influence transcription of cellular genes.

18.8 Proto-oncogenes

The normal cellular oncogenes (c-onc) are the host genes, whose product form
important constituents of various signaling pathways which control the proliferation,
division, and growth of cells. Viral oncogenes are homologues of these cellular
oncogenes, such as c-src which is the cellular homologue of v-src. Proto-oncogenes
are the representatives of normal genes of cells, which show similarity with the
nucleotide or protein sequences that are tumorigenic or have the potential of
transforming. A plethora of circumstantial evidence now points to the fact that
alteration in either copy number, expression, or structure of one of these genes is
responsible for the several malignancies of humans. The proteins encoded by these
genes include transcription factors, signal transducers, growth factors, and growth
factor receptors. Epigenetic and genetic changes are responsible for the conversion
of these proto-oncogenes to oncogenes. Scientists have isolated many c-onc from
different organisms including humans, by probing them using v-onc. The
18 Molecular Genetics of Cancer 917

Table 18.10 List of retroviral oncogenes (Vogt 2012)

Host
S. no. General class Oncogene Virus species Predicted function
1. Non-receptor abl Abelson murine Mouse Tyrosine-specific
protein fes leukemia Cat protein kinase
tyrosine fps ST feline sarcoma Chicken Tyrosine-specific
kinase src Fujinami sarcoma Chicken protein kinase
fgr Rous sarcoma Cat Tyrosine-specific
ros Gardner-Rasheed Chicken protein kinase
yes feline sarcoma Chicken Tyrosine-specific
URII avian sarcoma protein kinase
Y73 sarcoma Tyrosine-specific
protein kinase
Tyrosine-specific
protein kinase
Tyrosine-specific
protein kinase
2. Receptor erbB Avian Chicken Truncated version
protein fms erythroblastosis Cat of epidermal
tyrosine McDonough feline growth factor
kinase sarcoma receptor
Analog of colony-
stimulating
growth factor
receptor
3. Serine/ mil (mht) MH2 Chicken Serine/threonine
threonine mos Moloney sarcoma Mouse protein kinase
protein kinase raf 3611 Murine Mouse Transcription
sarcoma factor
Serine/threonine
protein kinase
4. Growth factor H-ras Harvey murine Rat GTP-binding
G protein K-ras sarcoma Rat protein
sis Kirsten murine Monkey GTP-binding
sarcoma protein
Simian sarcoma Analog of platelet-
derived growth
factor
5. Transcription erbA Avian Chicken Analog of thyroid
factor fos erythroblastosis Mouse hormone receptor
jun FJB osteosarcoma Chicken Transcriptional
myb Avian sarcoma Chicken activator protein
myc Avian myeloblastosis Chicken Transcriptional
rel MC29 Turkey activator protein
myelocytomatosis Transcription
Reticuloendotheliosis factor
Transcription
factor
Transcription
factor
918 B. Chuphal

conservation of these genes across different species suggests that the proteins
encoded by them are involved in vital cellular events. HMGA1 proto-oncogene
reactivation has been reported in several cancers. The protein encoded by these
genes is responsible for carcinogenesis under both in vitro and in vivo
conditions (Choudhuri & Chanderbhan 2007). After their expression was discov-
ered, Rat1a fibroblasts and lymphoid cells were proved to be transformed by their
overexpression. Under in vitro transgenic animal models, HMGA1 overexpression
results in pituitary tumor, benign mesenchymal tumors, and lymphomas. Impor-
tantly, these proteins may also disrupt the tumor suppressive pathways such as pRb
and p53 pathway.

18.9 Mutant-oncogenes

The proteins encoded by proto-oncogenes regulate key cellular activities, and,

therefore, a single mutation in any of these genes can disturb the normal functioning
inside a cell pushing it onto the highway of becoming cancerous. The first ever proof
linking mutant c-onc to cancer was given in case of human bladder cancer by Robert
Weinberg and colleagues using transfection test (Fig. 18.13).
The team identified a DNA fragment that carried an allele of c-H-ras oncogene
from the bladder cancer which had the reproducible capacity for transformation of
cells into the cancer cells. Mutant c-ras oncogene involvement has been reported in a
variety of tumors in humans (Fig. 18.14) (Bos 1989).

Fig. 18.13 Flowchart showing steps involved in transfection test for identifying nucleotide
sequences capable of making cells cancerous. DNA from tumor cell is transferred into normal
cells leading to its integration. The DNA is then isolated based on its marker
18 Molecular Genetics of Cancer 919

Fig. 18.14 Ras protein signaling and cancer. Normal Ras protein is regulated in presence of
extracellular signal and the mutated Ras protein leads to uncontrolled cell division

The amino acid change in one of the three positions, 12, 59, and 61, impairs the
mutant Ras protein to come out of its activated state, thereby stimulating cells to
divide continuously.

18.9.1 Phosphatidylinositol 3-Kinase (PI3K)

The lipid kinase PI3K constitute a heterodimer of adaptor/regulatory (p85) and

catalytic subunit (p110), resulting from the transcription from different genes and
undergoing alternative splicing during the translation. The interactive binding of
growth factors with the receptor tyrosine kinase is responsible for PI3K activation,
subsequently production of phosphatidylinositol 3,4,5 trisphosphate (PIP3) and
diacylglycerol (DAG) from phosphatidylinositol 4,5 bisphosphate (PIP2), leading
to the Akt pathway activation, thereby regulating the cell survival, cellular growth
and motility, apoptosis, and cell adhesion and transformation. With the discovery of
avian hemangiosarcoma virus (belongs to avian retrovirus family), ASV16, the
kinase activity and oncogenic activity of the PI3K have been reported. Many people
920 B. Chuphal

were found to have mutations in the gene encoding the p110α catalytic subunit of
PI3K (Karakas et al. 2006). The gene encoding the PIK3CA (PI3K catalytic subunit)
is located on chromosome 3 (3q26.3) with 34 kb gene size and consists of 20 exons,
which translate into 124 kDa protein having 1068 amino acid residues. The B-cell
defects, colorectal cancer, liver necrosis, and embryonic lethality are associated with
the mouse model with knockout of both PI3K subunits. The PTEN (phosphatase and
tensin homologue deleted on chromosome ten) acts as PI3K negative regulator, by
dephosphorylating the PIP3 and thus disrupting the PI3K signaling.

18.9.2 Protein Kinase B (PKB)

The PKB is also known as the Akt serine/threonine kinase and hyperactivated in
several human cancers. The Akt hyperactivation is responsible for the increased cell
growth, proliferation and energy metabolism, and development of resistance against
apoptosis. Activated PI3K generates PIP3, to which Akt PH domain binds, thereby
stimulating the translocation of Akt to the plasma membrane. Akt phosphorylation is
mediated by the PI3K-dependent kinase-1 (PDK1) at threonine (Thr 308) and by
PDK2 at the serine amino acid residue (Ser 473), which are essential for Akt full
activation. There is still a contradiction between FOXO and mTOR, as which
downstream effector molecule of Akt is responsible for the cancer development.
Akt inhibits the stimulation of the forkhead family of transcription factors (FOXO),
which is responsible for cell proliferation inhibition, whereas activation of mTOR
stimulates cell proliferation (Hay 2005). The TSC2/TSC1 (tuberous sclerosis com-
plex 2/tuberous sclerosis complex 1) acts as a negative regulator of Akt by inhibiting
the mTOR activity. The development of benign tumor has been reported with the
germline mutation in the TSC2 and TSC1 encoding genes. Several Akt-mTOR target
molecules have been under development which can be used in cancer therapy.

18.9.2.1 B-RAF
The oncogene B-RAF encodes a serine/threonine protein kinase B-RAF. Three
paralogs of RAF serine/threonine protein kinase are C-RAF, B-RAF, and A-RAF.
Binding of cytokines, hormones, and growth factors to the RAS, a membrane-bound
G protein, leads to activation of RAF kinase, subsequently MEK (mitogen-activated
protein kinase) activation, which activate ERK (extracellular signal-regulated pro-
tein kinase), thereby regulating the apoptosis, cell proliferation and differentiation
via cytoskeleton rearrangement, metabolism regulation and gene expression. RAF
protein structure constitutes the N-terminal domain with two conserved regions,
CR1 and CR2, and C-terminal containing the third conserved region, CR3 and
kinase domain. It has been reported that 7% of cancers contain B-RAF mutation.
The incidence of mutation in the B-RAF as shown in Fig. 18.15 is highest among the
malignant melanoma (27–70%), serous ovarian cancer (~30%), papillary thyroid
cancer (36–53%), and colorectal cancer (5–22%), while a variety of cancers has low
frequency of B-RAF mutation. The most frequent mutation found in B-RAF gene is
the transversion of thymidine to adenosine at 1796 position of nucleotide, which
18 Molecular Genetics of Cancer 921

Fig. 18.15 Schematic representation of B-RAF oncogene mutations associated with the cancer

Fig. 18.16 Diagram showing RET gene with codons identified in MEN 2 families

leads to conversion of valine at 599 amino acid position to the glutamate in the
B-RAF protein. This mutation, V599EB-RAF, is linked to 90% mutations of mela-
noma and thyroid cancer, while in non-small cell lung cancer, the rate of mutation is
very low. However, several other sites of mutation are also reported with potential to
cause cancer (Garnett & Marais 2004).

18.9.2.2 RET
The RET oncogene encodes a RET protein (Fig. 18.16), which is a membrane-bound
tyrosine kinase having intracellular tyrosine kinase, transmembrane, and
922 B. Chuphal

extracellular domain. On binding with the artemin, neurturin, and GDNF (glial cell
line-derived neurotrophic factor), RET kinase is activated and has been implicated in
multiple endocrine neoplasia type 2 (MEN 2) and papillary thyroid carcinoma. Upon
interaction with various activating genes (such as ELKS, H4, HTIF, ELE1), consti-
tutive expression of RET protein kinase is reported in papillary thyroid carcinoma,
whereas mutation in the RET proto-oncogene (Jhiang 2000) has been observed in the
multiple endocrine neoplasia type 2 (MEN 2). MEN 2 has been classified in three
subtypes depending on the organ affected: FMTC, MEN 2B, and MEN 2A
(Fig. 18.16). The parathyroid hyperplasia (in MEN 2A), medullary thyroid carci-
noma (in FMTC, MEN 2B, and MEN 2A), and pheochromocytoma (in MEN 2B,
MEN 2A) are characteristics of the inherited MEN 2 cancer syndrome. Mutation in
the RET gene region, which encodes for the tyrosine kinase domain and cysteine-
rich domain of RET protein are reported, thereby responsible for the constitutive
activity of tyrosine kinase and results in transformation of the cell.

18.10 Tumor Suppressor Gene

Tumor suppressor genes are responsible for regulating cell growth, proliferation, and
differentiation. The gain-of-function mutation results in proto-oncogene conversion
into the oncogene, whereas loss-of-function mutation in tumor suppressor genes
leads to various cancer developments. The tumor suppressor gene includes p53, Rb,
PTEN, BRAC1, BRAC2, APC, NF1, p27 (Kip1), and p16 (Ink4). Mutation in these
genes leads to cancer development. Loss of p16 gene is involved in prostate cancer.
The gene silencing of p27 and p16 gene through methylation results in carcinogene-
sis. The detailed analysis of few tumor suppressor genes is discussed below.

18.10.1 PTEN

PTEN (phosphatase and tensin homologue deleted on chromosome 10) or MMAC-1

gene is located on chromosome 10 (10q23), which encodes a PTEN protein with
403 amino acid residues and responsible for the downregulation of PI-3 K-Akt
pathway (Fig. 18.17) (Carnero et al. 2008). Under the mitogenic factor influence,
Ras protein kinase is activated, resulting in PI-3 K activation. The PI-3 K is
responsible for the conversion of PIP2 into PIP3, thereafter activating the Akt
signaling, resulting in cell growth and proliferation. PTEN, a tumor suppressor
gene, regulates this pathway, through dephosphorylation of PIP3 and inhibiting
the activation of the Akt signaling, thus preventing the excessive proliferation of
cells. Inactivation of PTEN either through mutation or homozygous deletion at both
the alleles results in loss of PTEN protein function (Wang et al. 1998). The familial
neoplastic syndrome, Cowden disease, is associated with the germline mutation in
the PTEN tumor suppressor gene. The large deletion at 10q region of PTEN gene is
observed in a variety of cancers such as endometrium melanoma and glioblastoma,
breast cancer, prostate cancer, renal cancer, and NSCLC (Chen et al. 2018; Leslie &
Downes 2004).
18 Molecular Genetics of Cancer 923

Fig. 18.17 Schematic representation of the interaction of PTEN and PI3K-Akt pathway

18.10.2 NF1

NF1 (neurofibromatosis type 1) gene is located on the chromosome number

17 (17q11.2) and encodes a tumor suppressor protein, Neurofibromin (Cichowski
& Jacks 2001). Loss of both alleles of NF1 gene results in familial tumor syndrome;
Neurofibromatosis type 1 is well known as von Recklinghausen, because of its
discovery by Friedrich von Recklinghausen in 1882, with a frequency of 1 in 3500
individuals. Defects in the NF1 gene are associated with tumors in the nervous
system, glioblastoma, and myeloid leukemia. In addition to cancer syndrome,
neurofibromatosis type 1 disease is associated with abnormal pigmentation, bone
deformations, and mental retardation. Neurofibromatosis acts as a GTPase-
activating protein and results in inhibition of Ras signaling activation.
Overexpression of the Ras pathway is observed in neurofibromatosis type 1.

18.10.3 BRCA1 and BRCA2

BRCA1 (breast cancer 1) gene is located on the chromosome number 17 (17q21.13)

and encodes a 1863 amino acid residue long protein. The structure analysis of
BRCA1 protein revealed the presence of the RING domain at the N-terminus and
BRCT domain on the C-terminus. The interaction of RING domain with the BARD1
(BRCA1-associated RING domain) and also by binding to the Rad51 mediate the
DNA repair through homologous recombination. The interaction of p53 protein with
the BRCT domain of BRCA1 and BRCA1 regulates the p53 transcription. Mutation
in the N-terminal and C-terminal of BRCA1 protein leads to cancer of the breast,
ovary, prostate, and colon. With the increase in age, chances of breast cancer through
924 B. Chuphal

mutation in the BRCA1 gene increase. BRCA1 also regulates various cell cycle
proteins (Baer & Ludwig 2002).
The BRCA2 gene is located on the chromosome number 13 (13q12-q13) and
encodes BRCA2 protein. Despite having the structural dissimilarity from the BRCA1
gene, BRCA2 gene share may feature with the BRCA1. By binding to the Rad51,
BRCA2 helps in DNA repair. Mutation in the BRCA2 gene leads to prostate cancer,
gastric cancer, and melanoma.

18.10.3.1 APC
APC (adenomatous polyposis coli) gene is located on chromosome number 5 and
encodes a 312 kDa protein consisting of multiple domains (Fig. 18.18), which serves
as a binding site for various proteins, including β-catenin, CtBP (C-terminal binding
protein), Asefs, IQGAP, axin, microtubule, and EB1, resulting in regulation of
spindle formation, chromosome segregation, cell adhesion and migration, and cyto-
skeleton organization. Germline mutation in APC gene causes familial adenomatous
polyposis (FAP), which is characterized by the presence of various polyps in the
intestine. Mutation in APC gene leads to colon cancer and lung cancer. Two APC
genes are present in humans: APC and APC2, which encodes for APC (containing
2843 amino acid) and APC2 (containing 2303 amino acid). The APC protein
consists of oligomerization domain, an armadillo domain, mutational cluster region
with 15–20 amino acid residue repeats essential for β-catenin binding and SAMP
repeats involved in axin binding, and CtBP binding region with basic region, EB1
binding region, and DLG binding domain. As regards binding of various proteins to
the APC protein, functions of APC are mediated. APC plays the suppressive effects

Fig. 18.18 Schematic representation of APC protein and its functions

18 Molecular Genetics of Cancer 925

on the Wnt signaling, which is responsible for the cell proliferation and differentia-
tion (Aoki & Taketo 2007; Senda et al. 2005).

18.11 Two-Hit Hypothesis

Analysis of cancer following a dominant pattern of inheritance helped researchers to

identify many tumor suppressor genes. Like all genes, mutations occur in tumor
suppressor genes, although almost all loss-of-function mutations occurring are rather
recessive in nature. Cancer is mostly bound to happen if only a second mutation
knocks out the normal functioning when it hits the somatic cells. Hence, cancer
development requires two loss-of-function mutations, “two hits,” one for each of the
two copies of the tumor suppressor gene (Fig. 18.19) (Chial 2008). In 1971, Alfred
Knudson explained this idea for the occurrence of retinoblastoma.

18.12 RB1 Tumor Suppressor Gene

The two-hit hypothesis was idealized way before the human genome project was
completed. Among the firstly isolated tumor suppressor genes was the RB1 gene
(Dyson 2016) responsible for causing retinoblastoma in 1985 by a pair of scientists,
Raymond White and Webster Cavenne. They showed that chromosome 13 was
missing huge segments in retinoblastoma cells and eventually the gene was isolated.

Fig. 18.19 Knudson’s two-hit hypothesis in case of retinoblastoma. Inherited and sporadic
retinoblastoma are the two types of conditions which lead to eye tumor formation
926 B. Chuphal

The gene causing retinoblastoma is located on chromosome number 13 (13q14.2).

Over the decades, scientists trying to characterize and identify the function by
deleting the Rb1 gene using mouse models have shown that RB1 can block cell
division and also regulate cell death. It has the ability to interact with various
proteins for carrying out many other functions. This gene encodes a protein product
pRb, having 948 amino acids, belonging to the RB gene family along with the
retinoblastoma-like 2 gene (RBL2) and retinoblastoma-like 1 gene (RBL1). RBL1
and RBL2 are present on chromosome 20 (20q11.2) and 16 (16q12.2) encoding
p107 and p130, respectively, which are involved in slowing the various neoplastic
growths. Till now, it is not clear as to why the retina is so susceptible for RB1 gene
mutation. There are ~300 cases arising every year in the USA. The mortality rate
being very low in developed countries is reported to be very high (approximately
70%) in developing countries.
Two forms of retinoblastoma are reported: hereditary form and non-hereditary
form. In the hereditary form of retinoblastoma, retinoblastoma is present in the
family history, through which one mutated allele is passed on to the child, while
the other allele is mutated during gamete formation or in developing retina. In the
non-hereditary form of retinoblastoma, mutation in one allele occurs either during
gamete formation or in developing retina, and the other allele is mutated at any time
during the life because of exposure to any radiation or genetic alterations. In other
case of non-hereditary retinoblastoma, the allele is mutated during the lifetime
resulting from epigenetic effects or genetic mutations. Hereditary form of retinoblas-
toma includes ~40% of the cases, while non-hereditary form accounts for ~60% of
retinoblastoma cases. Retinoblastoma can be unilateral or bilateral. Bilateral retino-
blastoma is generally hereditary and unilateral retinoblastoma is usually
non-hereditary. Bilateral retinoblastoma leads to multifocal tumors and malignancy
to various parts of the body; benign tumor with less severe form of disease is
observed in the unilateral retinoblastoma. Early detection and treatment of retino-
blastoma are essential, otherwise the tumor can spread to the optic nerve and further
to the brain and can metastasize to the bones also. “Trilateral retinoblastoma,”
characterized by the development of primary midline intracranial neoplasm, has
been observed in 10% children with hereditary retinoblastoma (Dimaras et al. 2015).

18.12.1 Clinical Characteristics and Diagnosis of Retinoblastoma

Leukocoria is the most common presentation of the retinoblastoma. It is the first sign
which is usually observed by parents/guardians. Leukocoria is characterized by the
presence of white appearance in one or both pupils. It results when a large tumor or
smaller tumor associated with the retinal detachment is present. Retinoblastoma is
generally characterized by strabismus (misalignment of eye). The least common
signs may also include orbital cellulitis, hyphema (blood in anterior chamber),
glaucoma (increased intraocular pressure), heterochromia (different color of pupils),
visible extraocular growth, and decreased vision. Patients with advanced stages of
retinoblastoma also show symptoms such as orbital swelling and proptosis because
of extraocular invasion.
18 Molecular Genetics of Cancer 927

Ophthalmoscope is the simplest test to detect leukocoria. Under the anesthesia

effect, portable slit lamp examination is done to check hyphema and iris
neovascularization; indirect ophthalmoscopy and fundoscopy are used to detect
various lesions. Further imaging methods of diagnosis include ultrasonography,
computed tomography (CT), and magnetic resonance imaging (MRI). MRI is
preferred over CT scan to reduce the exposure effects of ionizing radiations on the
patient. MRI is done for the brain and eye orbitals to detect any malignancy.
Trilateral retinoblastoma has been only detected through the MRI. Early diagnosis
of retinoblastoma increases the survival chances of patients.

18.12.2 Structure and Function of RB1 Gene

Human RB1 gene consists of 178,143 bp with 27 exons and 26 introns. The RB1
gene encodes 928 amino acid protein, pRb. pRb protein contains three domains:
N-terminal domain, A/B pocket domain, and C-terminal domain (Fig. 18.20). RB
gene family proteins, pRb, p130, and p107, belong to the “pocket” proteins family,
because of the presence of large pocket domains. These pocket domains contain
binding sites for many of their interacting proteins. In addition to the presence of
binding sites, A/B pocket domain of pRb also contains a LXCXE-binding cleft,
which serves as a separate binding site, and through this cleft pRb interacts with

Fig. 18.20 Schematic representation of pRb protein domains

928 B. Chuphal

Fig. 18.21 Regulation of E2F target genes by the pRb

proteins that share an LXCXE-binding motif. Being highly evolutionary conserved,

LXCXE-binding cleft also interacts with LXCXE sequence of various viral
oncoproteins (human papilloma virus E6 and E7, adenovirus E1A protein, and
simian virus 40 large T antigen). These viral oncoproteins bind to the LXCXE-
binding motif of pRb protein and lead to its inactivation. Discovery of these viral
oncoproteins plays important roles in the understanding of retinoblastoma (DeCaprio
2009). pRb contains 16 CDK (cyclin-dependent kinase) phosphorylating sites,
through which activation and inhibition of pRb activity are controlled. Since the
dephosphorylated/hypophosphorylated state of pRb is the active state, which is
controlled by the CDKI (cyclin-dependent kinase inhibitors) of the Cip/Kip families
and Ink4, phosphorylated/hyperphosphorylated state of pRb is the inactive state of
pRb. Ink4 family contains p19, p18, p16, and p15 proteins, and Cip/Kip family
consists of the p57, p21, and p27 proteins.
The product of RB1 gene, pRb protein, is responsible for the terminal differenti-
ation of the myocytes by promoting the functional interaction of MyoD (myoblast
determination protein 1) with the coactivator MEF2 (myocyte enhancer factor 2). By
binding with the histone deacetylase 1 (HDAC1), pRb inhibits its association with
the MyoD. HDAC1 is involved in gene silencing by deacetylation of histone
proteins of target gene. As binding of HDAC1 with MyoD is prevented, MyoD
indirectly promotes the expression of MyoD transcriptional target genes. The
LXCXE motif of A/B pocket domain of pRb protein is responsible for the interaction
with HDAC1. pRb plays an indispensable role in the fate determination, differentia-
tion, survival, and migration of developing neurons. pRb protein is involved in
regulation of arresting the cell at G0 phase of cell cycle, G1 cell cycle arrest,
senescence, apoptosis, and transcriptional control. pRb regulates these functions
through E2F transcription factor and Skp protein. The A/B pocket domain of pRb
has the binding site for E2F target genes and Skp protein.
E2 promoter binding factor (E2F) regulates the transcription of various cyclins
(cyclins A and E) and cyclin-dependent kinases (CDKs), by binding to their pro-
moter region. These CDKs and cyclins are essential for the transition from G1 to S
phase of cell cycle. The unphosphorylated or hypophosphorylated state represents
the active state of pRb, which either alone or through recruitment of histone
deacetylase 1(HDAC1), ATPases, and DNA methyltransferases binds to E2F gene
promoter region, thereby blocking the E2F target gene transcription (Fig. 18.21).
18 Molecular Genetics of Cancer 929

Fig. 18.22 Regulation of pRb. (a) Mitogens/growth factors lead to phsophorylation of pRB thus
progressing the cell in S-phase. (b) Growth inhibitory factors result in cell cycle arrest at G1 phase

The phosphorylated or hyperphosphorylated state of pRb is its inactive state (Nevins

2001).
Skp2 (S phase kinase associated protein), human F-box protein, regulates the
transition from G1 to S phase of cell cycle and cellular senescence through interac-
tion with cell cycle inhibitory protein, p27. p27 inhibits the cyclin E-CDK2 complex,
which are responsible for phosphorylation of pRb. In growth-promoting signals
(EGF, PDGF, TGF-β), Skp2 binds with the p27 and leads to its degradation, thereby
activating the cyclin E-CDK2, which phosphorylates pRb, resulting in S phase
transition of cell cycle. When growth-promoting signals are absent, pRb binds
with the Skp2, which further interacts with the APC/Ccdh1 and results in Skp2
degradation. p27 will accumulate in the cell, which inhibits pRb phosphorylation
by inhibiting the cyclin E-CDK2 complex. The active form pRb (unphosphorylated
form) leads to G1 cell cycle arrest.
Since activity of pRb is controlled by the CDKs and CDKIs; these CDKs and
CDKIs respond to various mitogen factors, DNA damage, senescence, and differen-
tiation signals, thereby regulating the pRb activity. Growth stimulatory signals lead
to the phosphorylation of pRb through CDKs, thereby inactivating the pRb, resulting
in S phase of cell cycle. When growth inhibitory signals such as DNA damage,
senescence, and antiproliferative signals are present, CDKIs inhibit the CDKs,
thereby dephosphorylating the pRb and cell arrest at G1 phase, or undergo senes-
cence, quiescence, and apoptosis (Fig. 18.22).
Additional functions of RB1 include its involvement in heterochromatin forma-
tion, thus ensuring the chromosomal integrity by stabilizing the histone methylation
through interaction with two LXCXE containing histone methyltransferase, Suv4-
20 h1 and Suv4-20 h2. Any disruption in these interactions results in delayed mitosis
process and centromere fusion, abrupt chromosomal segregation, and genomic
930 B. Chuphal

instability. pRb inhibits the intrinsic kinase activity of TAF1 (TATA binding
protein-associated factor 1). pRb promotes the centromeric localization of the
CAP-D3/condensing II (Condensin-2 complex subunit D3) protein complex,
thereby regulating the chromatin condensation, cohesion, and stability. In heritable
retinoblastoma samples, it has been found that there is aneuploidy of 6p and 1q
chromosome. Deletion in the 16q chromosome has been also reported in the
retinoblastoma samples. Genes present in these regions may also involve retinoblas-
toma development. The Mdm2 (Mouse double minute 2) related protein, MdmX, is
responsible for inhibition of p53 functions. The MDMX gene is located in the 1q32
region. In 65% and 10% of retinoblastoma cases, MDMX and MDM2 have been
upregulated, since these proteins inhibit the p53-mediated apoptosis, resulting in
tumor development. Deletion or inactivation of pRb leads to abrupt expression of
E2F target genes, such as MAD2 (mitotic arrest deficient 2), which is responsible for
encoding mitotic spindle checkpoint protein. Thus, deregulated expression of
MAD2 leads to tumorigenesis (Di Fiore et al. 2013).

18.12.3 Treatment Therapy

On the basis of International Retinoblastoma Staging System, retinoblastoma has

been classified among five groups: E (enucleation, advanced), D (diffuse, large) C
(confined, medium), B (medium), and A (small) (Table 18.11). Till now various
therapies have been used to treat retinoblastoma, like focal therapy (external beam
radiation, brachytherapy, cryotherapy, thermotherapy), chemotherapy (vincristine +
low-dose carboplatin, vincristine + high dose of carboplatin + etoposide + G-CSF,
subtenon carboplatin, and prophylactic 3-agent chemotherapy), and enucleation.
These treatments result in severe side effects. Various research have been undertaken
to develop a targeted approach for treatment of retinoblastoma with less side
effects (Lin & O’Brien 2009; Sachdeva & O’Brien 2012; Teixo et al. 2015).

18.12.3.1 External Beam Radiation

It is the most effective method to treat the retinoblastoma. It is generally advisable to
the patients with bilateral retinoblastoma. When chemotherapy fails and there is
recurrence of disease, then EBR is used to treat the tumor. EBR is effective in cases
of small tumors in the macula, where chemotherapy does not work. The range of
dose from 42Gy to 46 Gy is prescribed, with the 58–88% protection of the eye.
Patients with heritable retinoblastoma receiving EBR treatment lead to several
secondary malignancies, such as osteosarcoma and cancer of the nasal cavities and
the brain. Children below 1 year of age have high risk of developing undesired
effects like cataract, facial and temporal bone hypoplasia, vitreous hemorrhage, and
orbital hypoplasia.

18.12.3.2 Brachytherapy
Brachytherapy involves the implantation of a radioactive agent in the sclera, near the
tumor base. In this therapeutic approach, several radioactive agents have been used,
such as ruthenium-109 (109Ru), iridium-192 (192Ir), iodine-125 (125I), gold-198
18 Molecular Genetics of Cancer 931

Table 18.11 Different stages of retinoblastoma

Group Characteristics Treatment Prognosis Toxicities
E 1 mm tumor in 1. Enucleation Morbidity Neurotoxicity,
(advanced, anterior segment 2. Chemotherapy high from hyponatremia,
enucleation) and ciliary body, no with the nephrotoxicity,
vision, neovascular prophylactic-3 treatment ototoxicity,
glaucoma, vitreous agent and no secondary
hemorrhage, visual leukemias
phthisical eye, potential
orbital cellulitis,
extraocular disease
D (large, >3 mm from tumor 1. Chemotherapy Variable Neurotoxicity,
diffuse) margin with diffuse with three agents visual hyponatremia,
vitreous or (vincristine, prognosis; nephrotoxicity,
subretinal seeding, carboplatin, high ototoxicity,
and subretinal fluid etoposide) for six morbidity secondary
cycles from leukemias
2. With or without treatment Decreased ocular
subtenon or motility, optic
carboplatin atrophy with
3. External beam ischemic necrosis,
radiation (EBR) pseudo-preseptal
cellulitis
Midfacial
hypoplasia, soft
tissue and
osteosarcoma,
brain tumors
C (medium, 3 mm from tumor 1. Chemotherapy Visual Neurotoxicity,
confined) margins with with three agents prognosis hyponatremia,
vitreous or (vincristine, variable nephrotoxicity,
subretinal seeding carboplatin, ototoxicity,
etoposide) for six secondary
cycles leukemias
2. Treatment with Decreased ocular
or subtenon or motility, pseudo
carboplatin preseptal cellulitis,
3. Focal therapy optic atrophy with
ischemic necrosis
Vitreous seeding,
radiation
retinopathy, retinal
tears
B (medium) >3 mm height, 1. Vincristine + Good Neurotoxicity,
subretinal fluid clear low-dose visual hyponatremia,
3 mm from tumor carboplatin up to prognosis nephrotoxicity,
margin six cycles ototoxicity,
2. Focal therapy Vitreous seeding,
from two to six radiation
cycles retinotherapy,
retinal tears
(continued)
932 B. Chuphal

Table 18.11 (continued)

Group Characteristics Treatment Prognosis Toxicities
A (small) 3 mm height, Focal therapy Good Tissue damage,
2 mm disk (argon/YAG visual and vitreous seeding,
diameter from laser, overall retinal tears,
fovea, 1 mm disk brachytherapy, prognosis; chorioretinal
diameter from optic hyperthermia, or usually atrophy, radiation
nerve cryotherapy) eradicated retinotherapy

(198Au), cobalt-60 (60Co), yttrium-90/strontium-90 (90Y/90Sr), palladium-103

(103Pd), and rhodium-106/ruthenium-106 (106Rh/106Ru). In this therapy for
2–4 days, the tumor is exposed to 40–45 Gy ionizing radiation. Brachytherapy is
used in patients who stop responding to chemotherapy and EBR. Brachytherapy is
only advisable to the small tumor; however it is not recommended to large tumor size
or which is associated with the macula. This therapy has lesser side effects compared
to the EBR and includes cataract formation, radiation retinopathy, and optic
neuropathy.

18.12.3.3 Thermotherapy
Thermotherapy process consists of applying heat directly to the tumor through the
use of infrared radiation. In this therapeutic approach, temperature ranges from 45  C
to 60  C that does not result into blood coagulation in retinal vessels. It is advisable
for the small dimensions of retinoblastoma.

18.12.3.4 Laser Photocoagulation

Laser photocoagulation is associated with the delivery of diode laser or argon laser
or a xenon arc to the tumor. This technique is recommended to the small posterior
tumor, in which blood supply to the tumor is prevented by coagulation of blood.
Normally at monthly intervals, two or three sessions are required for the effective
treatment. Side effects of this treatment include the preretinal fibrosis, retinal vascu-
lar occlusion, and retinal detachment.

18.12.3.5 Cryotherapy
The principle of cryotherapy is the complete destruction of the vascular endothelium
that supplies the tumor, through the freezing process. This therapy is recommended
for the small peripheral tumors. On a monthly interval, one to two sessions with three
times per session of cryotherapy are done to treat the tumors. The side effects of this
therapy include retinal detachment and vitreous hemorrhage.

18.12.3.6 Chemothermotherapy
In this therapeutic approach, chemotherapy and thermotherapy are applied at the
interval of a few hours to combat the large-sized tumors. This treatment is most
effective in small-sized tumors present near the optic nerve and fovea. The major
disadvantage of this treatment is the atrophy of focal iris, retinal detachment, and
optic disk and corneal edema.
18 Molecular Genetics of Cancer 933

18.12.3.7 Chemotherapy
Chemotherapy consists of the administration of drugs through different routes such
as intravenous, intra-arterial, periocular, or intravitreal to reduce the tumor size so
that disease can be eradicated completely using other therapies. For intraocular
retinoblastoma or unilateral retinoblastoma, intravenous chemotherapy is
recommended. Carboplatin, vincristine, and etoposide are the main chemotherapeu-
tic agents, which are used in combination for six cycles based on the patient’s body
weight. The major side effects of these drugs are high risk of bacterial infections and
development of new tumors in various body parts. In view of side effects, less toxic
chemotherapeutic agents, such as topotecan and 2-deoxy-D-glucose (2-DG), are
used. Topotecan is an inhibitor of DNA topoisomerase-1 and 2-DG is a glycolytic
inhibitor. In intra-arterial chemotherapy, infusion of melphalan drug into the oph-
thalmic artery is done. It is safe and very effective for the unilateral retinoblastoma.
This technique is recommended for medium- and large-sized tumors. This approach
leads to local ocular toxicity. Injections of carboplatin are given in periocular
chemotherapy with systemic chemotherapy to increase the dose in the vitreous for
controlling retinoblastoma. The side effects of this approach include optic atrophy,
strabismus, and orbital and eyelid edema. Melphalan drug is used in intravitreal
chemotherapy against the retinoblastoma. It is one of the most effective methods, but
numerous injections in the eye for a period of 1 year lead to vitreous seeding.

18.12.3.8 Enucleation
Enucleation is applied in the advanced cases of retinoblastoma. In this approach the
eye is replaced with orbital implants of plastic, silicone, or hydroxyapatite. Enucle-
ation leads to complete vision loss. It is only effective when timely done, otherwise
chances of malignancies increase.

18.12.3.9 Targeted Approach with Small Molecules

Since all the above therapeutic approaches lead to severe side effects, there is a need
for the development of targeted approaches with less side effects. With the improve-
ment in the understanding of the different interacting molecular pathways, new
approaches have been developed. It is a well-known fact that p53 is involved in
apoptosis, so by stimulating the p53-mediated apoptosis pathway, tumor growth can
be inhibited. A small molecule, Nutilin-3, inhibits the Mdm2 and MdmX interaction
with the p53, resulting in apoptosis in retinoblastoma through p53-mediated path-
way. When nutilin-3 is used in combination with the topotecan, a p53 inducer, it
leads to synergistic effect on the reduction of retinoblastoma cells. The side effect of
nutilin-3 and topotecan is negligible. It is reported that the cells with upregulation of
E2F1 activity are responsive to the HDAC inhibitors. Thereby, another targeted
approach in which HDAC inhibitors (HDACi) are used leads to apoptosis in
retinoblastoma cells. These molecules are still under clinical trials. To combat the
retinoblastoma, targeted approaches with less side effects need to be developed.
934 B. Chuphal

18.13 p53 Tumor Suppressor Gene

The 20 kb gene encodes for a nuclear phosphoprotein, p53, with 53 kDa molecular
weight and containing 11 exons and 10 introns, located on chromosome 17pl3. The
other members of the family to which p53 gene belongs include p63 and p73.
Although being related structurally and functionally, p53 has evolved as a tumor
suppressor gene in higher vertebrates, whereas p73 and p63 are involved in devel-
opmental biology. p53 was first discovered in 1979, as bound to viral oncoprotein in
SV40 transfected cells. p53 has DNA-binding protein property and is usually found
in low quantities inside a normal cell’s nucleus, whereas 5–100X quantities can be
localized in both transformed and tumor cells.
Human p53 protein consists of 393 amino acids and comprises of various
domains (Fig. 18.23): an amino-terminal domain (1–42 amino acids) and proline-
rich region (61–94 amino acids) at N-terminal, a middle domain (102–292 residues),
and an oligomerization domain (324–355 residues), strongly basic regulatory
domain (363–393) at C-terminal (301–393 residues) along with nuclear export
signal sequence and nuclear localization signal (Fig. 18.23). The amino-terminal
region regulates the interaction with transcription factors (such as acetyltransferase
and MDM2) and transactivation activity. The stability of p53 is controlled by the
proline-rich region. Any mutation or deletion in this region increased the suscepti-
bility of p53 to degradation by MDM2. The central region of p53 is evolutionarily
highly conserved. The negative regulatory role is played by basic C-terminus, which
is involved in cell death induction. Various structural studies have revealed that the
majority of p53 mutations are missense mutations in the central DNA-binding
domain found in cancers, and mostly 126–130 residues are focused to study p53
mutation (Bai & Zhu 2006; Choisy-Rossi et al. 1999; Harris 1996; Joerger & Fersht
2010; Kamaraj & Bogaerts 2015; Sigal & Rotter 2000).

18.13.1 The Physiological Functions of p53

Following genotoxic stress, p53 functions as a tumor suppressor gene, thereby

preventing unregulated cell proliferation and maintaining the genome

Fig. 18.23 Diagrammatic representation of the p53 protein structure

18 Molecular Genetics of Cancer 935

Fig. 18.24 Schematic representation of p53 at the middle of a complex signaling network under
stress

integrity (Finlay et al. 1989). Following various stimuli both extracellular and
intracellular in nature, such as DNA, hypoxia, heat shock, and overexpression of
oncogene, activation of p53 occurs and hence triggers various biological responses.
The activation of this protein involves overall fold-change in protein level as well as
via extensive post-translational modification, eventually activating the several
p53-targeted genes. For instance, in case of double-strand DNA damage, activation
of protein kinase ATM (ataxia-telangiectasia mutated) results in Chk2 kinase acti-
vation. In turn, both the proteins lead to phosphorylation of p53, thereby resulting in
p53-dependent apoptosis or cell cycle arrest. Damage to DNA leads to blocking of
replication process and activates the ATR (ATM and Rad3-related) and Chk1,
subsequently resulting in p53 phosphorylation and activation. Activation of genes
by wild-type p53 results in DNA repair, senescence, cell cycle arrest, and apoptosis,
by regulating the downstream signaling molecules involved in these processes
(Fig. 18.24), such as p21waf1/Cip1, Bcl-2 family, and Gadd45 (growth arrest and
DNA damage inducible protein 45). In addition, p53 elevated level leads to inhibi-
tion of various gene expressions such as cyclin B1, bcl-X, bcl-2, MAP4, and
survivin. Interestingly, it was seen that in ovarian cancer cells having
p53-expressing adenovirus infection, 80% of putative p53-responsive genes are
repressed.
936 B. Chuphal

18.13.2 Cell Cycle Regulation

Cell cycle arrest in the G1, G2, and S phases can be induced by p53. The arrest at G1
and G2 subsequently allows cell genomic damage repair before entering into the S &
M phase of cell cycle. Once repair is done, the arrested cells enter into the
proliferating phase through the biochemical function of p53. The primary mediator
of G1 cell cycle arrest following DNA damage is p21waf1/Cip1. In response to
stimuli such as stress, upregulation of endogenous Cip1/p21waf1 mRNA and protein
levels takes place via p53. ZRXL motif of p21waf1/Cip1 in turn binds to cyclin-
CDK complexes. It is reported that p21waf1/Cip1 overexpression blocks Rb phos-
phorylation, resulting in arrest at G1 phase and E2F release which is critical for gene
expression involved in the entry of S phase. Similarly, Gadd45 prevents cyclinB/
CDK1 complex formation by binding to CDK1 (CDC2), which leads to kinase
activity inhibition, and 14–3-3δ separates the cyclinB/CDC2 from the target
proteins, leading to G2 arrest.

18.13.3 Induction of Apoptosis

p53-induced apoptotic genes include p53AIP1 (p53-regulated apoptosis-inducing

protein 1), Scotin, Bax (Bcl-2-associated X protein), DRAL, PIDD (p53-induced
protein with death domain), PIG3 (p53-inducible gene 3), Fas/CD95 (cell death
signaling receptor), Noxa (from the Latin word for “harm” or “damage”), Puma
(p53-upregulated modulator of apoptosis), Apaf-1 (apoptotic protease-activating
factor-1), PERP (p53 apoptosis effector related to PMP-22), DR5/KILLER (death
receptor 5), etc. (Fig. 18.25). Bax plays a critical role in apoptosis mediated through
Puma and also reportedly participates in response to death indirectly via Puma.
However, p53-mediated apoptosis seems to require Bax in a cell-dependent manner.
Loss of Bax results in 50% of accelerated brain tumor growth which are related to the
loss of function in p53. In addition to this, Bax is involved in the apoptosis in
intestinal epithelial cells and thymocytes. In addition, several Bcl-2 family members
and mitochondrial proteins are also involved in apoptosis induced by p53. During
apoptosis, p53 is seen to regulate the genes encoding Apaf- 1, apoptosome compo-
nent, and PIG3, involved in depolarization of mitochondria and can also promote
apoptosis through death receptor activation located at the plasma membrane, includ-
ing DR5, DR4 and Fas/CD95, and often IAP (inhibitor of apoptosis proteins)
inhibition. Apoptosis is also induced by the ER-dependent mechanism by
transactivating Scotin expression, a protein present in the ER and nuclear membrane.
p53 also promotes apoptosis in transcription-independent manner, in certain cell
types, and under certain conditions. For example, in mitochondria, p53 is reported to
directly bind Bcl-XL/Bcl-2 and thus displace Bax or BH3 domain-only
pro-apoptotic proteins, thereby facilitating Bax-dependent apoptotic changes in
mitochondria. p53 induces oligomerization by binding to Bak in mitochondria,
which facilitates plasma membrane permeabilization and release of cytochrome
18 Molecular Genetics of Cancer 937

Fig. 18.25 Schematic representation of p53-associated genes and pathways involved in apoptosis

c. This is a tissue-specific response. This is a very fast response (30 min), which
takes over the transcriptional response (taking more than 2 h).
Studies have shown that tumorigenesis is accelerated in the mouse brain when
there is loss of p53-dependent apoptosis. Transcriptional factors such as ASPP
(apoptotic-stimulating protein of p53) family, JMY (junction-mediating and regu-
latory protein), c-Myc, p73, and p63 affect the balance between the cell cycle arrest
and apoptosis. The balance between p21Waf1/Cip1 and Puma identified in human
colorectal cancer cells regulates the apoptosis and cell cycle arrest in response to
p53.

18.13.4 p53 Level and Activity Regulation

p53 exists mainly in an inactive form and is maintained at a very low concentration
under normal conditions. Crucially, p53 low basal level has to be even tightly
controlled during cell cycle progression. A complex cellular protein network is
required for regulation of p53 level including PARP-1, MDM2, JNK, HPV16 E6,
SV40 T-antigen, E1B/E4, Pirh2, and WT-1. p53 stability increases when it binds to
E1B/E4, WT1, or SV40 T antigen, whereas its degradation accelerates when it
associated with MDM2 or E6. MDM2 blocks p53 transcription or stimulates its
export to the nucleus, or its degradation, thereby, inhibits p53 activity. MDM2
protein interacts with transactivation domain of p53 at N-terminal and blocks its
interaction with the transcription components. Because of its intrinsic E3 ubiquitin-
ligase activity, it mediates p53 ubiquitylated-dependent degradation. It recruits the
histone deacetylase 1 (HDAC1) at the p53 C-terminal, thereby marking it for
938 B. Chuphal

ubiquitination. However, under stress, p53 regulates MDM2 gene transcription

through p53-dependent promoter present in MDM2 gene. In this manner, an
autoregulated network is established between the activity of p53 and MDM2 gene
expression. Under genotoxic stimuli, p53 degradation mediated by MDM2 was
abolished by various mechanisms, leading to p53 response maintenance (Shaikh &
Niranjan 2015).
In addition to the protein complexes, stability and activity of p53 are also highly
regulated via post-translational modifications, such as phosphorylation, acetylation,
ADP-ribosylation, ubiquitylation, sumoylation, etc. Acetylation and phosphoryla-
tion are two important post-translational modifications, which leads to p53 stabiliza-
tion and promotes its activity as a transcription factor for its target genes. These
modifications also prevent p53 degradation by abolishing its interaction with
the MDM2.

18.13.5 p53 Mutation

A few common missense mutations in the DNA-binding domain (102–292 amino

acids) of p53 subsequently results in both the loss of tumor suppressor function and
accumulation of a mutant protein, having its own oncogenic life. The p53 loss of
function in cancers is brought about by several mechanisms, including lesions, gene
mutations, or mutations in the downstream mediators of p53’s function. Approxi-
mately 18,000 p53 mutations are identified in the majority of human cancers.
Majority of these tumors are either because of loss or mutation in p53, resulting in
its activation. For example, 70% of lung cancer, 45% of stomach cancer, and 60%
head and neck, colon, bladder, and ovary cancer are because of p53 mutation. In
addition, mutations in the downstream signaling molecules of p53 function result in
50% of human cancers. In Li-Fraumeni syndrome, inherited germline mutation is
present in one allele of p53, and people with this syndrome are more susceptible for
cancer development during lifetime. Brain, breast, adrenal gland, hematological
system, and connective tissue tumors develop because of loss of p53 alleles. It has
been reported that mice deficient in p53 allele are associated with tumorigenesis. In
addition, p53 inactivation is also associated with the tumor’s development, because
of its inactivation by defects in upstream signals or oncoproteins.

18.14 Cell Cycle and Cancer

There exist a confined number of vital events which propel tumor cells and its
daughter cells into uncontrolled proliferation and invasion throughout the body. One
such vital event is cell proliferation gone haywire which along with suppressed
apoptosis gives a platform to tumor cells for further neoplastic progression. Nor-
mally, a cell cycle consists of three important events, namely, growth, DNA synthe-
sis, and division, where the duration of each event is tightly controlled by chemical
signals provided to or by the cells. In addition, the transition between each phase
18 Molecular Genetics of Cancer 939

Fig. 18.26 Schematic representation of START checkpoint in the cell cycle

demands selective chemical signals and timely responses which if gone wrong such
as in cases where signals are not correctly sensed or when the cell is not prepared for
the response can give rise to cancerous tissue. The major phases involved in the cell
cycle are G1 phase, S phase, G2 phase, and M phase which are regulated at various
checkpoints and halt the progression of cell until it has all the necessary machinery
such as DNA synthesis or repair of faulty DNA. A cell can only progress further into
the division process when all the checkpoints are satisfied. Cyclins and cyclin-
dependent kinases (CDKs) are critical during cell cycle. CDKs are catalytically
active cell cycle components, which can transfer the phosphate group, thus
regulating the activity of other proteins (Malumbres & barbacid 2009). However,
their activity majorly depends on their interaction with cyclins forming cyclin/CDK
complexes leading to CDK activation. Normal cell cycle requires cyclic pattern in
the formation and degradation of cyclin/CDK complexes.
G1 phase involves the most important checkpoint, START (Fig. 18.26), to decide
the appropriate time to proceed to S phase. The cell is said to be committed to cell
division only if it passes this checkpoint after which DNA replication is initiated.
There exist inhibitory proteins which while sensing issues in G1 phase such as DNA
damage can halt the cyclin/CDK complex, thereby preventing the cell to enter S
phase.
In tumor-inflicted cells, these checkpoints are deregulated often due to defects in
genetic machinery such as mutation in genes encoding cyclin or CDKs (Table 18.12)
or by modified proteins during cell cycle disruption (Table 18.13).
Normal cells have been programmed in such a way that they pause at the START
to ensure all the machinery is working before proceeding for DNA replication. On
the other hand, cells in which this checkpoint is faulty move to S phase without
repairing the DNA damage. Over a period of time, these accumulated mutations
cause further cell cycle deregulation, thus leading to the formation of aggressive
cancerous cells.
940 B. Chuphal

Table 18.12 List of cyclins and CDKs along with their function involved during cell cycle
Protein Chromosome Function Relevance in human cancer
Cyclin 4(q25-q31) Regulation of S phase and G2- Hepatocellular carcinoma and
A M transition by forming breast carcinoma showed
complex with CDK2 and 1 overexpression
Cyclin 5(q13-qter) Regulation of G2-M transition Breast carcinoma showed
B1 by forming complex with overexpression
CDK1
Cyclin 11q13 Early G1 phase regulation by Overexpression in various tumors
D1 forming complex with CDK4/ including lymphoma, breast
6 cancer, and parathyroid adenoma is
reported
Cyclin 12q13 Early G1 phase regulation (in Overexpression in few colorectal
D2 some cells) by forming cancers
complex with CDK4/6
Cyclin 19q12 Regulation of late G1 and G1- Overexpression is reported in
E S transition by forming various cancers including colon,
complex with CDK2 breast, prostate carcinomas, and
leukemia
CDK1 10 Regulation of G2-M transition Overexpression in breast cancer
by forming complex with
cyclin B1
CDK4 12q13 Early G1 phase regulation by Mutation in case of melanomas and
forming complex with cyclin overexpression in brain tumors
D

18.14.1 Cell Cycle and Signal Transduction

Cells at quiescent stage are stimulated by appropriate signals, so that they enter into
the cell cycle. These signals enter the cell by molecules like growth factors and
hormones that have the ability to bind to receptors present on the cell surface
relaying the signals into the cytoplasm via a process called signal transduction.
The final result is certain gene expression activation which can propel a cell out of
a quiescent phase into cell cycle. By contrast to normal cells, cancer cells are
reported to have defects in signal transduction pathways ranging from abnormal
growth signals or molecules present at downstream signaling. In addition, cancerous
cells have also been shown to stop responding to external growth inhibitory signals.

18.14.2 Cell Cycle Checkpoints

To deal with exogenous and endogenous sources of DNA damage, cells have cell
cycle checkpoint and conserved DNA repair mechanisms (Funk 2001; Kastan &
Bartek 2004; Visconti et al. 2016). In order to regulate events in cycle progression
such as case of genome damage, complex signal transduction pathways play a role
which together constitute “checkpoints.” These checkpoints allow cell DNA damage
repair by arresting cell cycle. In order to avoid risking generation of altered progeny,
some of the cell types prefer to undergo the programmed cell death (apoptosis).
When damage to the genome is detected, cell cycle checkpoints delay the cell
18 Molecular Genetics of Cancer 941

Table 18.13 Types of proteins involved in cancer due to cell cycle disruption
Role in human Mouse knockout
Protein Chromosome Function cancer models
p21CIP1 6p21 Block G1 and S Rare mutations in Defect in G1-S
phase by binding to the breast, bladder, checkpoint, no
multiple cyclin/ and prostrate spontaneous
CDK complexes and carcinomas tumors, no tumor
proliferating cell suppressor
nuclear antigen
(PCNA); induced by
p53
p27KIP1 12p13 Induce G1 arrest by Variable loss in Pituitary
binding to multiple expression of hyperplasia/
cyclin/CDK protein in several adenoma,
complexes and malignancies and organomegaly,
inhibit them heterozygosity loss gigantism; haplo-
insufficient tumor
suppressor
p57KIP2 11p15.5 Induce G1 arrest by Mutation found in Adrenal
binding to multiple Beckwith- hyperplasia,
cyclin/CDK Wiedemann developmental
complexes and syndrome patients, defects, neonatal
inhibit them few inactivation lethality; no
identified spontaneous
tumors
p61NK4a 9p21 Induce G1 arrest by Often inactivated Carcinogen-
binding to CDK4/6 in bladder and lung induced increase
and inhibit its carcinomas, in melanomas, low
function pancreatic chances of
adenocarcinomas, spontaneous
melanoma mutations;
cooperative effects
with haplo-
insufficient
p14ARF status
p14ARF 9p21 G1 and G2 arrest by Mutation in High incidence of
blocking MDM2 gliomas, melanoma induced and
inhibition of p53 cell lines; targeted spontaneous
in acute T-cell mutations;
leukemia p16INK4a/
p19ARF/ show
very similar
phenotype

progression, by affecting the critical cell cycle regulator activity. The checkpoints
are essential for the genetic stability maintenance, and therefore any mutations in
their components result in the aberrant cell cycle progression under perturbing
stimuli. How a cell responds to any DNA damage type is one essential constituent
of the cancer biology field. From in vivo and in vitro studies using animal models,
and from the fact that mutations in genes implicated in DNA damage responses, it
can be well concluded that damage to cellular DNA leads to cancer. Also,
942 B. Chuphal

Fig. 18.27 Cell cycle checkpoint pathways. ATM(ATR)/CHK2(CHK1)–p53/MDM2-p21 path-

way is shown to stop the G1-S phase transition of cell. P phosphorylation

surprisingly DNA damage is often used for cancer treatment. Many of the therapeu-
tic approaches used to cure malignancies by targeting the DNA includes chemo
agents and radiation therapy. In addition, DNA damage itself leads to various side
effects, for example, hair loss, gastrointestinal toxicities, and bone marrow suppres-
sion. So, weirdly, DNA damage is crucial for the disease cause as well as treatment
of disease and also for the toxicities for the same disease. A variety of different repair
mechanisms exists for the plethora of DNA lesions. The cell, besides undergoing
toward DNA damage repair, undergoes apoptosis or blocks the progression of cell
proliferation. Although we still have a limited understanding about the coordination
of cell cycle arrest or programmed cell death with DNA repair, this coordination is of
utmost importance for cell outcome optimization. In addition to the DNA damage,
cells are also required to cope with other stresses such as deficiency in nutrients or
oxygen levels. The term “cell cycle checkpoint” referred to the process in which cell
cycle progression is halted until the cell ensures that earlier steps, such as mitosis or
DNA replication, are completed.
Cell cycle progression is a highly regulated process in normal cells to ensure that
each step should be completed before proceeding to the next. There are three well-
known cell cycle checkpoints, the G1/S, the G2/M, and M checkpoints (Fig. 18.27),
in the whole cell cycle where equilibrium between external and internal signals is
checked before a cell enters the next stage.

18.14.2.1 The G1 and G1/S Checkpoint Responses

G1/S checkpoint monitors the cell size and DNA damage and halts the cell cycle
progression until these faults are corrected. After the cell size and DNA integrity
pass the normality test, the checkpoint is traversed, and cell proceeds for S phase. In
mammalian cells, the most essential response toward the DNA damage while
traversing through the G1 phase is ATM(ATR)/CHK2(CHK1)–p53/MDM2-p21
pathway, resulting in induction of G1 arrest.
18 Molecular Genetics of Cancer 943

Although there appears to be a relatively constant ATM and CHK2 expression

during cell cycle, it is the concentration of CHK1 and ATR that fluctuates with low
levels till mid-G1 and starts their important activities once G1/S transition
approaches. ATR/ATM acts by phosphorylating the p53 at serine 15 which falls
under the amino-terminal transactivation domain. Threonine 18 and serine 20 within
the same domain of p53 are reported to be targets of CHK1/CHK2. In addition,
MDM2, ubiquitin ligase which normally binds to p53, is targeted after the DNA
damage by both ATM/ATR and CHK2/CHK1. These modifications aid stabilization
and accumulation of p53 protein and increase in its activity. The main target of p53 is
the transcription of p21CIP1/WAF1 inhibitor of CDKs, which inhibits the G1/S-
promoting cyclin E/Cdk2 kinase causing G1 arrest of the cell. This results not only in
the inhibition of DNA synthesis initiation but also leads to safeguard of RB/E2F
pathway in its active, growth-suppression mode and eventually causes G1 blockade
sustain. In humans, the most commonly deregulated tumor suppressor pathways,
governed by p53 and pRB, are targeted by G1 checkpoint. Increased CHK1 and
ATR expression along with induction of Cyclins E and A, and CDC25A phospha-
tase, cyclin E(A)/CDK2 kinase activator occurred during late G1. Under genotoxic
stress, enhancement in the physiological mechanism via CHK1 and CHK2 increased
activity and leads to CDC25A downregulation and eventually cyclin E(A)/CDK2
complex inhibition. Interestingly, despite the p53 and CDC25A constitutive phos-
phorylation by checkpoint kinases, CDC25A-degradation cascade is faster as com-
pared to the p53 pathway. Thus, the CHK1/CHK2-CDC25A checkpoint activated
quickly, independent of p53, and results in delay of G1/S transition, unless
p53-dependent mechanism sustains and leads to prolonged G1 arrest.

18.14.2.2 S Phase Checkpoint

Activated by genotoxic insults, the intra-S phase causes transient and reversible
inhibition of those DNA replication origins that have not been initiated yet. Two
independent, ATM/ATR controlled pathways are responsible for the entry into S
phase. One of the mechanisms is operated through the CDC25A-degradation cascade.
CDC45 protein required for DNA polymerase recruitment into pre-replication
complexes is blocked by CDK2 activity inhibition downstream of CDC25A pathway;
hence CDK2 activity inhibition leads to prevention of the new origin initiation. The
other part of checkpoint involves the ATM-mediated NBS1 phosphorylation, in
particular at serine 343 and serines 966 and 957 of the SMC1 cohesin protein. The
concept of these two branches of intra-S phase checkpoint reported for ionizing
radiation and to ultraviolet light responses. Apart from origin of replication inhibition,
another essential function of S phase checkpoints (replication checkpoint) is mainte-
nance of the stalled replication fork integrity. Such fork stability maintenance, by
undiscovered mechanisms, helps the prevention of primary lesions converting into
DNA breaks and facilitates recovery of DNA replication subsequently. The primary
function of G1/S CDKs is activation of E2F. For the S phase induction and to
overcome a G1 arrest, cyclin E-associated kinase and E2F overexpression are
required.
944 B. Chuphal

18.14.2.3 The G2 Checkpoint

When a cell experiences DNA damage during G2 phase or when it progresses into
G2 with unrepaired damage during S or G2 phases, G2 checkpoint (G2/M check-
point) prevents the cell from mitosis initiation. In addition, the G2 arrest reflects the
S/M checkpoint contribution, after sensing some persisting DNA lesions from the
previous S phase. The important target of the G2 checkpoint is the cyclin B/CDK1
kinase and its mitosis-promoting activity specifically whose activation during stress
is inhibited by CHK1/CHK2, ATM/ATR, and/or sequestration, degradation of
subcellular components mediated by p38-kinase, and/or CDC25 phosphatase inhi-
bition which under normal conditions activate CDK1 at the boundary of G2/M. BP1
and BRC1 are involved in the G2 checkpoint response regulation as being analogous
to S phase checkpoint mediator role. Cell cycle inhibitors such as CDK inhibitor
p21, 14–3–3 sigma proteins, and GADD45a (growth arrest and DNA damage
inducible 45 alpha) upregulation are controlled by BRCA1 and p53 which maintain
the G2 checkpoint. The fact that tumors with p53 mutation tend to accumulate in G2
due to DNA damage and indicate sustenance of G2 arrest is brought about by
p53-independent mechanisms. Moreover, this phenomenon has put forward new
scope to interfere with this checkpoint to sensitize cancer cells as a potential
treatment, with G1/S checkpoint deficient, to DNA damage due to irradiation or
drug usage.
Mutation of any kind in the genes involved in checkpoints of the cell cycle
contributes to cancer development. For example, mutation in genes controlling the
first two checkpoints of the cell cycle might result in continuous growth and division
without DNA repair. Likewise, if genes for cyclins are expressed at a wrong time or
at incorrect levels, the cell may never be able to exit the cell cycle and enter the
quiescent phase, the result being the cancerous cell.

18.14.3 Cyclin D1 and Cyclin E Proto-oncogenes

Three homologues of Cyclin D are found in mammalian cells: Cyclin D1 (Bates &
Peters 1995; Diehl 2002), cyclin D2, and Cyclin D3, which are responsible for the
formation of active protein kinase by binding with either CDK6 or CDK4. The
structure analysis of Cyclin D revealed the presence of LXCXE-binding cleft near
N-terminus that binds with the LXCXE motif of pRb and cyclin box in the middle
that interacts with the CDKs and PEST sequence at the C-terminal. The induction
and assembly of Cyclin D1 with CDK4 are regulated by the growth factors via
Ras-mediated pathways. Under the influence of mitogenic factors, Ras kinase is
activated, which further activate downstream molecule Raf, subsequently activation
of mitogen-activated protein kinases (MEK1 and MEK2), resulting in continuous
extracellular signal-regulated protein kinase (ERK) activation, which regulates the
transcription of Cyclin D1 and its association with CDKs. In another signaling, Ras
leads to the activation of PI3K (phosphatidylinositol 3-kinase), thereafter Akt
activation, which results in glycogen synthase kinase-3β (GSK-3β) inhibition. The
GSK-3β leads to phosphorylation of the specific threonine (Thr-286) residue present
18 Molecular Genetics of Cancer 945

Fig. 18.28 Schematic representation of restriction point control

near the C-terminal of Cyclin D, which is essential for Cyclin D polyubiquitination-

mediated proteolysis and nuclear export. The accumulation of Cyclin D in the
nucleus induces a cell cycle G1 phase. To cross the G1 phase checkpoint, Cyclin
D leads to the pRb protein inhibition through its phosphorylation, thereafter release
of E2F transcription factor that regulates Cyclin E (Hwang & Clurman 2005) and
other proteins involved in the G1-S phase transition (Fig. 18.28). In addition, Cyclin
D1/CDK4 complex bind with the Cip/Kip family (CDK inhibitory protein) proteins
and stabilize Cyclin D1 in the nucleus, inhibits the cyclin D1 nuclear export, and
induces activation of the Cyclin E/CDK2.
PRAD1 is considered a putative proto-oncogene usually activated via gene
arrangement or gene amplification in various cancer types. It was isolated as gene
rearranged in parathyroid tumors by Motokura et al. in 1991. This gene (aka
CCND1) encodes for cyclin D1 which acts at the G1/S cell cycle transition leading
to pRB phosphorylation by sequestering to CDK4. The gene encoding the cyclin D1
is located on the chromosome number 11 (11q13). The inversion between the 11q13
region and the gene encoding region (11p15) for the parathyroid hormone leads to
the parathyroid adenoma. Because of the inversion between these genes, an alterna-
tive name for the cyclin D1 encoding gene (CCND1) is named PRAD1. In addition to
being overexpressed in various neoplasms, Cyclin D1 has also been identified in
acral melanomas (hand and feet occurrence). The translocation between the 11q13
region and gene region encoding for the heavy chain of immunoglobulin present on
chromosome 14 (14q32) accounts for the 70% B-cell lymphoma, because of the
upregulation of Cyclin D1 gene. This translocation has been reported in 15–20%
multiple myeloma. However, amplification of the Cyclin D1 encoding gene is
documented in 30–46% of NSCLC, 30–50% of neck and head squamous cell
carcinoma, 25% of pancreatic tumor, 49–54% of pituitary adenoma, 13% of breast
946 B. Chuphal

Fig. 18.29 Subtypes of

breast cancer based on
immunohistochemical profile.
TNBC triple-negative breast
cancer, HR hormone receptor

cancer, and 15% of bladder cancer. Mutation in the p16 also results in dysregulation
of Cyclin D and induction of tumor development. The Cip/Kip family is associated
with the tumor suppression; however, loss of one allele in Kip1 gene results in tumor
progression via overexpression of Cyclin D. The expression of Wnt-1 transgene in
association with the heterozygosity at the Cip1 locus is demonstrated with
upregulation of Cyclin D and increased tumor progression. The truncated form of
Cyclin D resulted from the A/G single nucleotide polymorphism (A870G) in the
Cyclin D encoding gene is reported in the development of lung colon and cancer.
Further, insertion of retroviral gene near the cyclin D gene region is also associated
with numerous cancers. Treatment with the synthetic inhibitor of Cyclin D and its
associated kinase, knockdown of Cyclin D, and ectopic expression of CDK inhibitor
proteins represent a potential therapeutic agent against the cancer.
The other putative proto-oncogene, cyclin E, has been isolated with the help of its
ability to complement the triple CLN mutant in S. cerevisiae. So far, it is unclear as
to whether the abnormal expression will perturb the normal cell cycle machinery; it
has been reported that the cyclin E mRNA levels dramatically rise and reach its peak
at the G1/S boundary. With the fact that very little information is available regarding
cyclin E protein, it would be important to show that the protein follows a periodic
expression pattern during cell cycle. The upregulation of Cyclin E expression has
been demonstrated in leukemia, sarcoma, lymphoma, and breast, lung, gastrointesti-
nal tract, cervix, and endometrial carcinoma. The nonfunctional pRb protein is also
associated with the overexpression of Cyclin E and Cyclin D via the upregulation of
E2F target genes. Till now, the exact mechanism of Cyclin E dysregulation is not
known; however, mutation in the p16, Cyclin D, pRb, and E2F may be associated
with the overexpression of Cyclin E.
18 Molecular Genetics of Cancer 947

Fig. 18.30 Role of CDKN2A and CDK 4/6 in cell cycle progression (Adapted from Sekulic et al.
2008)

Box 18.1: Scientific Concept: Cell Cycle Checkpoint Pathway Alterations

in Breast Cancer (Gargi Dan Basu et al.)
Breast cancer subtypes are molecularly determined based on immunohisto-
chemical profile defined by estrogen receptor (ER), progesterone receptor
(PR), and human epidermal growth factor receptor (HER2) status
(Fig. 18.29). Dysregulation in the cell cycle is a major hallmark of cancer
and has been reported in the various breast cancers as well.

(continued)
948 B. Chuphal

Box 18.1 (continued)

The communication between cyclins, cyclin-dependent kinases (CDKs),
and cell cycle inhibitors is reported in cell cycle progression through various
stages. During the progression, cyclins are involved in activating respective
CDKs by binding to them, thereby causing phosphorylation of downstream
target proteins. The cell cycle inhibitors such as p16INK4A and p14ARF, also
known as tumor suppressor proteins, block cyclin-CDK complex activities,
thereby suppressing the downstream processes. The p16INK4A and p14ARF
proteins are implicated in the RB1 and p53 pathway, respectively, and the loss
of any of these proteins can cause uncontrolled growth and proliferation inside
a cell. RB1, a tumor suppressor gene, is acting as a gatekeeper of the cell cycle
pathway. Unphosphorylated RB1 binds with and inhibits the E2F transcription
factor, hence preventing the E2F from activating the target gene expression
involved in G1/S progression. Cyclin D1 and CDK4 or CDK6 complex
phosphorylates RB1 protein which in turn releases E2F and allows the induc-
tion of target gene expression (Fig. 18.30).
In the recent past, outstanding understanding in the management of breast
cancer leads to expeditious detection and hence development of more effective
treatments with remarkable death rate decline. Breast cancer is considered as
multifaceted rather than a single disease consisting of a variety of biological
subtypes with diverse natural histories, presenting a varied spectrum of molec-
ular, pathological, and clinical features with different prognostic and therapeu-
tic implications. According to preclinical data, palbociclib, a CDK4/6
inhibitor, induced growth arrest in RB-positive cell lines and xenograft, and
higher RB1 and cyclin D1 expression levels as well as low levels of
p16INK4A were also associated with sensitivity to palbociclib. Based on
phase II trial results, the inhibitor was granted accelerated approval by the
FDA in February 2015 as the first-line treatment in postmenopausal women
with ER+/Her2- metastatic breast cancer. However, currently ER status is
considered as the most reliable predictive biomarker for palbociclib response;
fewer than half of the ER-positive breast cancer patients in the phase II
PALOMA-1 trial responded to this drug, thereby highlighting the need for
identification of additional biomarkers to help guide treatment decisions. Next-
generation sequencing, mutation analysis, and somatic structural
rearrangements involving genomic tumor DNA have identified dysregulation
of the cell cycle pathway to occur in HR + HER2+ and HR + HER2- cohort of
breast cancer patients, thus helping to identify potential targets for cell cycle-
related treatment options. Genomic profiling of 48 breast cancer samples has
revealed CDKN2A loss in 11% of the total population and was found to
co-occur with CCND1 amplification or CDK4 amplification which was not
observed in HR + HER2+ breast cancer.

(continued)
18 Molecular Genetics of Cancer 949

Box 18.1 (continued)

In conclusion, HER2-positive patients might be sensitive to CDK4/6
inhibitors due to the cell cycle pathway activation as evidenced by key gene
alterations. CCND1 gain was found to be most frequent in HR + HER2+
cohort (40%), followed by HR + HER2- cohort (29%), and then by TNBC
cohort (9.5%). Loss of RB1 was localized in the TNBC cohort which may
indicate resistance from CDK4/6 inhibitor therapy.

18.15 Summary

• Cancer is considered to be a group of disorders in which the normal cell cycle

regulation is lost. In fact, it is well established today that cancers usually arise
from a single cell which has dramatically undergone a series of genetic mutations.
• Histological type of cancer as established by the International Classification of
Diseases for Oncology includes hundreds of different cancer types which have
been primarily grouped into six major categories, namely, carcinoma, leukemia,
lymphoma, myeloma, sarcoma, and mixed types.
• Cancer treatment generally includes procedures such as surgery, chemical ther-
apy, radiation therapy, or therapy including drug targeting.
• There exist a limited set of “mission-critical” events propelling the tumor and its
progeny cells into uncontrolled division and invasion such as unregulated cell
proliferation, which, along with suppression of apoptosis required for support,
gives a minimal “stage” inevitable to support further neoplastic progression.
• Two initiation pathways, (a) mitochondrial (intrinsic) and (b) death receptor
(extrinsic), eventually lead to a common pathway or execution phase of apopto-
sis. Perforin/granzyme pathway is a third pathway; however a lesser known
fourth intrinsic endoplasmic reticulum pathway is also present for apoptosis.
• Telomeres are the protective structures present at the end of chromosomes and
consist of repetitive nucleotide sequences (TTAGGG in case of vertebrates) along
with linked proteins, commonly termed as shelterin.
• As regards overexpression of shelterin genes, such as TRF1, TRF2, TIN2, and, in
some cases, POT1, many cancer types have been reported as compared to the
non-cancerous tissues. Interestingly, telomere length is inversely correlated with
the high expression of these shelterin genes and TERT or even telomerase
activity.
• IARC has classified carcinogens under six groups: biological agents; arsenic,
fibers, metals, and dust; pharmaceuticals; chemical agents and related occupation;
radiation; and personal habits and indoor combustions.
• Viral oncogenes are homologues to the cellular oncogenes, for instance, c-src is
the cellular homologue of the v-src. Proto-oncogenes are the representatives of
950 B. Chuphal

normal genes of cells, which show similarity with the nucleotide or protein
sequences that are tumorigenic or have the potential of transforming.
• Cell cycle progression is a highly regulated process in normal cells to ensure that
each step is completed before proceeding to the next. There are well-known three
points: the G1/S, the G2/M, and M checkpoints in the whole cell cycle where
equilibrium between external and internal signals is checked before a cell enters
the next stage.

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 Part IV
Population Genetics
Developmental Genetics
19
Divya Vimal and Khadija Banu

19.1 Genetic Approach of Development

The foundation of the genetic approach to understand the development of an

organism is that all the information is already encoded in an organism’s DNA.
These approaches help to understand how cells achieve different fates and what
combinations of intercellular signaling and intracellular regulatory circuits generate
spatially and temporally encoded patterns along the body axis. With the advent of
enormous numbers of techniques in the field of genomics, molecular biology, and
biochemistry, it is now possible to study the process of development at each and
every step along with the role of individual gene, gene function, genetic pathways,
and genome sequences. In order to unravel the mystery of developmental biology,
integrative perspective is required where systems level approach combining compu-
tational and genomic approaches with cell and molecular biology techniques is
required. Differential pattern of gene expression along with different transcription
factors, which bind to promoters of genes thereby activating or repressing them,
regulates the development of an organism. During development, there are control
genes that regulate this cascade of genes and transcription factors.

Both authors have contributed equally.

D. Vimal (*)
Columbia University Irving Medical Center, New York, NY, USA
K. Banu
Yale University, New Haven, CT, USA

# The Author(s), under exclusive license to Springer Nature Singapore Pte 955
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_19
956 D. Vimal and K. Banu

19.1.1 Significance of Model Organism

Model organisms are non-human species that help scientists understand about a wide
array of biological processes. The results obtained from these organisms are
extrapolated to higher organisms like humans where experimentation is not possible
due to ethical or practicality constraints. This extrapolation is possible due to the
conservation of basic biological processes in all organisms during the course of
evolution ranging from single-celled organisms to most complex humans.
Researchers can control the variables that can affect the outcomes of the experiment
by using varying animal models and by regulating the living conditions. Model
organisms are generally selected based upon their unique characters like short life
cycle, less breeding time, large litter size, and availability of mutant lines. These
unique features of model organisms make them amenable to different types of
manipulation under in vitro conditions (Müller 1997). Use of model organisms in
research started with the realization that the data obtained from studies on these
organisms can be used to understand the complex mechanism behind basic physiol-
ogy and molecular mechanism of higher organisms (Fig. 19.1). Early uses of model
organisms include discovery of germ theory by Louis Pasteur, natural selection by
Charles Darwin, and genetics of heredity by Gregor Mendel. Model organisms can
be categorized into genomic, experimental, and genetic model organisms. Genomic
model organisms have a particular genomic size or the arrangement of genes in a
specific manner which can be used for reference or manipulation during experiment.
An experimental type of model organism has specific characteristics well suited for
the particular type of experiment, while genetic model organisms are particularly
useful in molecular manipulation as well as genetic studies where different mutants
are generated by genetic crosses. The genome sequencing of these organisms has
revealed the conservation of genes and cellular pathways which allows the manipu-
lation of genes and studying their effect. In addition, it helps to apply reverse
genetics to answer the functional roles of genes which can be extrapolated to the
higher organism due to biological conservation. Discovery of many homologous
genes of human diseases has allowed researchers to mimic the human disease
pathological conditions and study them in simpler experimental systems. Knowl-
edge of whole genome provides the opportunity to create genetic screen studies on a
large scale that covers all genes of an organism. Libraries of gene knockouts,
knockdowns, as well as overexpression are available which permit the study of
function of each and every gene involved in a particular process.
Frequently used model organisms include Escherichia coli, yeast (Saccharomy-
ces cerevisiae), nematode worm (Caenorhabditis elegans), fruit fly (Drosophila
melanogaster), zebrafish (Danio rerio), Western clawed frog (Xenopus tropicalis),
mouse (Mus musculus), etc. Libraries of different mutants along with a wide range of
genetic tools are commercially available owing to the fact that all model organisms
have been genetically sequenced. Model organism database (MODs) is a database
dedicated to provide all the information available for a particular model organism
like precise location of genes as well as regulatory regions present in the genome,
gene expression patterns and phenotypes of individual genes, gene ontology
annotations, pathway information, DNA/RNA/protein sequences, and stock centers
19 Developmental Genetics 957

Fig. 19.1 Model organisms. Model organisms are the key players of biological research, and
studies carried out on different organisms such as yeast, worm, fly, fish, and mouse have provided
valuable insight into the biology of developmental as well as disease pathogenesis. (Adapted from
https://biology.uiowa.edu/model-organisms)

(Table 19.1). These databases are also cross-referenced with many other useful tools
and techniques which can be applied to carry out experiments feasibly. Some
common keywords such as gene ontology are used to collect information regarding
a specific gene like its function, genomic location, protein product, and biological
process it is involved in.
For the study of development, nematode, fruit fly, frog, zebrafish, chick, and
laboratory mouse are extensively investigated. In order to elucidate the biological
mechanisms underlying any cellular process, different genetic approaches are
employed. Studying the defects in single-gene mutants and comparing it with wild
type allow to identify the new genes and their functions. This gene obtained in this
way is then mapped to its genomic location, cloned using a specific vector, and thus
identified at the molecular level. The protein product of this gene is then studied
958 D. Vimal and K. Banu

Table 19.1 List of model organism databases

Common name Scientific name Database name
Baker’s yeast Saccharomyces cerevisiae Saccharomyces Genome Database (SGD)
Fission yeast Schizosaccharomyces pombe PomBase
Clawed frog Xenopus Xenbase
Fruit fly Drosophila melanogaster FlyBase
Mouse Mus musculus Mouse Genome Informatics (MGI)
Nematode Caenorhabditis elegans WormBase
Rat Rattus norvegicus Rat Genome Database (RGD)
Social amoeba Dictyostelium discoideum dictyBase
Thale cress Arabidopsis thaliana The Arabidopsis Information Resource
(TAIR)
Zebrafish Danio rerio Zebrafish Information Network (ZFIN)
– Candida albicans CGD
– Escherichia coli EcoCyc

using different methods of cell biology and biochemistry. Applying this method on
model organisms has allowed mapping, cloning, and subsequent study of many
heritable human diseases like breast cancer and cystic fibrosis. This genetic analysis
method can be applied in two ways in order to dissect the mechanisms of action of
developmental process: forward and reverse genetics. In forward genetics the inves-
tigation starts with a mutant phenotype (organism) which then leads to the gene. The
first step in forward genetic is selecting a specific defective phenotype of interest
which is easily recognizable. Next, using these mutagenized populations, saturation
screens are carried out, and all the genes that are involved in producing a given
phenotype in an organism or species are elucidated. In this way the screens are
carried out until all the genes of a mechanism are determined and no new genes
remain. This process was first used by Eric Wieschaus and Christiane Nüsslein-
Volhard in Drosophila to study the genes involved in body plan patterning. Genetic
mapping and complementation tests are then used to identify the genes responsible
for these mutations. Effect of complete absence of gene function in null mutants
either by deletion or abnormal function of gene is also determined. Further, in order
to elucidate the sequence of function of these identified genes, double mutants are
generated. In addition, modifier genes are identified that either reduce or worsen the
phenotype of an existing mutant by employing screens for enhancer and suppressor
sequences for a secondary mutation in a sensitized genetic background. Clones of
individual gene in the form of complementary DNA (cDNA) libraries are generated
for molecular analysis through genetic mapping. cDNAs corresponding to each gene
are then sequenced, and respective sequence of each encoded protein is determined.
This sequence is then used for similarity search in different databases to distinguish
different domains and motifs which disclose the functional class of particular
protein. To determine the developmental stages during which the transcripts and
translated product of particular gene are expressed, nucleic acid probes, antibodies,
and reporter constructs are used. The last step in the process is to express the
19 Developmental Genetics 959

candidate gene in the mutants to comprehend if the encoded protein product reverts
the defective phenotype of mutant back to wild type.
In contrast to forward genetics, reverse genetic approaches are carried out from
gene sequence to a phenotype. As the genomes of most organisms are fully sequenced,
this vast information can be used to study biological processes using reverse genetics.
In higher organisms such as humans where forward genetic approaches are not
feasible, reverse genetics comes as rescue. The first step in reverse genetics process
is cloning of gene of interest which is then used to generate mutant organisms with
defective gene or abnormal expression studying its function. The gene of interest is
inactivated or silenced by targeted inactivation or permanently by producing mutant
organisms carrying null mutation called knocking out. This is achieved by inactivating
the gene of interest by the process of RNA interference where a double-stranded
antisense mRNA is injected that specifically prevents gene expression of that particu-
lar gene. The phenotypic consequences of this inactivation are then observed for
function disruption. Different strategies can be followed in different model organisms
for the inactivation of gene of interest; RNA interference using miRNA is widely used
in C. elegans, whereas in Drosophila random transposition event or mutagen
generates mutants which are then screened from a large population. In contrast, the
most common and efficient method to generate mutants is by injecting the mutant
DNA constructs into the germline cells which recombine with the host DNA.
Mutations thus obtained are subjected to a wide array of genetic analyses either
using traditional approaches or rapid large-scale approaches using microchips.
The genes identified in different model animals from both forward and reverse
genetic approaches are then applied on higher organisms like humans where
orthologous genes are isolated and characterized. Various tools and data from
Genome Project Comparison are available for this task. For example, comparative
analysis between genomic maps of human and mouse has revealed widespread
conservation of linkage called synteny; arrangement of genes (orthologous) over a
large region of DNA has been conserved similar to the last common ancestor. Much
of the understanding and the key genes in human development and physiology have
been studied in this way through the study of model organisms (Perlman 2016).
The most widely studied prokaryotic model organism is Escherichia coli owing
to the ease of growing and culturing it inexpensively. E. coli is gram-negative
bacteria with a rod shape, 2.0 μm in length and 0.5 μm in diameter inhabiting
warm-blooded animals in the gastrointestinal tract. Different strains of E. coli like
E. coli K12 are available that have been well improved and adjusted according to the
lab environment. E. coli is called a molecular biologist tool box due to the availabil-
ity of extensive molecular tools for different purposes. Discoveries made in E. coli
have contributed a lot to the basic understanding or cellular biological processes and
has also helped scientists to win well-deserved Nobel Prizes. Major keystone
discoveries like DNA replication, genetic code, genetic regulation, gene organiza-
tion, basis of mutation, evolution of organisms, and development of genetically
modified organism have been carried out in E. coli. Manipulation of E. coli genome
plays the most significant role in biotechnology.
Major advantages that E. coli provides as an ideal model organism include fast
growth in relatively cheap chemically defined media; industrial scalability;
960 D. Vimal and K. Banu

Fig. 19.2 E. coli. It is an ideal model organism for studies related to various aspects of molecular
biology and biochemistry due to its simplicity. Any experiment utilizing E. coli as a model
organism includes three stages, design, build, and test; firstly a design-specific strain is selected
followed by suitable genomic modifications to produce a mutant strain which is then tested using
various techniques. (Adapted from Adamczyk and Reed 2017)

enormous number of tools for genetic alterations; expansive knowledge of its

genome; wide information of its transcriptomics, proteomics, as well as
metabolomics; and lastly, availability of various strains that are nonpathogenic
(biosafety-1). The most important feature of E. coli is that unlike multicellular
model organism especially mammalian models, there are no ethical concerns
(Fig. 19.2). Small size allows their usage in large numbers in a confined laboratory
space. The doubling time of E. coli is about 20 min resulting in doubling of its
population which helps researchers to study multiple generations within a short
period of time. Another important advantage is that E. coli can endure a wide
range of environmental conditions and survive under in vitro conditions. Culture
19 Developmental Genetics 961

media of different compositions depending upon the experimental conditions and

consisting of various components and nutrients are sufficient for the growth of
E. coli. The most common ingredients of any culture medium include carbon source
generally glucose, nitrogen source like yeast extract, various salts, and different
organic compounds. Some E. coli strains are well known for serious problems and
diseases in humans like fever, diarrhea, and abdominal pain. E coli is a common
inhabitant of the intestine constituting the gut flora and is essential for several
functions like production of vitamin K2 and restricting the growth of harmful
bacteria. In 1997, the first report of E. coli genome sequencing of a lab strain,
K-12 derivative MG1655, was published. E. coli genome consists of approximately
4.6 million base pairs consisting of ~4000 genes with 7 rRNA and 86 tRNA genes,
respectively.
Saccharomyces cerevisiae, also called as budding yeast, is the organism of choice
for eukaryotic studies as it provides the basic eukaryotic features. Similar to any
eukaryotic system, yeast cells have a nucleus enclosed by nuclear membrane
containing its genome which is ~12.1 million base pairs organized in
16 chromosomes and consists of ~6500 genes. In addition, it also contains all the
subcellular organelles suspended in cytoplasm. It is single-celled yeast commonly
used in the bread-making industry with a genome size of 12,157,105 base pairs in
length containing 6692 genes. It is a free-living organism residing primarily on
substances having sugar like fruits and flowers. Yeast cells can survive a variety of
environmental conditions ranging from freezing temperatures to ~55
C with
proliferative temperature of 15–35
C. Yeast can grow in varying pH conditions
ranging from pH 2.8 to 8.0, and after almost complete drying it can regenerate and
thrive on any sugar-containing medium with osmotic pressure as high as 3 M sugar
concentration and ferment it. The sequencing of Saccharomyces cerevisiae genome
was completed and published in 1996 and was among the first eukaryotic organisms
to be completely sequenced. Approximately 20% genes of human genome involved
in various diseases have functional equivalents in yeast suggesting similar basic
cellular machinery. S. cerevisiae offers advantages in various aspects such as small
size, short generation time, easy accessibility, manipulation tools, known DNA
sequences and regulatory regions, and conserved molecular mechanisms with higher
organisms along with economic use. S. cerevisiae and S. pombe are the two most
studied Saccharomyces species and provide excellent systems for studies related to
DNA damage and its repair.
The life cycle of S. cerevisiae contains both haploid and diploid vegetative stages
which allow generating mutant strains in haploid condition and studying its effect
directly (Fig. 19.3). Furthermore, yeast offers an important tool in the form of
complementation test used to assign a particular mutation to gene by studying the
diploid organism with heterozygous condition for a mutation. In contrast, by study-
ing the haploid progeny during meiosis, genetic relationship can be understood and
used for gene mapping and to define the relationship between different genes on the
basis of their function. The most valuable tool that yeast offer is the incredibly
efficient systems for homologous recombination by which two DNA molecules
having similar sequences or transformed DNA recombine. The recombination
962 D. Vimal and K. Banu

Fig. 19.3 Life cycle of Saccharomyces cerevisiae. Yeast is an extensively used eukaryotic model
organisms, and studying homologs in yeast leads to the discovery of many vital proteins like those
involved in cell cycle and signaling which are of utmost importance in human biology. Both diploid
and haploid yeast cells undergo mitosis through budding producing daughter cells; however,
diploid cells sometimes divide by meiosis into four haploid spores. (Adapted from Duina et al.
2014)

between DNA sequences in this way is highly precise and efficient and allows
researchers to alter the genome with ease. The altered DNA thus generated is then
used for transforming the yeast cells which then locate the precise location for
incorporation based on the sequence similarity of few bases and bring out the
predicted genetic change. S. cerevisiae has been extensively studied in order to
understand the biology of aging and has led to the discovery of many genes
involved. In corroboration, yeast cells provide excellent system for aging studies
as it exhibits both chronological and replicative aging. In chronological aging the
amount of time that a cell has survived is studied, while in replicative aging the
number of progeny cells generated by a parent cell before senescence is generally
studied. Yeast cells undergo replicative ageing where the cells divide finitely (30–40
division) by mitosis before the cell dies which is analogous to aging profile of human
stem cells. S. cerevisiae is industrially manufactured and used in several ways, for
example, as a probiotic, or used in numerous digestive tract-related problems.
Another important advantage of using yeast is the microarray analysis which can
be used to determine the expression of multiple genes at the same time. Microarray
analysis combined with chromatin immunoprecipitation (ChIP sequencing) reveals
19 Developmental Genetics 963

the binding of transcription factors to specific sites. A complete set of more than
6000 deletion mutants (yeast deletion analysis) is available for research; the pheno-
typic analysis of these mutants can be done in high throughput to study the genetic
networks. Yeast cells can be used to test the effect of new drugs as it has many genes
common with humans. Mutated yeast cells carrying diseases of human gene can be
used to study the effect of numerous drugs on their capability to rescue the normal
function. Two mismatch repair system (MMR) genes of yeast that are almost
identical in terms of sequence as well as function with humans are mutL homolog
(Mlh1) and mutS homolog (Msh2). Both of these genes are well studied with respect
to one of the most common types of cancer in humans, hereditary non-polyposis
colorectal cancer (HNPCC). Mutations in either of the genes cause HPNCC, and
their study in yeast has helped gain insight in their role in cancer.
The Saccharomyces Genome Database (SGD) is a database consisting of all the
information on the biology of budding yeast Saccharomyces cerevisiae. In addition,
it provides various searching and analyzing tools that can be used to carry out
comparative studies with higher organisms at genomic as well as phenotypic level.
S. cerevisiae has been used to study the role of α-synuclein during Parkinson’s
disease, dementia, and other neurodegenerative diseases. Different drugs or test
compounds can be used to test their potential in reversing the adverse effects of
α-synuclein in nervous system and thereby treating Parkinson’s disease.
C. elegans is a nonparasitic, free-living soil nematode of ~1.3 mm in length
belonging to the family of roundworms and can be found at various places. The use
of C. elegans in research as a model system started since the nineteenth century with
the completion of its genome sequencing published in 1998 (Marsh and May 2012).
Earliest studies on C. elegans were carried out by Sydney Brenner due to various
features like short duration of life cycle, large number of offspring, and ease of
genetic manipulation. C. elegans has contributed a lot to the fundamental aspects of
developmental and neuronal biology (Fig. 19.4). C. elegans development starts with
an egg which then molts to L1 larvae followed by successive molts to L2, L3, and L4

Fig. 19.4 Anatomy of C. elegans. Lateral view of a hermaphrodite (a) and male (b) showing nerve
ring, vulva, gonads, intestine, and pharynx. (Adapted from Corsi et al. 2015)
964 D. Vimal and K. Banu

Fig. 19.5 Developmental stages of the nematode, Caenorhabditis elegans. Development of

C. elegans starts from an egg which then develops through four larval stages, L1–L4, to the
reproductive adult animal. (Adapted from Pandey 2014)

larvae and finally to an adult worm; this process takes about 3.5 days at 20
C.
C. elegans can be grown in large numbers inexpensively on nutrient plates which
contain bacteria as food. C. elegans is a very small organism having high fecundity
(~1000 eggs every day) but short life span (~2–3 weeks) which makes it very
feasible for the developmental studies. Despite their short life span, under unfavor-
able conditions, worms adapt a unique developmental stage producing dauer larvae
which resist and survive extreme unfavorable conditions like drying and absence of
food even up to many months (Fig. 19.5). C. elegans cultures can be stored for
indefinite time by freezing it and when needed can be defrosted, revived, and used.
C. elegans is a diploid organism having five pairs of autosomal (1–5) and one pair
of sex chromosomes. C. elegans can exist in two sexes, hermaphrodite (XX) which
can self-fertilize producing both sperms and oocytes, and a male (X0) which is
formed due to the spontaneous loss of X chromosome and is present at a very low
frequency (~0.2%). Hermaphrodites produce more than 300 progeny by asexual
reproduction and are identical to each other, thereby providing an invaluable tool for
genetic analysis. Hermaphrodites when exposed to heat shock give rise to males
which can then mate with hermaphrodites producing cross-progeny. Another
fascinating characteristic of nematode is that the complete development of fertilized
egg to the adult worm along with cell lineage can be easily studied due to the simple
19 Developmental Genetics 965

body structure and limited number of somatic cells (1031 in male and 959 in
hermaphrodite). The nervous system of C. elegans is relatively simple containing
~300 neurons in adult, thereby providing an excellent system for neurological
studies as compared to much complex nervous system in other organisms. In
C. elegans, most of the nerve cells are present in a large nerve ring, ventral and
dorsal nerve cord, and a complex head sensory system. Additionally, well-known
signaling components and neurotransmitters that function in mammalian nervous
system can be seen in C. elegans nervous system. The genome size of C. elegans is
~100 million base pairs consisting of ~20,000 genes which make it feasible to both
forward and reverse genetic approaches. In addition, C. elegans has ~43% of gene
that have human homologs, including numerous disease genes. The transparent body
makes studying the behavior of individual cells very easy throughout its develop-
ment. In addition, C. elegans is an excellent system to study the expression patterns
of genes under in vivo condition, and determining the localization of proteins within
the cell is very feasible. The proteins can be tagged with various reporter enzymes
like β-galactosidase or constructs like green fluorescence protein (GFP) either by
injecting or bombarding the germ cells. The anatomy and development of C. elegans
can be examined easily under a microscope, and each cell can be traced back to the
embryo due to specific and specialized pattern of development. One of the most
important tools that were discovered in C. elegans is RNA interference (RNAi)
which can be used to generate thousands of mutants by gene silencing through
double-stranded RNA (dsRNA) at transcriptional and post-transcriptional levels in a
sequence-specific manner. There are various ways of delivering double-stranded
RNA (dsRNA): injection of dsRNA into the worm, feeding the nematode bacterial
food which produces dsRNA or directly soaking the worms into the dsRNA solu-
tion, as well as production of dsRNA using transgenic promoters in vivo.
C. elegans shares important molecular signaling pathways regulating its develop-
ment with humans (Leung et al. 2008). Mutants with single-gene mutations can be
easily produced to study the functions of specific genes. C. elegans genome has
functional equivalents of many genes of humans that allow producing different
mutants and disease models with single-gene mutations and studying their function.
Different C. elegans mutant models are available to study various human diseases
including cardiovascular diseases, neurological diseases, and renal disorders.
Thousands of potential drugs for the treatment of several severe diseases can be
screened using C. elegans mutants. By studying the mechanism behind apoptosis in
C. elegans, the effects of aging can be offset in humans. In addition, studies in
nematodes regarding the molecular mechanisms and hallmark gene that are gener-
ally found to be mutated in several diseases can provide important insights for curing
these diseases.
Another important widely used model organism is zebrafish (Danio rerio) which
is a freshwater fish of the Cyprinidae family. Zebrafish is one of the ideal model
organisms for studying vertebrate development. Zebrafish derives its name due to
the presence of zebra-like horizontal stripes on the side of the body which are five in
number and of blue color. Zebrafish is used as an animal model due to various
advantages including its regenerative abilities, small size and robustness, rich
966 D. Vimal and K. Banu

Fig. 19.6 Life cycle of zebrafish (Danio rerio). Zebrafish is one of the best studied vertebrate
animal models that has contributed much of our understanding in the field of developmental
biology, mis-regulation of molecular mechanisms in cancer development, toxicological studies,
drug discovery, and screening. (Adapted from Willemsen et al. 2011)

genetics, and rapid and transparent development. The zebrafish is ~2.5 cm to 4 cm

long and are cheaper to maintain in a laboratory (Fig. 19.6). Where most fishes lay
eggs in the dark, zebrafish mates in daylight producing a large number of progeny
every week providing excellent system for embryological studies. In addition,
zebrafish embryo is an outstanding model system as it develops outside the adult
worm and can be observed as well as altered at all stages of development. The
embryos develop and grow at an exceptionally rapid rate and are almost transparent
allowing the examination of internal structure development. Due to the transparent
body, specific cells and their components can be tagged with fluorescent proteins,
and the fate of the cells can be studied in the presence or absence of different genes.
Zebrafish genome share 70 percent of genes with humans while containing 84 per-
cent of genes that are known to be associated with various human diseases. Being a
vertebrate organism, major organs and tissues similar to higher organisms like the
kidney, eyes, muscle, and blood can be seen in zebrafish. One unique feature of
zebrafish is its episodic memory associated with explicit memory systems where
individuals can remember the context associated with respective locations,
occasions, and objects. Another interesting feature of zebrafish is the capability to
19 Developmental Genetics 967

repair heart muscle in a matter of weeks which can be useful for patients with
cardiovascular diseases. Completely sequenced zebrafish genome was published in
2001 and was carried out by the Sanger Institute, UK, which provided invaluable
information for the generation of numerous mutant lines with single- or multiple-
gene mutations.
Zebrafish is one of the most popular model systems for carrying out the behav-
ioral studies and has helped researchers to understand the complex mechanism
behind learning, sleep wake cycle, and depression. Recent advances in neuronal
studies paired with behavioral analysis provide the understanding of the role of
neural regulatory pathways in behavioral changes. Zebrafish is also being studied as
a regenerative model as it can either restore or replace the damaged cells or tissues
including major organs like the spinal cord, heart, as well as appendages. The repair
can occur by either of the two mechanisms: dedifferentiation, proliferation of the
neighboring cells, and replacement of the damaged cells or through the activity of
stem cells. By unraveling the mechanisms and regulatory pathways behind the
regeneration capacity of zebrafish, the knowledge might be applied to the mammals
as well.
Being a fish, zebrafish can detect toxins present in the water and hence can be
used as a predictive model for screening of toxic compounds to study the adverse
effects of xenobiotics. In addition, zebrafish is widely used for studying a variety of
diseases including cancer, kidney disease, diabetes, pigment cell disorders, aging-
related disorders, nervous system-associated diseases, epilepsy, blood disorders, as
well as addiction. Furthermore, zebrafish models of disease are used to carrying out
pharmacological screening on a large scale by mixing the drug with water.
Fruit fly, Drosophila melanogaster, is one of the most extensively studied model
organisms for development, behavior, neurobiology, genetics, diseases, and
molecular-related studies. Flies have been used for basic research for more than a
hundred years. Earliest uses of flies date back when Thomas Hunt Morgan carried
out a series of experiments on the eye color and its gene providing the chromosomal
theory of inheritance for which he won the Nobel Prize in 1933. This was followed
by many more keystone discoveries in the field of mutations, genetic control of
embryonic development, immunology, olfactory system, and molecular mechanisms
of circadian rhythms which helped scientists bag five more Nobel Prizes.
Drosophila has 60% of the counterpart genes of the human diseases which allows
the scientists to mutate, amplify, or delete diverse set of human disease-related genes
and study their function in different scenarios. In addition, other attributes like short
life cycle with high fecundity, ease of maintaining the cultures, manageable number
of chromosomes, small genome size, and giant salivary gland chromosomes (poly-
tene chromosomes) allow carrying out research with ease. The less number of
chromosomes is an important feature of the fly that allows easy manipulation during
genetic studies. Discoveries made using this feature of flies has helped to understand
the mechanism behind transmission of genetic material from one generation to the
next. Another major benefit of using Drosophila in research is that there are no
ethical issues which are a major problem with mammalian models such as monkeys,
rats, dogs, cats, and pigs. Behavioral studies such as eating, mating, and sleeping can
968 D. Vimal and K. Banu

also be done with ease in flies which allows observing the possible effect of genetic
manipulation upon behavior and can be applied to humans also.
The fruit fly is ~3 mm in length; their small size allows raising them in a large
number at once. Fruit flies are cultured in quarter-pint bottles using ripened banana
or a maize and agar mixture as food. A vast range of mutant stocks (~80,000
Drosophila stock variants) are commercially available, and numerous experiments
using only a few flies can be done in a limited lab space. The largest public collection
of Drosophila lines is available at the Bloomington Drosophila Stock Center
(BDSC), Indiana University, USA. Other major centers like RNAi library, Vienna
Drosophila RNAi Center (VDRC), and the TRIP-RNAi Harvard collections from
Japan, China, and Europe are all made available through BDSC. Another major
stock center is at the Drosophila Genomics Resource Center (DGRC) which
provides cDNAs and vectors.
A large number of embryos, larvae, or adults can be harvested at a time, and the
material can be frozen in liquid nitrogen which can be later used to extract DNA,
RNA, enzymes, or proteins. The completed sequence of Drosophila melanogaster
genome was published in 2000, and a year later comparative studies with human
genome were done which unraveled various aspects of similarity between the two
organisms on both genetic and molecular levels establishing fruit fly as an excellent
model organism. The fruit fly has four pairs of chromosomes: chromosome 1 is the
sex chromosome (two X chromosomes in females while one X and one Y in males)
and chromosomes 2–4 are autosomes (non-sex chromosomes). The smallest chro-
mosome is the fourth and is called dot chromosome which represents only 2% of the
total genome. Fly genome contains 132 million base pairs containing ~15,000 genes
on 4 chromosomes as compared to the 3.2 billion base pairs containing ~22,000
genes on 23 chromosomes of humans. The Drosophila genome is 60% homologous
to humans with ~75% of the homolog genes associated with various human diseases.
Drosophila is a holometabolous insect which undergoes many body plan changes
throughout its development. Life cycle of a Drosophila completes in about
10–12 days at room temperature (25
C) with the adult female fruit fly laying
~750–1500 eggs in her lifetime. After the fertilization of egg (~0.5 mm in length),
embryo emerges in ~24 h (Fig. 19.7) and undergoes successive molts to become the
first, second, and third instar larva which is a voracious eater consuming food in a
large amount leading to rapid development which is then followed by a quiescent
pupal stage. During the process of development through different stages, fly
undergoes vivid reorganization of the body plan (metamorphosis) followed by the
emergence of adult fly. Interaction between the two important hormones,
prothoracicotropic and ecdysone, along with Drosophila insulin-like peptides
(Dilps), ensures the proper development of the fly.
Arabidopsis thaliana belonging to Brassicaceae family is also called as rockcress
or thale cress, is a small plant with white flowers, and is a very popular model
organism for plant studies. It is a member of the mustard (which also includes
cabbage and radish) (Fig. 19.8). Arabidopsis utilizes basic nutrients like water and
few minerals to carry out the process of photosynthesis in the presence of light and
complete its life cycle starting from germination to mature seed which takes
19 Developmental Genetics 969

Fig. 19.7 Life cycle of fruit fly (Drosophila melanogaster). Being a holometabolous insect,
Drosophila undergoes complete metamorphosis. The life cycle of fruit fly consists of an egg that
hatches into first instar larva which successively molts into second and third instar larva which
further molts into dormant pupa from which the adult fly eclosed. The development takes ~10 days
and varies depending upon the temperature with rapid development at higher temperatures.
(Adapted from Ong et al. 2014)

~6 weeks. Other important features of this model organism includes abundant seed
production which can be easily cultivated in a small space, small genome size
~114.5 Mb which is well characterized, availability of extensive genetic and physi-
cal maps of all five chromosomes, easy transformation using Agrobacterium
tumefaciens as vector, and availability of numerous mutants and wide range of
genetic tools. The genome of A. thaliana is completely sequenced and was published
in the year 2000. Diploid genome of the plant makes analysis of recessive mutations
easy.
Flower development studies in A. thaliana have provided valuable insights on the
mechanisms involved. Flower in Arabidopsis contains two whorls: outer whorl
consisting of four sepals and inner whorl containing four petals and six stamens
and a carpel in the center. Two scientists, E. Coen and E. Meyerowitz, studied
homeotic genes in A. thaliana and found that mutations in these genes result in the
change of one organ to another which formed the basis of formulation of the ABC
970 D. Vimal and K. Banu

Fig. 19.8 A. thaliana.

Arabidopsis is one of the most
popular plant model
organisms and is widely
studied in the field of
molecular biology and
development. (Adapted from
https://nl.wikipedia.org/wiki/
Bestand:Zandraket.jpg)

model of flower development which is applicable to most flowering plants (Murai

2013). ABC model of flower development states that the genes responsible for
defining floral development can be categorized into class A genes responsible for
sepals and petals, class B genes for petals and stamens, and class C genes regulating
stamen and carpel identity. The transcription factors encoded by these genes regulate
the specification of individual region into the respective floral organ. Moreover
A. thaliana have also been studied extensively to understand the genetics behind
leaf morphogenesis. In addition A. thaliana is the system of choice to understand
various aspects of development and flowering, genome organization, as well as gene
regulation.
Although all the abovementioned organisms are excellent model organisms to
gain insight into various cellular processes at molecular level, certain characteristics
like placentation, intrauterine development, lactation, and aspects of immunology
and carcinogenesis are restricted to mammals and hence demand a mammalian
model. Being a mammal, intricate processes like aging and immunity can be studied
at the molecular level. Their endocrine systems are very similar to humans with
secreted hormones affecting a wide range of processes. The mice is among one of the
first mammalian species to be completely sequenced. The mouse genome has a
19 Developmental Genetics 971

Fig. 19.9 Mouse as a mammalian model organism. Mouse is one of the most widely used
mammalian model systems to understand the basic aspects of cellular and molecular machinery.
Many groundbreaking discoveries have been done in this model system which unraveled
mechanisms behind disease susceptibility and progression and has also helped to develop treatment
for it. (Adapted from http://rotarynoidabloodbank.com/en/blog/2016-05/why-use-mice-medical-
research.html)

similarity of ~85% with the human genome with a size of ~2.5 Gbp. In order to
understand different aspects of biomedical research, various mammalian model
organisms have been used, but among all of them, the mouse (Mus musculus) is
the most flexible and extensively studied mammalian model organism (Fig. 19.9).
Advantages of using mice include small size, low cost of maintenance, short
generation time of around 10 weeks, prolific breeding, large litter size, and repro-
ductive cycles that can be easily monitored especially during pregnancies (Monica
et al. 2016). One of the most common problems with many model organisms is that
in order to study a disease, it needs to be induced using artificial means; however,
using mice model solves this problem as it develops many diseases like cancer,
diabetes, and hypertension naturally. Studies from several years on different mouse
models have culminated in our understanding of complex mechanisms underlying
many grave diseases and the effectiveness of candidate drugs on these diseases as
well as predicted the patient response against these drugs. Humanized mice models
are being widely used in the field of biomedical research with the aim to minimize
the risk in human therapeutics. Humanized mice models express human gene or
contain human cells and tissues and are used to understand the involvement of that
particular gene, cell, or tissue in disease development and biological response. Many
breakthrough discoveries that have helped scientists win many Nobel Prizes have
been done on mice models. Studies in mouse models have led to the discovery of
vitamin K, vaccine development for various diseases, monoclonal antibody technol-
ogy, and tuberculosis vaccine. Studies on cancer mouse models provide microscopic
details about the process of metastasis and potential treatments. In the field of
medical science, studies on various severe diseases like blood cancer have provided
valuable insights which have helped to produce a treatment. In addition, studies on
972 D. Vimal and K. Banu

mouse model of cystic fibrosis have facilitated to create a gene transfer protocol
which is being used to treat the condition. Another example of use of mice models in
disease includes development of meningitis Hib (Haemophilus influenzae type b)
vaccination. Further, drug tamoxifen is a widely used drug for the treatment of and
prevention against breast cancer which was tested in mice to study its role in
blocking hormone action. In strike contrast to various other model systems, mice
provide an in vivo system to study the disease development as well as to study the
response to different drugs. Additionally, a wide diversity of commercial strains are
available with different exclusive features from which a researcher can select from,
for example, the CBA mouse which is an inbred strain made from a cross between
Bagg albino female and DBA male. CBA mouse is selected for its characteristic
feature of low incidence of mammary tumors (breast cancer). Another specific mice
strain is BALB/c nude mouse which lacks a thymus and is therefore immunode-
ficient. Mdx mice models lack mature dystrophin muscle protein and are used to
study Duchenne muscular dystrophy. To develop new treatments for autoimmunity,
non-obese diabetic (or NOD) mice models are generally used. Such function-specific
mouse strains are produced by inbreeding of different mouse models and are used to
study a specific disease. Furthermore, several genomic modification tools like
CRISPR gene editing and Cre/lox system allow adding or removing a specific
gene in a gene casket, thereby producing disease in the model system which helps
to study its progression and developing new ways to treat it.
Apart from all the advantages of using mice as a model organism, there are some
disadvantages associated with it, for instance, these organisms are complex animals
with a genome as large as humans. Another point is that the embryos develop in
utero hidden from view restricting access to study the developmental process. In
addition, embryo culture is very difficult and limited. Moreover, the generation
interval is long, ~3 months. It is difficult to search for the key genes involved in
many important cellular processes and find genes through mutational screens.
Despite all these difficulties, mouse is still an important model system as it allows
researchers to study the development in a mammal unraveling many aspects of
human development.

19.1.2 Analysis of Developmental Mechanism

Development is an intricate process leading to the remodeling of cellular differenti-

ation where cells respond to different signaling pathways allowing specific localiza-
tion of specialized cell subtypes in developing embryo and thus in adult. During the
differentiation process, group of cells commits to a particular fate to differentiate into
a given cell type; however the commitment is reversible. In the absence of retrogres-
sive factors, inductive signals evoke commitment to convert into determination
which is irreversible sealing the cell fate. Cells acquire specific behaviors according
to distinct fates which induce growth and morphological changes in embryo. Studies
on developmental biology are based upon the fact that most of the complex cellular
processes along with the key molecular regulators are conserved throughout the
19 Developmental Genetics 973

animal kingdom. Completion of whole genome sequences and the advent of molec-
ular tools over this time have allowed evolutionary developmental biologists to
prove this by comparing the genes and their functions involved in the development
of one organism to another. In order to dissect and gain an deep insight into the
developmental systems, researchers utilize various tools like genetic maps, forward
and reverse genetic screening, cell differentiation, etc.
During the development of an adult from zygote, the journey of any specific cell
starting from origin to destination can be tracked, and a map can be made called the
fate map. An extreme example of fate map comes from the completion of C. elegans
cell lineage studies. This is achieved by asymmetric cell divisions in the daughter
cells which obtain different fates from one another, either by segregation of some
cytoplasmic determinants or through signaling pathways. Studies on the green algae,
Volvox, has provided much of the information on evolution of multicellularity and
asymmetric divisions. Further, signals from one cell or tissue induce another cell or
tissue to affect its developmental fate. One model organism that has been extensively
studied to understand this process is Astyanax mexicanus, which is a tropical
freshwater fish. It is an inhabitant of dark caves which gave rise to completely
different varieties or morphs that lack eyes and have several other unique physical,
behavioral, and physiological changes. In the first few days of development, the
expression of eye genes is inhibited through epigenesist resulting in loss of eye. The
visual system of any organisms utilizes a big portion of energy; therefore the loss of
eye adaptation to dark offers an energy advantage to these organisms. In the absence
of visual system, these organisms depend on sucking instead in order to sense their
environment. It was found that these cavefish have altered Pax6 expression and
higher levels of a DNA methyltransferase called DNMT3B in their developing eyes.
It was assumed that this occurred during the course of evolution of cavefish, leading
to epigenetic suppression of eye development genes. Small genetic changes in a
subset of genes may have a large impact in the evolution of organisms. A cell or
tissue is also highly affected by extrinsic factors or signals often received from the
neighboring cells or tissue which is called competence. Typical example of compe-
tence can be seen in nematodes where the position of the vulva is highly variable
which results due to the change in size of equivalence group during evolution.
Another feature of developmental process is genetic redundancy where multiple
set of genes are responsible for a single function and silencing of any one of them has
no or negligible effect on the respective phenotype. One such example is the function
of bicoid, hunchback, and orthodenticle proteins of insects which are responsible for
establishing the anterior-posterior axis. Moreover, during the development different
mRNA and proteins make a blueprint that a cell can detect and use it for specification
and pattern formation. This can also be observed during the development of insect is
changes in the Hox gene, Ultrabithorax, Ubx, results in altered morphology of insect
wings and in restricting the segments that can bear limbs. Determination is the
capacity of a cell to acquire different fates, for example, a region of the embryo
became committed to form a particular part of the body at a particular stage of
development. Although the zygote is totipotent and has the capacity to make all the
cells and tissues of a future organism, it is considered a highly polarized cell. In most
974 D. Vimal and K. Banu

of the organisms like insects, nematodes, and amphibians, the polarity of the zygote
and cell fate potential of blastomeres is already highly restricted at the two-cell stage.
In contrast, the mammalian embryo does not show polarity as the individual
blastomeres retain totipotency through the four-cell stage such that an isolated
blastomere is capable of forming a viable embryo. Additionally, blastomeres can
be removed or added up to the eight-cell stage without affecting the viability of
embryo. The last step in developmental process is lateral signaling by which
neighboring cells inhibit each other from developing in a similar way. Excellent
example of lateral inhibition is changes in bristle patterning in Diptera where nascent
bristle precursors prevent neighboring cells from developing into bristles through
achete-scute complex (AS-C) genes which regulate Notch lateral signaling. More-
over, in order to execute developmental processes, different genetic networks func-
tion as maps that represent interactions between discrete genes and modules.

19.1.3 Developmental Genetics: Overview

Developmental genetics explains how the genes control growth and development of
an organism throughout its life cycle. A newly fertilized egg cell has all the
necessary genes that carry information needed to transform it from a single cell
into an embryo and then an adult. During the course of development, single cell
transforms itself into an adult organism by developing complex structures. A vast
variety of life forms and the intricate details of adult body plans arise from a
unicellular stage through the process of embryonic development. This includes
three key processes: cell division, cell differentiation, and morphogenesis where
cells divide to produce more cells and change into different types of cell to do
specific jobs in the body and groups of cells produce different structures of the
organism, respectively. After the determination of cell into a specific type, different
sets of genes are activated in the cells which are responsible for producing particular
types of proteins which in turn carry out specific functions. There are some 350 dif-
ferent types of cells in an adult human being all express different sets of proteins.
Some genes are activated (switched on), while others are inactivated (switched off)
through DNA regulatory mechanisms. Similar cells express different sets of protein
which differentiates them into different cell types and is regulated at the level of gene
transcription, nuclear RNA processing, mRNA translation, and protein modification.
For example, a nerve cell only produces the proteins required for the nervous
system-related functions. There are master control genes or regulatory genes that
produce proteins which in turn control the activity of other genes. The development
of body plans in all animals is controlled by a remarkably small number of genes
which are virtually identical in all animals. For example, homeotic or homeobox
genes are responsible for the basic body plan of the embryo in most of the organisms
by regulating different sets of genes, thereby differentiating specific body
structures (Lawrence and Morata 1994). In addition to body plan, a common axial
patterning system and other general architectural features in both vertebrates and
invertebrates also appear to be controlled by common genetic mechanisms. These
19 Developmental Genetics 975

regulatory genes produce proteins called transcription factors that bind to specific
DNA sequences called promoter and enhancer regions making a gene to switch on or
off depending upon the requirement. In case of eukaryotes, RNA polymerase in
addition with basal transcription factors binds to promoter sequences of the genes,
thereby initiating transcription. Moreover these genes contain enhancer sequences
that regulate their transcription in time and space. The transcription factor binds to
the promoter of its own gene in order to maintain its activation. In order to inhibit the
expression of nonspecific genes in any region of organism, enhancer sequences play
an important role during transcription. Transcription factors act in different ways to
regulate RNA synthesis. These transcription factors act in different ways; some
stabilize the binding of RNA polymerase to DNA, some disrupt nucleosomes,
while others increase the efficiency of transcription. One common mode of suppres-
sion of genes is through methylation of promoter and enhancer regions of genes.
Difference in methylation pattern results in a process called genomic imprinting
where the same gene transmitted through sperm and egg is expressed differentially.
Different RNAs are selected which are transported to the cytoplasm from the
nucleus, while others remain in the nucleus. Moreover, RNA splicing and combina-
tion of different exons and introns create a family of related proteins that function
differently. During oogenesis, many mRNAs are localized at certain regions of the
oocytes regulated by 30 untranslated region of the mRNA. Translation of these
mRNAs is timely regulated and is carried out only at a specific time during the
development of oocyte either by inactivation of inhibitory proteins or by mRNA
polyadenylation.

19.2 Genetic Control of Eye Development

The eye is one of the most fascinating organs of the living organisms and has been
widely studied as well. The eye is an important organ for the functioning of the body
but not essential for the survival of the organism, thereby allowing the study of lethal
genotypes. Eye formation is a complex process which includes eye territory specifi-
cation, polarity axis patterning, and regional specifications which are regulated by
multiple genes. Further, morphogenetic movements, cell proliferation, and cell
differentiation occur in the prospective eye, and lastly neural connections are
established allowing visual function. Specification of eye organ primordia is a
well-conserved developmental patterning pathway where mechanisms underlying
the formation of eyes and photoreceptor cells in different animals exhibit striking
similarity as shown by a series of experiments.
Studies on two mutations, aniridia defect in humans and small eye (Sey) mutation
in mice and rats, played key role in unraveling the molecular pathways underlying
the eye development. In aniridia defect in humans, the eyes become reduced in size
and iris is absent in them, while in small eye mutation, there is complete absence of
eyes and the mice dies in utero. Genetic and molecular analysis of both aniridia and
the small eye mutants revealed problems in the same gene, Pax6, which belongs to
the paired box/homeodomain family of transcriptional regulators. Pax6 protein is
976 D. Vimal and K. Banu

abundantly expressed in the eye cells from the early stages (optic sulcus) to later
stages (eye vesicle, lens, retina, and finally cornea) of eye morphogenesis. Two
genes, eyeless (ey) and twin of eyeless (toy), in Drosophila encode proteins that are
homologs of Pax6. Both ey and toy are expressed at high levels in the eye primordial
cells that form the photoreceptor cells of Drosophila eye. Heterozygous mutations in
ey lead to the reduction or complete loss of compound eyes, whereas homozygous
mutations are lethal. Ectopic expression of mouse Pax6 gene in various tissues led to
the formation of small ectopic eyes on the wings, legs, and antennae of Drosophila.
Pax6 and eyeless genes are considered as master control genes for eye morphogene-
sis as homologous genes are present in vertebrates, ascidians (sea squirts), insects,
cephalopods (squids and octopus), and nematodes (worms), throughout the meta-
zoan necessary for promoting eye development.
Visual system of flies has been studied extensively and represents an excellent
model for studying the development, differentiation, and specification process of the
retina and photoreceptors. Drosophila eyes develop from the posterior part of the
monolayer epithelium called eye imaginal disc. During late larval and early pupal
stages, neural as well as non-neural cell types in the retina are specified. Drosophila
has compound eyes with each of them consisting of ~700 hexagonal unit eyes called
ommatidia. One ommatidium comprises of light-sensing neural cells [photoreceptors
(PRs)], 12 supporting non-neuronal cells (cone and pigment cells), and
interommatidial cells (tertiary pigment cells and bristle complexes). The ommatidial
structure is very precisely repetitive in normal individuals; therefore even the subtle
abnormalities may be recognized. Rhabdomeres which are the microvillar structures
extend toward the center of the ommatidium and bind to the six of the eight PRs
(R1–R6) which are called outer PRs (Fig. 19.10). Rhabdomeres of each PR consist
of the light-sensitive pigments called Rhodopsin (Rh). Outer PRs are arranged in a
trapezoid shape and accompany the two other PRs, R7 and R8, also called as inner
PRs and are positioned in the center of the ommatidium with R7 on top of R8. Outer
PRs (R1–R6) express the broad-spectrum Rh1 and are thought to be functionally
similar to vertebrate rod cells required for dim-light vision and motion detection. In
contrast, inner PRs (R7 and R8) are considered similar to the vertebrate cone cells
and function in mediating color vision and perception. Inner PR, R7, expresses the
ultraviolet-sensitive Rh3 and Rh4, while R8 cells express either the blue-sensitive
Rh5 or the green-sensitive Rh6. The expression of Rhs in both R7 and R8 cells is
coupled in such a way that the expression of Rh3 in R7 is coupled to the expression
of Rh5 in the R8, whereas Rh4 expression in the R7 cell is coupled to Rh6
expression in R8 cells. The ommatidia coupling of Rh3/Rh5 is called pale, while
that of Rh4/Rh6 is called yellow. The distribution of pale to yellow is highly
conserved among different fly species in a 30:70% ratio in the retina (Fig. 19.10).
Eye formation in Drosophila occurs at the posterior margin of the eye imaginal
disc in response to a differentiation wave called morphogenetic furrow (MF) which
proceeds from posterior to anterior end (Fig. 19.11). MF initiates and progresses
with the help of secreted molecules and directs cell-cell signaling resulting in the
sequential differentiation of PRs. MF initiation on the posterior margin of the eye
imaginal disc occurs due to the Egfr and Notch signaling which further induces
19 Developmental Genetics 977

Fig. 19.10 Arrangement of photoreceptors during eye development. Transverse section of an adult
ommatidium (left) showing six outer PRs and one of the inner PRs (R7). PRs specified simulta-
neously are represented in the same color. Rhabdomeres, light-sensing structures, are attached to
PRs and are shown in black color. R3 rhabdomere is located further apart from the inner PR, giving
rise to a trapezoid shape. Secondary and tertiary pigment cells surround the PRs with evenly
localized bristle cells. Longitudinal section of an adult ommatidium (right), covered by lens and a
pseudo-cone. (Adapted from Sahin and Celik 2013)

Antenna Eye

Morphogenetic
furrow

Anterior Posterior

Fig. 19.11 Morphogenetic furrow (MF) in the eye-antennal imaginal disc. Eye imaginal disc is an
epithelial tissue, where the eye is originated from the posterior part while the antenna and maxillary
palps are formed from the anterior part. The morphogenetic furrow (MF) moves from the posterior
to the anterior and is responsible for sequential differentiation of PRs. (Adapted from http://
slideplayer.it/slide/999922)
978 D. Vimal and K. Banu

Hedgehog (Hh) expression. Hh is one of the key players in the eye development
process as evident by initiation of more than one MF in case of ectopic expression of
Hh while arrest of MF progression in the absence of Hh. The MF is further
progressed to the anterior parts of the eye imaginal disc by the expression of
proneural gene, atonal (ato), as well as Hh. Hh has a short-range effect; therefore
Hh induces the expression of a morphogen, Decapentaplegic (Dpp), member of the
transforming growth factor-b family which has a long-range effect. Hh along with
Dpp regulates the expression of Homothorax (Hth), Ato, and Notch ligand Delta
(Dl) to induce the proneurogenesis in specific cells. Dpp activity is regulated by the
expression of Wingless (Wg) which represses the retinal development. The balance
between Dpp and Wg controls the MF wave in thin column of cells. In addition,
Notch, receptor tyrosine kinases, and Ras-MAPK signaling define the PR patterning.
Hh is in a positive feedback loop with Pointed (Pnt) and Sine oculis (So) while in
negative feedback with Egfr ligand Spitz (Spi) which limits the MF to a thin line of
cells. As the MF progresses, retinal differentiation occurs resulting in the generation
of PRs.
In addition to the abovementioned signaling molecules, retinal determination
genes which include eyeless (ey), eyes absent (eya), twin of eyeless (toy), teashirt
(tsh), and dachshund (dac) play key roles in eye development. Ey, a homeodomain
transcription factor, is the Drosophila ortholog of Pax6 and is considered the master
regulatory gene for retinal development. Ey along with its transcription factor toy
(an activator of ey) is expressed during embryogenesis which results in formation of
the eye and antenna. Ey expression is restricted to only limited row of cells that
become proneural at once; this is regulated by interplay between Hh and Dpp
signaling which in turn control So and Ato. As the MF progresses, the epithelial
cells in the imaginal disc proliferate asynchronously and form an evenly spaced
cluster of ~20 cells called rosette. In order to complete the ommatidial assembly, first
the posterior-most cells of the rosette are specified as the PR 8 (R8) which is then
joined by pairs of PR cells, R2/R5, R3/R4, R1/R6, and R7 cells, followed by four
cone cells as well as different types of pigment cells. This specification is mainly
regulated by the transcription factor, Ato, through Wnt and Notch signaling in
addition to Ey and So. The rosette cells now constitute the intermediate group,
which starts to reduce due to apoptosis in some cells reducing the cluster to, first,
five-cell cluster and, later, to three-cell cluster. The three-cell cluster is called the
equivalence group with each cell equally equipped to become an R8 cell. R8 cell fate
choice is determined by the Ato-Notch signaling through two transcription factors,
Senseless (Sens) and Rough (Ro). Ato induces the Sen which in turn represses the
Ro in one of the cells of the equivalence group specifying the cell as R8. Ro is
expressed at a higher level in two other cells of the cluster repressing Sens and giving
rise to R2 and R5 while repressing R3/R4 and R1/R6 fates by suppressing a nuclear
receptor, Seven-up (Svp). After the specification of R8, Egfr signaling through its
ligand Spi is responsible for the specification of all other PRs except R7. Moreover,
Spalt complex, which consists of the two transcription factors, Spalt major and Spalt
related, is responsible for inducing Svp resulting in the specification of R3/R4.
Further, two actions in the cells surrounding the rosettes, first suppression of Spalt
19 Developmental Genetics 979

genes by Svp and second induction of another transcription factor, Lozenge (Lz),
result in the generation of R1/R6 cells which are recruited to the cluster. Sev is
expressed in other PR cells, R3/R4 and R1/R6; however, Svp represses its activity,
thereby preventing them to differentiate into R7 cells. The last cell in the assembly of
PRs to be specified is R7, which starts to express a receptor tyrosine kinase,
Sevenless (Sev), which binds to the transmembrane ligand Bride of Sev (Boss) on
the R8 cell. Activation of Lz as well as another transcription factor, Prospero (Pros),
which is regulated by several zinc-finger transcription factors, Lz, So, Eya, and
Glass, gives rise to R7. After the specification of all the PRs, support cells
constituting of cone cells, pigment, and bristle complexes from the surrounding
undifferentiated cell pool join the ommatidium. First, four cone cells join the
ommatidium just above the PRs in order to minimize the surface area and secrete
the pseudolens and the lens. Toward the basal membrane lies two primary pigment
cells between the cone cells and PRs. Finally, in order to complete the visual cellular
specification, secondary and tertiary pigment cells and bristle complexes join the
ommatidium.
The eye consists of D-V polarity as the dorsal and ventral sides of the fly eye are
different from each other in their cellular layout which is called planar cell polarity
(PCP). Both the dorsal and ventral halves of the ommatidia are aligned as mirror
images of each other around the line of symmetry called the equator, which is
perpendicular to the MF along the A-P plane (Fig. 19.12). During early develop-
ment, the entire disc has a ventral identity by default. During the first instar larval
stage, several signaling pathways and transcription factors including Pannier (Pnr),
Wg, Iro-C (araucan, caupolican, and mirror homeobox genes), Notch, and Janus
kinase-signal transducer establishes the D-V polarity. Pnr functions at the dorsal side
of the eye imaginal disc, whereas Wg functions at both sides. The dorsal identity is
obtained via specific signals, such as Pnr, a zinc-finger transcription factor, Notch
ligand Dl, and Iro-C, whereas the ventral identity is obtained with the help of
glucosyltransferase Fringe (Fng) and Ser. Notch and its effector Eye gone (Eyg)
play key roles to establish the dorsal-ventral polarity. Overexpression of Eyg results
in the formation of additional eyes on the ventral side of the head, while Eyg mutants
lose their eyes entirely. After the MF, symmetrical organization of ommatidia starts
where the cell clusters start to rotate in opposite directions around the midline, which
cuts the D-V axis into two halves. After the first 450 rotation, the clusters rotate
another 450 in order to create a proper image. The PRs are arranged in an asymmetric
trapezoidal arrangement in such a way that the distinct position of R3 and R4 gives
rise to the chirality in the retina. Ommatidia exist in either of two chiral forms,
depending on their position such that ommatidia in the dorsal half of the retina adopt
one chiral form and the ventral half adopt the other. Establishment of chirality starts
at the equator where one of the two anterior cells in the five-cell precluster, the cell
with higher Frizzled expression, is destined to adopt the R3 fate, whereas the other
cell adopts the R4 fate.
After the establishment of D-V polarity, the developing eye imaginal disc
undergoes several morphological changes like establishment of cell junctions in
the retina, attachment of PRs to each other through cell junctions, change in cell
980 D. Vimal and K. Banu

Fig. 19.12 Sequential

assembly of ommatidia.
Photoreceptor specification
starts after the dorsal and
ventral identities in the eye
disc are obtained. The
symmetrical organization of
ommatidia starts right after the
MF passes. The eye disc is
symmetrical around the
equator. This opposing
alignment of the ommatidia
should be precise in order to
create a proper image.
(Adapted from Sahin and
Celik 2013)

shape of photoreceptors during early pupal stages, and extension of axons from PRs
to different layers of the optic lobe to form proper connections with the brain.
Among the cell junctions in the eye, the main homophilic adhesion molecules are
the adherens junctions (AJs). In the fly, all three cadherin homologs function in the
development of the retina. Drosophila E cadherin (DE-cad) is ubiquitously
expressed, in all cone and pigment cells, whereas the expression of Drosophila N
cadherin (DN-cad) is limited to different subsets of cells at each developmental
stage. The asymmetric specification of R3/R4 in dorsal and ventral halves initiates
polarization and ommatidia turn perpendicular to the equator in opposite directions
in such a way that the R8 cell faces the equator. This motion requires cadherin
function, DEcad promotes the rotation, whereas DN-cad has a specific function in
R3/R4 rotation with both DN-cad and DN-cad2 having redundant functions. Further,
the apical surfaces of PRs turn inside toward the center of the ommatidium detaching
them from other cells. This leads to the formation of light-sensitive microvillar
19 Developmental Genetics 981

structures called rhabdomeres. Zonula adherens plays an important function of tissue

integrity during this process. During the late pupal stages, PR cells start to elongate
basally leading to the remodeling of zonula adherens junctions in the process to
reach the brain. Before pupariation, larval photoreceptor neurons extend axons down
the optic stalk toward the brain finding their target zones in the lamina and medulla,
while glial cells migrate in the reverse direction, following photoreceptor axons from
the brain back to the eye disc. R7 cells elongate sending their axon to the optic lobe,
while R8 stays beneath the R7. Transmembrane protein, Crumbs, plays the most
important role during this elongation event. In addition, cadherin-mediated adherens
junctions play a key role in achieving the stereotypic arrangement as well as peculiar
shape of the cone cells and pigment cells. Finally, PRs send their axons to the optic
lobe of the brain in order to establish a functional connection with the brain. Axons
of all the PRs send their axons through a fenestrated membrane which is formed by
the cone cells and secondary and tertiary pigment cells at the basal part separating the
retina from the brain. Thus, proper assembly and remodeling of AJs is absolutely
required for different aspects of eye development to ensure the generation of a
functional retina.
The last step in the eye morphogenesis is the specification of the photoreceptor
subtypes which is dependent on the distinction between inner and outer PRs. This
specification is dependent upon the transcription factors of Spalt family, for exam-
ple, Sal is necessary for determining the inner cell fate. In addition, there is activity
of additional transcription factors, Pros in R7 and Sens in R8, which then terminally
differentiates these two inner cells into R7 and R8, respectively. Further, the next
fate-determining step is the generation of the two major ommatidial subtypes pale
and yellow. The yellow fate is determined by the transcription factor Spineless
which is expressed in 70% of ommatidia in the R7 cell, thereby inducing rh4
expression in them which communicate with the underlying R8 cell including
them to express rh6. All the remaining R7 cells that do not express spineless express
rh3 by default and induce the underlying R8 cell to express rh5. Orthodenticle (otd)
belongs to the paired class of homeobox genes and is expressed in all PRs which
leads to the expression of pale Rhs, rh3 in R7 and rh5 in R8, as well as represses the
expression of rh6 in outer PRs by activating the repressor defective proventriculus
(dve). After the expression of correct subsets of Rh genes, two proteins, Melted, a
Pleckstrin-homology (PH) domain protein, and lats, a serine/threonine kinase, func-
tion in a regulatory loop to ensure the maintenance of the correct Rh choice. The
expression of both lats and melt is regulated by the Hippo signaling, through its
members Mer and Kib. Melted is expressed in pale R8 cells promoting the expres-
sion of Rh5 and repressing lats, while lats is expressed in the yellow R8 cells
promoting Rh6 in turn and represses melted expression. In addition to pale and
yellow subtypes, the dorsal part of the eye contains two additional ommatidial
subtypes called the dorsal rim area (DRA) and the dorsal y. The DRA is present at
the two dorsal-most rows of the retina with R7 and R8 cells expressing Rh3 and is
required for polarization vision. In contrast, dorsal y ommatidia are present in the
dorsal third of the retina (with R7 cells expressing Rh3 and Rh4, whereas R8 cell
expressing Rh6) and are required to discriminate between the solar and non-solar
982 D. Vimal and K. Banu

parts of the sky for proper navigation of insects. The expression of UV-sensitive Rh3
and Rh4 that are coexpressed in the R7 cells of dorsal ommatidia is through the
action of iro-C. Once all the cells are specified, fly head eversion moves the eye and
head tissues into their adult configuration before the end of pupation.

19.3 Drosophila: Embryonic Development

Drosophila is an excellent model organism for the study of developmental processes

as the basic framework of early development between human and Drosophila
closely resembles owing to the highly conserved network pathways and the key
regulators in it. Numerous studies on Drosophila make the basis of our current
understanding on developmental processes and pattern.

19.3.1 Overview

The transition of the oocyte to embryo marks the onset of development which
employs complex and stringent regulations of the developmental signals, mRNA
translation, as well as cell cycle. In almost all organisms, embryogenesis occurs in
the absence of zygotic transcription, utilizing the maternal mRNAs, translational
machinery components, and nutrients which are accumulated in the eggs during
oogenesis well before embryogenesis. In drosophila, there are two meiotic arrests
during oogenesis, first at the prophase I and a secondary meiotic arrest at metaphase
I. This developmental strategy allows the maternal stores to be deposited into the
oocyte during oogenesis. Further, several changes in protein levels, mRNA transla-
tion, polyadenylation, and egg activation lead to oocyte-to-embryo transition called
maternal-to-zygotic transition (MZT) allowing activation of expression of the
zygotic genome.
Accumulation of maternal mRNAs and proteins is a prerequisite for the normal
production of functional oocyte embryo development. Drosophila oogenesis is
extensively studied to understand the process of ovarian development, embryogene-
sis, as well as the underlying mechanism due to simplicity of oocyte development
and the ease of studying it. Drosophila female contains a pair of ovary which in turn
each has 20–30 ovarioles (Fig. 19.13). Each ovariole contains a germarium at the
most anterior part and a mature egg at the most posterior end with 14 progressively
more developed follicles or egg chambers in between. Germarium is the production
house of the egg chambers, each containing 16 sister cells that share common
cytoplasm attached through cytoplasmic channels called ring canals, out of which
15 cells become nurse cells, while 1 cell acquires the oocyte fate during oogenesis.
Egg chamber is covered by follicle cells (FCs) which are polyploidy cells and are
required for the patterning of early stages as well as for depositing the multilayered
egg shell during late oogenesis (Fig. 19.13). An egg chamber provides a microenvi-
ronment for the development of the oocyte. Nurse cells are required for the synthesis
of the DNA and RNA that are stored in the egg and are required for the early embryo
19 Developmental Genetics 983

Fig. 19.13 Ovary morphology and oocyte development in Drosophila melanogaster. Each Dro-
sophila female has a pair of ovaries covered in peritoneal sheath connected through lateral oviduct
which descends into a common oviduct followed by the uterus and vulva. Spermatheca and seminal
receptacles are the sperm storage organs, while accessory organs also called as parovaria are
believed to have a secretory function. Each ovary is composed of 17 to 20 ovarioles each divided
into 14 stages of development with stage 14 mature egg as the last stage. In each ovariole, the egg
chambers are arranged in a developmental sequence with germarium at the most anterior end and
most mature stage at the posterior end. Germline stem cells present in the germarium undergo
karyokinesis simultaneously for four times with incomplete cytokinesis producing a 16-cell struc-
ture called cyst. In the cyst, 1 cell becomes the oocyte and the other 15 become the nurse cells.
(Adapted from Middleton et al. 2006 and Ables et al. 2016)

development post-fertilization. The oocyte undergoes meiotic maturation at stage

13 of oogenesis where all the contents of the nurse cells are transferred to the oocyte
in a process called dumping while they undergo apoptosis after that (Pritchett et al.
984 D. Vimal and K. Banu

Fig. 19.14 Different stages of Drosophila embryogenesis. In Drosophila, embryonic development

is divided into 17 stages and starts when male and female pronuclei fuse together (cycle 1). This
nucleus then starts to multiply at the anterior portion of the embryo (cycles 2–3) forming multiple
nuclei which spread along the A-P axis (axial expansion, cycles 4–6). These nuclei start migrating
toward the periphery of the embryo in a process called cortical migration (cycles 8–10) followed by
the formation of the pole cells on the posterior end (cycle 9). The embryo is enveloped by multiple
layers by the end of all nuclear divisions followed by invagination and formation of cellular
blastoderm. (Adapted from Tram 2011)

2009). Mature egg (stage 14 oocyte) is released from the ovary (ovulation)
descending into the oviduct, where it undergoes a process called egg activation
which prepares the oocytes for fertilization using the mechanical forces and hydra-
tion. The sperm then fertilizes the egg entering through a pore called micropyle
present on the anterior side of the oocyte. After fertilization, the meiotic arrest is
released, and the meiosis is resumed resulting in the formation of four female meiotic
products. In the absence of fertilization, development is arrested and the four female
meiotic products assemble to form a single polar body. After fertilization, the male
and female pronuclei fuse and undergo 13 rounds of mitosis in a common shared
cytoplasm (syncytia) which is regulated by maternal mRNAs and proteins.
The fused male and female pronuclei undergo 13 rounds of dynamic nuclear
divisions in the shared cytoplasm resulting in the formation of a syncytial blastoderm
containing 6000 nuclei (Fig. 19.14).
Activation of a cascade of genes sets up the Drosophila body plan. The first in
this sequence are maternal genes that are expressed in the ovaries and are
accumulated at different but specific regions of the developing embryos. Translated
products of these mRNAs act as morphogens and form a gradient along the embryo.
Bicoid and Hunchback regulate the production of anterior structures, while Nanos
and Caudal are responsible for the posterior parts of the embryo. Next in this
sequence are zygotic genes that are, depending on the condition, either activated
or suppressed by maternal genes. Zygotic genes include gap genes, pair-rule genes,
19 Developmental Genetics 985

and segment polarity genes in the same sequence of action. Gap genes are expressed
in broad domains throughout the embryo, and mutations in them result in gaps
between these segments. Next, pair-rule genes that are responsible for dividing the
embryo into seven bands perpendicular to the A-P axis are activated by the gap
genes. Further, segment polarity genes come in action that are regulated by the pair-
rule genes and are responsible for dividing the embryo in 14 equal segments. Once
the combined action of all three abovementioned set of genes has divided the embryo
in periodic segments, homeotic selector genes are activated and regulate the fate of
individual segments. In the next 24 h, the embryo converts to the larva that keeps
growing for the next ~4 days (at 25
C). The larva then molts two times to second
instar larva in ~24 h and third instar larva in ~48 h. The larva then converts into pupa
that is a dormant stage where it undergoes metamorphosis for ~4 days before the
adult or imago ecloses. The adult fly can be divided into head, thorax, and abdomen.
The head region contains the eyes, mouth, and antennae; the thoracic region is
divided into three segments T1 (contains a pair of legs), T2 (contains a pair of leg
and a pair of wings), and T3 (contains a pair of legs and a pair of halteres), while the
abdomen region is divided into eight segments (A1 to A8).

19.3.2 Anterior-Posterior Body Axis

The anterior-posterior (A-P) as well as dorso-ventral (D-V) body axes are deter-
mined way before the embryonic development during the egg development. The
anterior-posterior axis of the embryo is broadly specified by three sets of genes: the
first set defines the anterior organizing center, the second set defines the posterior
organizing center, and the third set defines the terminal boundary region. Different
mRNAs are localized at the different regions of egg predestining the embryo
development. The A-P patterning starts after the completion of gastrulation and is
regulated by two sets of genes, maternal effect genes and zygotic genes (Fig. 19.15).
Maternal effect genes are expressed when the egg is in the ovary of the fly and their
transcripts are distributed in the egg. Maternal effect genes include Bicoid, Nanos,
Hunchback, Caudal, Torso, and Toll. Among these genes, bicoid and hunchback
mRNAs determine the head and thorax formation, while nanos and caudal mRNAs
are required for the formation of abdominal segments. A-P patterning is established
due to the intercellular communication between the oocyte and the somatic follicle
cells. The nurse cells present in the egg chamber of ovary deposit the transcripts of
the different maternal effect genes in the egg. These transcripts are localized due to
the action of microtubules which are arranged in such a way that their positive and
negative ends are oriented toward the posterior and anterior sides of the egg
chambers, respectively. The localization of these maternal effect genes in the egg
predetermines the A-P polarity of the embryo. After fertilization, the proteins from
these maternal effect gene transcripts act as the transcriptional activators for the next
set of genes called zygotic genes.
During egg development in the ovary, bicoid mRNA from maternal bicoid gene is
accumulated in the anterior region of the egg cells by the nurse cells in the dormant
986 D. Vimal and K. Banu

Fig. 19.15 Action of maternal and zygotic genes in Drosophila pattern formation. Maternal effect
genes first establish the anterior-posterior (A-P) pattern with bicoid at the anterior tip and nanos at
the posterior tip of the fertilized egg. Gap genes are regulated by maternal genes and divide the
embryo into broad regions. Pair-rule genes are then activated by gap genes (hunchback and
Krüppel) which are responsible for the segment formation in embryo. Two pair-rule genes, fushi
tarazu and even stripped, are expressed in alternate strips, resembling the zebra stripes, along the
A-P axis of the embryo. All these genes regulate the expression of homeotic genes that define the
identity of each segment. Expression of segment polarity genes (engrailed) divides the embryo into
a repeated series of segmental primordia along the anterior-posterior axis. (Adapted from Mundlos
2010)

condition (Chang et al. 2011). Cytoskeleton (microtubule motor protein dynein) at

the anterior region of the egg anchors the bicoid mRNA through 30 untranslated
region (UTR). After fertilization, a polyadenylate (poly-A) tail is added to the bicoid
mRNA resulting in its translation forming a gradient with highest concentration in
the anterior of the egg and the lowest in the posterior third of the egg. Different
mutation studies have confirmed the function of bicoid in the anterior region of the
embryo. When bicoid is injected in the anterior region of the bicoid-deficient
embryos, normal anterior-posterior polarity was established in it. When bicoid is
19 Developmental Genetics 987

added in the center of the embryo, it developed in the head, while both ends became
thorax. In contrast, if bicoid is added in the posterior region of the wild-type embryo
(having normal bicoid level at anterior end) two heads develop at either side.
Another evidence for bicoid function was observed in the exuperantia and swallow
mutant embryo; both these genes are required for restricting bicoid in the anterior
region. In these mutants, bicoid diffuses toward the posterior end of the egg such that
the gradient cannot be formed resulting in the absence of anterior structures and
presence of extended mouth and thoracic region. Bicoid protein not only ensures the
formation of anterior organs but also ensures that the proteins required for the
posterior region are localized there only. One such protein is caudal which is
required for the formation of posterior domains of the embryo. Bicoid protein
binds to the 30 UTR of the caudal region inhibiting the translation of caudal mRNA
allowing its translation only in posterior region. In addition, bicoid works as a
transcriptional activator of the hunchback gene in the nucleus. The Bicoid and
Hunchback proteins act synergistically as the enhancers to promote the transcription
of the genes required for head formation.
Next in the array of maternal effect genes is nanos, which similar to bicoid is
synthesized by nurse cells during egg development and accumulated in the posterior
region of the egg. Similar to bicoid, nanos expression remains repressed by the
binding of the Smaug protein to its 3´ UTR. Nanos protein remains bound to the
cytoskeleton in the posterior region of the egg through its 3´ UTR. Nanos along with
oskar, valois, vasa, staufen, and tudor ensures normal embryonic abdomen forma-
tion. In addition, nanos along with pumilio protein binds with hunchback mRNA
and prevents its translation in the posterior region. As a result, early Drosophila
embryo shows a gradient of four proteins, Bicoid and Hunchback proteins at anterior
and Nanos and Caudal proteins at the posterior end. Bicoid, Hunchback, and Caudal
proteins function as transcription factors which further activate or repress different
zygotic genes.
Another maternal effect gene is torso (encodes a receptor tyrosine kinase) which
is required for the formation of extreme ends in the embryo; the most anterior head
segments are called acron and most posterior abdominal segments are called telson
(tail). Similar to all maternal effect genes, torso mRNA is synthesized by the ovarian
cells, deposited in the oocyte, and translated after fertilization. The ligand for the
activation of Torso protein is trunk protein which is secreted in an inactive form.
Trunk is activated by the proteolytic cleavage carried out by Torso-like protein
which is activated by trunk-like protein present only at the two poles of the oocyte.
Thus, Torso protein is expressed only at the extreme anterior and posterior regions of
the oocyte membrane. Torso protein functions through the Receptor tyrosine kinase
cascade which results in the activation of tailless and huckebein gap genes. These
gap genes along with bicoid specify the termini of the embryo. In the presence of
bicoid, these genes form acron, while in its absence the terminal regions differentiate
into telson.
988 D. Vimal and K. Banu

19.3.3 Embryogenesis

Embryonic development also called embryogenesis is the process from fertilization

of the egg followed by embryo formation and its development. The study of
Drosophila embryogenesis has contributed most of the understanding of the molec-
ular basis of animal development. Drosophila and human development are homolo-
gous processes. Drosophila utilizes conserved set of genes and regulatory networks
which makes the studies on this animal model to be utilized to understand the
process in higher organisms. The embryonic development of Drosophila
melanogaster has been subdivided into 17 stages (Fig. 19.16, Table 19.2). Drosoph-
ila egg is bilaterally symmetrical where only difference between the two sides is flat
dorsal side and slightly convex ventral side. The average length of the egg is

Fig. 19.16 Different stages of embryonic development. After the fertilization, embryonic devel-
opment starts and comprises of 17 stages of development marked by specialized process occurring
in that stage. Earlier stages are characterized by nuclear divisions without division of cytoplasm
(cytokinesis), after ten rounds of synchronized nuclear divisions, nuclei start to migrate to the
periphery of embryo where they become encapsulated by actin-based furrow canals (stages 3–4).
Stage 5 is characterized by cellularization followed by gastrulation which determines the three germ
layers (stage 8). Stage 9 is characterized by germband extension which remodels the body plan as
cells from the posterior end of the embryo migrate toward the anterior end. Germband retraction in
stage 12 is followed by the migration of epithelial cells toward the dorsal midline called dorsal
closure (stage 13). Further, in stage 15, head involution occurs where head structures mature, and
finally the larva reaches its mature state (stage 17) and hatches from the eggshell. (Adapted from
Hales et al. 2015)
19 Developmental Genetics 989

Table 19.2 List of Drosophila embryogenesis stages with key process

Stage Developmental events
Stage 1 First two nuclear divisions
Stage 2 Syncytial divisions 3–8, cytoplasmic clearing
Stage 3 Syncytial division 9, polar bud formation
Stage 4 Syncytial divisions 10–13, pole cell formation
Stage 5 Cellularization
Stage 6 Onset of gastrulation, formation of ventral and cephalic furrow, dorsal shift of pole
cells
Stage 7 Completion of gastrulation, pole cells in a pocket
Stage 8 Rapid phase of germ band extension, to 60% egg length
Stage 9 Germ band elongation to 70% egg length, early neuroblast delamination
Stage 10 Germ band elongates to 75% egg length, stomodeum invaginates
Stage 11 Segmentation, tracheal pits arise, posterior midgut invagination reaches the posterior
pole
Stage 12 Onset of germ band retraction, fusion of anterior and posterior midgut
Stage 13 Completion of germ band shortening
Stage 14 Dorsal closure to 80% along the dorsoventral axis, head involution
Stage 15 Dorsal closure of epidermis and midgut
Stage 16 Gastric caeca are formed, somatic musculature becomes visible
Stage 17 Completion of organogenesis, movements of the embryo within the vitelline
envelope

~500 μm; the diameter is about 180 μm. The mature egg is covered by a tough,
opaque outermost layer called chorion. Below chorion is an additional transparent
homogeneous membrane called vitelline membrane. Multiple hexagonal and pen-
tagonal patterns can be seen on the chorion which is the impressions of the ovarian
follicle cells. A pair of filament is present at the anterior dorsal surface as an
extension of the chorion. There is an opening in the vitelline membrane called
micropyle required for the entry of sperms.
Embryogenesis in Drosophila starts with the fertilization, followed by multiple
rounds of nuclear division through mitosis without cytokinesis resulting in a multi-
nucleated cell called syncytium or syncytial blastoderm with shared cytoplasm.
Syncytial blastoderm allows different proteins to form gradient along the cytoplasm
which regulates pattern formation in embryo. After eight rounds of nuclear division,
256 nuclei are produced in the central portion of the egg. A group of cells reach the
surface of the posterior pole of the embryo and become enclosed by a cell membrane
forming pole cells which in the future give rise to the gametes of the adult fly. After
the tenth nuclear division, the nuclei in the center starts to migrate to the periphery of
the embryo where the nuclear divisions continue. The shared cytoplasm is not
uniform in nature, but each nucleus is surrounded by its cytoskeletal proteins
(microtubule and microfilament). The nuclei and its associated cytoplasmic islands
are called energids. At the 13th division, ~6000 nuclei, arranged at the periphery of
the embryo, are partitioned into separate cells by the invagination of the oocyte cell
990 D. Vimal and K. Banu

membrane. This process produces an embryo with peripheral cells and yolk center
and is called cellular blastoderm. At the 14th cycle of embryo development also
called as midblastula transition, division is asynchronous producing different types
of cells. Midblastula transition is followed by gastrulation where the presumptive
mesoderm, endoderm, and ectoderm are formed. Approximately 1000 cells of future
mesoderm forming ventral midline of the embryo start to fold inward to produce
the ventral furrow. The ventral furrow pinches off from the embryo surface forming
the ventral tube within the embryo. The endodermal cells form two pockets at the
anterior and posterior ends of the ventral furrow followed by pole cell internaliza-
tion. At this time, the embryo bends to form the cephalic furrow. The ectodermal and
mesodermal cells migrate toward the ventral midline forming the germ band. The
cells of germ band are destined to become the trunk of the embryo. The germ band
extends posteriorly and covers the dorsal surface of the embryo. At this stage the
cephalic furrow separates the future head region (procephalon) from the germ band
that will form the thorax and abdomen, and body segments start to appear. While the
germ band is in extended position, various important processes like organogenesis,
segmentation, segregation of imaginal discs, and nervous system formation occur.
All the developmental stages of Drosophila, embryo, larva, and adult have the
segmented body plan with three thoracic and eight abdominal segments. The first
thoracic segment contains legs, the second thoracic segment has legs and wings, and
the third thoracic segment has legs and halteres (balancers).

19.4 Drosophila: Zygotic Gene

The first set of genes that are expressed in the embryo are zygotic genes. This
includes segmentation genes (gap genes, pair-rule genes, and segment polarity
genes) that are responsible for the transition of the syncytial embryo to segmented
bodied fly. Maternal effect genes work as the transcriptional activators for the
zygotic genes. There are two steps for a cell to commit to its fate in Drosophila:
firstly a cell is specified and then later on its fate is determined. In Drosophila cell
specification is based upon its environment where different maternal effect
morphogens guide the cell. This process is reversible and can be altered by
modifying the surrounding morphogens. The next step in cell commitment is cell
determination, which is irreversible as well as cell intrinsic and occurs due to the
expression of segmentation genes. Expression of segmentation genes divides the
early embryo into a series of repeating segmental primordia along the A-P axis.
Segmentation genes divide the embryo into major anatomical divisions and
14 parasegments. Each parasegment includes the posterior part of an anterior
segment and the anterior portion of the posterior segment.
19 Developmental Genetics 991

19.4.1 Gap Genes

First in the array of segmentation genes are gap genes that are either activated or
repressed by the maternal effect genes. Gap genes were discovered in the mutant
embryos which lacked groups of consecutive segments. All gap genes function as
transcription factors and include hunchback, Krüppel, giant, knirps, tailless,
huckebein (zygotic), orthodenticle, buttoned, and empty spiracles. Gap genes are
responsible for the segment formation, and thereby they are expressed in the
overlapping domains (Fig. 19.17). A significant amount of hunchback protein
accumulation can be detected at the anterior portion of the embryo by the completion
of 12th cycle. The transcription pattern of the next gap genes is regulated by the
levels of the hunchback and bicoid. In the anterior region, high levels of hunchback
promote the expression of giant while suppressing the expression of posterior gap
genes such as knirps. Caudal protein at the posterior end activates the expression of
abdominal gap genes knirps and giant. Giant produces two bands, one anterior
expression band and another posterior expression band. In addition to maternal
effect genes, different gap gene expression itself establishes their expression
patterns. After gap gene expression, the early embryo has a broad anterior

Fig. 19.17 Expression

pattern of different gap genes.
The anterior gap genes
include Hunchback, Giant,
and Krüppel, while posterior
gap genes include Knirps and
Giant. The terminal gap genes
include Tailless and
Huckebein. These genes are
required for the formation of
the unsegmented terminal
regions of the larvae: acron at
the anterior and telson at the
posterior end. (Adapted from
Gilbert 2000)
992 D. Vimal and K. Banu

hunchback band, giant bands at middle anterior and middle posterior, Krüppel band
in the middle, tailless bands at middle anterior and extreme posterior, and knirps
bands at extreme anterior and middle posterior end.

19.4.2 Pair-Rule Genes

Next in the line of segmentation genes are pair-rule genes that are activated by gap
genes during the 13th division cycle and divide the embryo in seven vertical bands
perpendicular to the A-P axis. Pair-rule genes are expressed in the zebra stripe
pattern along the A-P axis, dividing the embryo into 7 transverse bands and
15 subunits. Pair-rule genes are of two types, primary pair rule genes which includes
hairy, even-skipped, and runt and secondary pair-rule genes which include fushi
tarazu, odd-skipped, odd-paired, and paired. Gap genes activate the primary pair-rule
genes which are essential for the formation of the periodic pattern in the embryo.
Gap gene protein concentrations regulate the function of pair-rule genes through the
enhancer sequences. Mutation in the enhancer of a particular pair-rule gene can
delete its particular stripe. Product of primary pair-rule genes acts as a transcriptional
activator for the secondary pair-rule genes. Expression of each pair-rule gene in
seven stripes divides the embryo into 14 parasegments, with each pair-rule gene
being expressed in alternate parasegments with particular and unique combination of
pair-rule products which in turn activate the segment polarity genes.

19.4.3 Segment Polarity Genes

In Drosophila, segment polarity genes carry out two important functions: first, they
reinforce the parasegmental periodicity, and second they establish the cell fates
within each parasegment due to cell to cell signaling (Lee et al. 2016). In the embryo,
segment polarity genes establish the A-P polarities within each embryonic
parasegment through Wnt and Hedgehog signaling pathway. Major segment polarity
genes include engrailed, wingless, hedgehog, fused, patched, cubitus interruptus,
dishevelled, frizzled, gooseberry, pangolin, and armadillo. The pair-rule genes either
positively or negatively regulate the expression patterns of segment polarity genes at
the transcription level. There are two phases of segment polarity gene regulation:
first, regulation by pair-rule genes, and second, cell to cell signaling. Pair-rule genes
like ftz or eve regulate the expression of engrailed as well as wingless in each
parasegment of the embryo. Evidence for cell to cell communication comes from the
mutation studies where in engrailed mutant embryos there is no detectable wingless
expression and vice versa suggesting both genes are required for each other’s
expression. Engrailed and wingless are expressed in different cells; therefore for
such regulation there must be cell-cell communication. The receptor for wingless
(secreted peptide ligand) is present on the posterior cells, which upon binding
regulates the expression of engrailed at transcription level. Engrailed and wingless
expression is also lost in another segment polarity gene mutant called hedgehog
19 Developmental Genetics 993

suggesting its involvement in the genetic circuit regulating wingless and engrailed
expression. Hedgehog ligand binds to its receptor on the wingless-expressing cells
which results in the maintenance of wingless transcription. The expression of
segment polarity genes acts as morphogens by accumulating in different
concentrations in individual segments where they regulate the cell differentiation
fate. The expression of paired as well as even skipped is required for the regulation
of odd engrailed stripes, whereas fushi tarazu along with the odd paired is required
for even engrailed stripes. The expression of Engrailed is required for the determi-
nation of the A-P compartment boundaries. Wingless and hedgehog expression is
responsible for the anterior and posterior compartment of each segment, respec-
tively. Mutations in the segment polarity gene result in the segment errors like
deletion, mirror image, duplication, and segment polarity reversal.

19.5 Drosophila: Homeotic Gene

Homeotic selector genes are a group of genes that control the pattern of body
formation as well as for establishing the characteristic structures of each segment
during early embryonic development of organisms (Mallo and Alonso 2013).
Homeotic genes include multiple subsets of Hox and ParaHox genes that are the
key regulators of segmentation in flies. These genes encode proteins which function
as transcription factors directing different cells to form various parts of the body.
Mutations or misexpression of homeotic genes causes displaced body parts or
transformation of one organ into another which is called homeosis. For example,
flies with antennapedia mutation (lethal mutation) have ectopic legs on the head in
the place of antennas.
The elucidation of homeotic gene function in the embryonic development can be
attributed to Edward B. Lewis, Eric F. Wieschaus, and Christiane Nüsslein-Volhard.
Their discovery of the genetic control of early embryonic development helped them
win the Nobel Prize in the field of Physiology or Medicine in the year 1995. During
the early research, Edward B. Lewis at the California Institute of Technology in Los
Angeles observed flies with occasional malformations. In one such case, a mutation
leads to the transformation of halteres (balancing organ of the fly) into an extra pair
of wings. In Greek, homeosis means malformations, and from there the homeotic
genes acquired their name. It was later found that the mutation in segmentation gene
of bithorax complex leads to the doubling of same body segment leading to the
development of additional pair of wings. According to the colinearity principle,
genes at the beginning of the complex controlled anterior body segments, while
genes further down the complex regulated posterior body segments (Gummalla et al.
2014) (Fig. 19.18). It was also found that the regions controlled by the individual
genes of this complex overlapped each other and a complex interplay between them
specified the individual body segments during development. The inactivity of the
first gene of the bithorax complex in the segment where halteres should be produced
leads to the formation of extra pair of wings resulting in a fly with four wings. This
inactivity caused other homeotic genes to re-specify this particular segment into one
that forms wings.
994 D. Vimal and K. Banu

Fig. 19.18 Conservation between the HOM-C (Drosophila) and HOX (human) gene clusters. In
terms of nucleotide sequence and relative position to each other (colinear expression), the two
Drosophila Hom-C complex clusters can be seen distributed over four Hox gene clusters in
mammals. (Adapted from Lappin et al. 2006)

Homeotic genes contain a unique DNA sequence of ~180 base pair length known
as a homeobox, which encodes a segment of 60 amino acids within the homeotic
transcription factor protein. Homeoboxes were first discovered in three Drosophila
homeotic and segmentation genes: (1) Antennapedia (Antp), ultrabithorax (Ubx),
and fushi tarazu (Ftz), mutations which caused homeotic transformations. The
homeodomain protein is composed of three alpha helixes; helix 2 and 3 form a
helix-turn-helix (HTH) structure, where the two alpha helices are connected by a
19 Developmental Genetics 995

short loop region. These two helices are present at the N-terminal in antiparallel
position, while helix 1 is present at the C-terminal in perpendicular position. Helix
1 directly interacts with the DNA by a large number of hydrogen bonds, by
hydrophobic interactions, as well as by indirect interactions between specific side
chains and the exposed bases within the major groove of the DNA. Due to the
DNA-recognition properties, homeodomain proteins induce cascade of coregulated
targeted genes which direct the formation of many body structures during early
embryonic development.
The homeodomain proteins bind to the DNA at a particular conserved nucleotide
sequence, TAAT present at the 50 terminal with the thymine being the most impor-
tant for binding. All homeodomain proteins recognize this initial sequence; however
the base pairs following this initial sequence are used to distinguish between
different homeodomain proteins. For example, amino acid lysine present at the
position 9 of homeodomain protein Bicoid recognizes the nucleotide guanine after
the initial sequence. Similarly, glutamine present at the ninth position in
Antennapedia recognizes and binds to adenine. If lysine in Bicoid is switched with
glutamine of Antennapedia homeodomain, the resulting protein starts to recognize
Antennapedia-binding enhancer sites. Moreover, Hox proteins bind to protein
cofactors that provide DNA sequence specificity. Two such examples of Hox
cofactors are Extradenticle (Exd) and Homothorax (Hth) which upon binding induce
conformational changes in the Hox protein, thereby increasing the sequence
specificity.
Homeotic genes homologous to those of Drosophila were later found in a wide
range of organisms, including fungi, plants, and vertebrates. In vertebrates, these
genes are commonly referred to as HOX genes. Humans possess ~39 HOX genes,
which are divided into four different clusters, A, B, C, and D, located on different
chromosomes, 7p15, 17q21.2, 12q13, and 2q31, respectively (Fig. 19.18). These
genes have been assumed to have arisen due to the duplication and divergence from
a primordial homeobox gene. On the basis of sequence similarity and relative
position within the cluster, each cluster consists of 13 paralog groups with 9 to
11 members. A high degree of homology can be seen between the human HOX
genes and the Hom-C genes of Drosophila. The human paralog groups 1–8 are more
closely related to antennapedia (Antp), while groups 9–13 are more closely related to
abdominal-B (abd-B).

19.5.1 Hox Gene in Drosophila

Hox genes are a subset of homeobox genes that specify regions of the body plan of
an embryo along the A-P axis of organism (Pavlopoulos and Akam 2007). The
products of Hox genes are Hox proteins that function as transcription factors by
binding to a specific nucleotide sequences on DNA called enhancers through their
homeodomain. The same Hox protein can act as a repressor for one gene and as
activator for another. Hox genes are arranged in clusters, and their order on the
chromosome is the same as the order in which they appear along the body such that
996 D. Vimal and K. Banu

Fig. 19.19 Hox gene clusters in Drosophila. The two Hox gene clusters Antennapedia complex
(left) and bithorax complex (right). The break mark (//) in the chromosome indicates that these two
clusters of genes are separated by a long intervening region. (Adapted from Hox genes of fruit fly by
PhiLiP, public domain)

the genes on the left control patterning of the head, while the genes on the right
control patterning of tail. Drosophila has eight Hox genes that are clustered into two
complexes, antennapedia complex (ANT-C) and bithorax complex (BX-C) collec-
tively called homeotic complex (HOM-C), both of which are located on chromo-
some 3 (Fig. 19.19). The ANT-C is also called anterior homeotic gene complex as it
controls the identity of parasegment present prior to five, while BX-C regulates the
identity of fifth to 13th parasegment and is called posterior homeotic gene. The
Antennapedia complex contains the homeotic genes labial (lab), Antennapedia
(Antp), sex combs reduced (scr), deformed (dfd), and proboscipedia (pb). The labial
and deformed genes of the antennapedia complex specify the head segments, while
sex combs reduced and antennapedia define the thoracic segments. The bithorax
complex contains ultrabithorax (ubx), abdominal A (abdA), and Abdominal B
(AbdB) genes. Ubx is required for the identity of the third thoracic segment; abdA
and AbdB genes are responsible for the segmental identities of the abdominal
segments.
The lab gene is the most anteriorly expressed gene of the Antennapedia complex.
It is expressed in the head, mainly in the intercalary segment between the antenna
and mandible as well as in the midgut. The lab gene was initially named because it
disrupted the labial appendage; however it was later found that it was due to the
broad disorganization resulting from the failure of head involution. Mutation in lab
results in defective head involution process where embryos fail to internalize the
19 Developmental Genetics 997

Fig. 19.20 Antennapedia expression and mutation. In Drosophila, antennapedia is expressed in

the second segment of a fly’s thorax (a). Ectopic expression of antennapedia gene into the fly’s head
results in the activation of normal, second-segment leg development program in the head producing
a mutation where legs grow from the fly’s head in place of antennae (b). (Adapted from Hox genes
of fruit fly by PhiLiP, public domain)

mouth and head structures that initially develops on the outside of the body. Failure
of head involution disrupts or deletes the salivary glands and pharynx. The pb gene
of the ANT-C is responsible for the formation of the labial and maxillary palps. The
Dfd gene is responsible for the formation of the maxillary and mandibular segments
in the larval head. Similar to lab, mutation in Dfd results in a failure of head
involution. The Scr gene is responsible for cephalic and thoracic development in
Drosophila embryo and adult. The Antp gene specifies the development of a pair of
legs and a pair of wings on the second thoracic segment, T2. The classical example
of homeosis is due to dominant Antp mutation caused by a chromosomal inversion
leading to the Antp expression in the antennal imaginal disc resulting in the
formation of leg coming out of the fly’s head in place of antenna (Fig. 19.20).
The first gene in the bithorax complex is the Ubx which is responsible for the
determination of a pair of legs and a pair of halteres, highly reduced wings that
function in balancing during flight, on the third thoracic segment, T3. Ubx functions
mainly by repressing the genes involved in wing formation like blistered and spalt
which play important role in wing development. Another classical example of
homeosis is the four-winged flies due to the loss-of-function Ubx mutation
(Fig. 19.21). In these mutants Ubx is no longer able to represses the wing develop-
ment genes resulting in the transformation of halteres as a second pair of wings
eventually resulting in four-winged flies. In contrast, upon Ubx misexpression in the
second thoracic segment, it represses wing genes and the wings develop as halteres,
resulting in a four-haltered fly also called as Cbx enhancer mutation. The next gene
in the BX-C is abd-A which is expressed from abdominal segments A1 to A8 and is
required for the specification of most of the abdominal segment identity. Moreover,
it also affects the pattern of cuticle generation muscle generation in the ectoderm and
mesoderm, respectively. One of the main functions of abd-A in insects is to repress
limb formation. In abd-A loss-of-function mutants, abdominal segments A2–A8 are
transformed into A1 similar segments. The last gene in the assembly of BX-C is
998 D. Vimal and K. Banu

Fig. 19.21 Ultrabithorax gene expression and mutation. Ubx is strongly expressed in the third
segment of the thorax. Inactivation of Ultrabithorax results in the conversion of halteres into a
second set of wings behind the normal set of wings producing a four-winged fly. (Adapted from
Hox genes of fruit fly by PhiLiP, public domain)

Table 19.3 List of Hox genes, target genes, their function

Target gene regulated
Hox genes by Hox genes Function of target gene
ULTRABITHORAX distal-less Activates gene pathway for limb formation
(represses distal-less)
ABDOMINAL-A distal-less activates gene pathway for limb formation
(represses distal-less)
ULTRABITHORAX decapentaplegic Triggers cell shape changes in the gut that are
(activates required for normal visceral morphology
decapentaplegic)
DEFORMED reaper Apoptosis: localized cell death creates the
(activates reaper) segmental, boundary between the maxilla
and mandible of the head
ABDOMINAL-B decapentaplegic Prevents the above cell changes in more
(represses posterior positions
decapentaplegic)

abd-B which is transcribed into two different forms, a regulatory protein and a
morphogenetic protein. Regulatory abd-B suppresses embryonic ventral epidermal
structures in the eighth and ninth segments of the Drosophila abdomen. Both the
regulatory protein and the morphogenetic protein are involved in the development of
the tail segment.
Hox proteins with identical homeodomains are assumed to have identical
DNA-binding properties as well as functions and are classified based on the phylo-
genetic inference, synteny, and sequence similarity. Hox genes regulate many genes
that in turn regulate large developmental signaling networks. In addition, they also
regulate realisator genes or effector genes which are directly responsible for forming
the tissues, structures, and organs of each segment (Table 19.3). Hox genes are
regulated by gap genes and pair-rule genes which themselves are regulated by
19 Developmental Genetics 999

maternally supplied mRNA. In this way, maternal factors activate gap or pair-rule
genes which in turn activate Hox genes which further activate realisator genes that
cause the segments in the developing embryo to differentiate.
Initial regulation of homeotic genes is carried out by the gap and pair-rule genes
as they act as transcription factors for homeotic genes by cis-regulatory elements
called initiator enhancer elements. For instance, Hunchback and Krüppel proteins
repress the expression of abdA and AbdB genes from the head and thorax restricting
their expression only in the abdomen. In contrast, Ultrabithorax gene is activated by
the Hunchback protein expressing it in a broad band in the middle of the embryo,
while Antennapedia is activated by Krüppel. The Fushi tarazu and Even-skipped
proteins confine the expression of homeotic genes to the parasegments only. In
addition, homeotic gene themselves act as the transcriptional factors, as ANT-C and
BX-C homeotic gene complexes repress the expression of each other in their
expression region. Once the expression pattern of the homeotic genes have become
stabilized, chromatin conformation occurs which locks them in their respective
positions. The repression of homeotic genes is regulated by polycomb family,
while chromatin conformation is regulated by Trithorax proteins.
Mutation in homeotic genes leads to the abnormal development of the fly body
parts. Normal fly body contains three thoracic segments, the first segment contains
only a pair of legs, the second thoracic segment contains both a set of legs and a set
of wings, and the third thoracic segment produces a set of wings and a set of
balancers known as halteres (Fig. 19.22). Upon deletion of ultrabithorax gene, the
third thoracic segment becomes transformed into another second thoracic segment

Fig. 19.22 Organization of the Drosophila body into segments. Drosophila adult as well as larvae
is broadly divided into three regions called head, thorax, and abdomen which further contains
segments. The bodies of both the larval and adult insect are divided into 14 segments along the A-P
axis. In the adult fly, each thoracic segment (T1-T3) contains a pair of legs with the middle segment
T2 having a pair of wings and the most posterior segment T3 containing a pair of halteres. (Adapted
from Gilbert 2000)
1000 D. Vimal and K. Banu

resulting in a fly with four wings. Similarly, mutation in the Antennapedia gene

results in the fly head, having legs in place of antennae in the head sockets. The
ParaHox gene cluster is an array of homeobox genes from the Gsx, Xlox (Pdx), and
Cdx gene families and is involved in morphogenesis and regulation of anatomical
development patterns.

19.5.2 Genetic Disorder in Humans

The Human Genome Project which was completed in April 2003 revealed that it is
composed of 46 chromosomes or 22 pairs of autosomal chromosomes and 2 sex
chromosomes made up of ~3 billion base pairs of DNA and contains ~20,500
protein-coding genes with coding region of only ~5%. Most genetic diseases are
the direct result of a single or multiple mutations in one or multiple genes.
Genetic disorder is any disease caused by the abnormalities in the genetic makeup
of an individual ranging from single-base mutation to chromosomal abnormality like
addition, subtraction, or inversion of an entire chromosome or set of chromosomes.
These diseases can be hereditary or acquired due to some mutation exposure to some
chemicals. There are four types of inherited genetic disorders: single-gene disorder,
multifactorial inheritance, chromosome abnormalities, and mitochondrial
inheritance.
Single-gene disorder is also called Mendelian or monogenetic disorders as there
is mutation in the DNA sequence of a single gene. Currently there are ~4000 single
gene disorders known which are caused by the mutation in one gene (Table 19.4).
Common examples of single-gene disorders include cystic fibrosis, alpha- and beta-
thalassemias, sickle cell anemia (sickle cell disease), Marfan syndrome, Fragile X
syndrome, muscular dystrophy, familial hypercholesterolemia (FH), Huntington’s
disease, and hemochromatosis. Single-gene diseases affect at least 1 in 500 people
around the globe. These diseases may follow autosomal dominant or recessive as
well as X-linked dominant or recessive inheritance pattern. Pedigree analyses of
large families with many affected members can be used for tracking the inheritance
of many diseases. Many databases provide the accumulated and comprehensive
information on diseases and related genes; one such database for genes following
Mendelian inheritance is Online Mendelian Inheritance in Man (OMIM™). This
database was initially started by Dr. Victor A. McKusick in the early 1960s for
recording the Mendelian traits and disorders and was called Mendelian Inheritance
in Man (MIM). OMIM till date report ~387 human genes with a known phenotype,
~2310 human phenotypes with a known molecular basis, ~1621 confirmed Mende-
lian phenotypes with unknown molecular basis, and ~ 2084 phenotypes with
suspected Mendelian basis. OMIM was made online by the collaborative effort of
National Library of Medicine and the William H. Welch Medical Library at Johns
Hopkins in 1985.
In addition to gene mutations, environmental factors also contribute to many
disease, and in combination they cause a specific set of disorder called multifactorial
19 Developmental Genetics 1001

Table 19.4 Examples of single-gene diseases in humans, their mode of inheritance, and associated
genes
Disease Type of inheritance Gene responsible
Phenylketonuria (PKU) Autosomal recessive Phenylalanine hydroxylase (PAH)
Cystic fibrosis Autosomal recessive Cystic fibrosis conductance
transmembrane regulator (CFTR)
Sickle-cell anemia Autosomal recessive Beta hemoglobin (HBB)
Albinism, oculocutaneous, Autosomal recessive Oculocutaneous albinism II (OCA2)
type I
Huntington’s disease Autosomal dominant Huntingtin (HTT)
Myotonic dystrophy type 1 Autosomal dominant Dystrophia myotonica protein kinase
(DMPK)
Hypercholesterolemia, Autosomal dominant Low-density lipoprotein receptor
autosomal dominant, type (LDLR), apolipoprotein B (APOB)
B
Neurofibromatosis, type 1 Autosomal dominant Neurofibromin 1 (NF1)
Polycystic kidney disease Autosomal dominant Polycystic kidney disease 1 (PKD1) and
1 and 2 polycystic kidney disease 2 (PKD2)
Hemophilia A X-linked recessive Coagulation factor VIII (F8)
Muscular dystrophy, X-linked recessive Dystrophin (DMD)
Duchenne type
Hypophosphatemic rickets, X-linked dominant Phosphate-regulating endopeptidase
X-linked dominant homolog, X-linked (PHEX)
Rett’s syndrome X-linked dominant Methyl-CpG-binding protein 2 (MECP2)
Spermatogenic failure, Y-linked Ubiquitin-specific peptidase 9Y,
non-obstructive, Y-linked Y-linked (USP9Y)

inheritance disorder which includes neurodegenerative diseases, heart-related

diseases, various cancers, etc.
Any change at chromosomal level either in the structure or addition or subtraction
of a part or whole chromosome can result in various severe genetic disorders as they
are the genetic carriers for the next generation. Chromosomal abnormalities typically
occur due to a problem during cell division. The most common examples of
chromosomal abnormalities include Down’s syndrome (trisomy 21), Prader-Willi
syndrome, chronic myeloid leukemia (CML), Turner syndrome (45, X0), Klinefelter
syndrome (47, XXY), and Cri du chat syndrome (cry of the cat syndrome; 46, XX or
XY, 5p-). Numerical and structural abnormalities are the two types of chromosomal
abnormalities. Addition or subtraction of one of the chromosomes from the pair
causes numerical abnormalities, whereas structural errors are caused due to the
change in the structure of a chromosome. Change in the structure of chromosomes
can be of many types: deletion where a portion of the chromosome is missing or
deleted or duplication which results in the copying of a segment. Translocation is the
transfer of a segment from one chromosome to another and can be of two types,
reciprocal and Robertsonian translocation. During inversion, a segment gets inverted
before attaching again, whereas a ringed structure is formed when a segment is
deleted or broken off.
1002 D. Vimal and K. Banu

Mutation in non-nuclear genetic material like in mitochondria and cytoplasm

forms another class of genetic disorders. Each mitochondrion contains 5–10 circular
DNA and is carried by egg cells from maternal side. Mitochondrial genetic disorders
include some rare diseases like Leber hereditary optic neuropathy (LHON), myo-
clonic epilepsy with ragged red fibers (MERRF), lactic acidosis, and mitochondrial
encephalopathy.
With the sequencing of human genome, maps of each chromosome were
generated showing the precise location of every gene. This also revealed the parts
of the genome that differ from one person to the next and called as polymorphic. The
human genome contains various single nucleotide changes throughout the genetic
material (one SNP per 1000 base pairs) and is called single nucleotide
polymorphisms (SNPs). The International HapMap Project provides the human
SNP map of all the chromosomes. High throughput screening can be done using
SNP chip and can be used to map disease-associated genes. A small silicon glass
chip is used on which short, single-stranded DNA molecules are adhered in a grid-
like pattern that corresponds to known SNP variants; this chip is called a SNP chip.
The isolated DNA from the individual is then converted into labeled probes (fluo-
rescently labeled single-strand segments) and is incubated with the SNP chip. The
probes then bind to their respective SNPs due to the complementarity between them;
the fluorescence is then read with a scanner to identify the specific SNPs. Whole
SNP map can be made accordingly for each individual person and can later be used
for various types of comparison. This comparison can be used to determine which
SNPs segregate with a particular disease. Because the SNP sequences have already
been mapped to specific chromosomal locations, researchers can use the obtained
information to tag the specific disease-related gene to individual chromosomes.
In addition to this, various bioinformatics tools are available that can compare the
sequence of a gene with unknown function to the rest of the genome and find similar
genes with known functions. Researchers can also try to predict the function of
unknown proteins from different genes. Large-scale studies of genes and their
expression levels in healthy and diseased individual can be done and are called
genomics.
In order to study gene expression, gene chips are created that are bound to small
single-stranded DNA segments. Next a sample from tissue of interest is produced
that contains the fluorescently labeled single-stranded complementary DNA
(cDNA). This sample is then incubated with the gene chip. Using the complementary
binding property, a laser then scans for the fluorescent signal producing an expres-
sion profile for the target tissue. This expression data from healthy individuals and
those with diseases can then be used for the comparison and identification of
disrupted process. Proteomics utilizes the similar approach through large-scale
gene chips to study thousands of proteins within a given cell population at the
same time. Similar to gene chips in order to carry out the translational studies,
protein chip grids are used. In this approach, fluorescently labeled protein sample
from the target individual is prepared and is incubated with the antibody-coated chip
so that the specific protein binds to their respective antibody which is read by a laser.
Using proteomic approach, the protein profile of any individual can be made and
19 Developmental Genetics 1003

compared with the diseased individual’s profile in order to determine the altered
proteins.
Owing to the conservation of key processes and their molecular components
across the evolutionary tree, model organisms like mice, frogs, worms, flies, and
yeast are studied to understand the basic cellular processes and their underlying
molecular mechanisms. In a similar fashion, genes associated with single-gene
diseases and the underlying molecular mechanisms can be elucidated using model
organisms. One such model organism is mice in which most of the homologous
human disease genes are found and much of our understanding of human disease
comes from these studies. Various disease models in mice have been generated using
mutation or deletion of disease-associated genes where detailed phenotypic as well
as functional analyses can be carried out.

19.5.3 Control of Hox Gene Expression

Expression of Hox genes is regulated by three mechanisms, transcriptional regula-

tion from earlier segmentation genes, a cellular memory system based on the action
of Polycomb (PcG)/trithorax (trxG) group proteins, and cross-regulatory interactions
among the Hox genes themselves. Hox genes regulate each other by posterior
prevalence; posterior Hox genes are able to repress the expression (and suppress
the function) of more anterior genes. In vertebrates, Hox genes are regulated by
temporal collinearity.
where these genes are kept globally silent but are activated during initial stages of
development through temporal sequence that correlates with the gene’s position
within the cluster in a 30 to 50 direction. After initial activation, PcG and trxG system
of proteins regulates the Hox genes to produce specific expression patterns. The
major determinant of Hox gene regulation is functionally autonomous transcrip-
tional cis-regulation at spatial level. Several studies have revealed a role of retinoic
acid in the regulation of Hox gene expression in higher animals including humans
along the anterior-posterior axis. Retinoic acid induces the expression of genes at the
30 ends of the Hox clusters, thereby regulating the body plan of anterior embryo
while having no effect on 50 end Hox genes. In addition, Hox clusters have also been
known to harbor various noncoding RNAs (ncRNAs). One such example is
HOTAIR which is negatively regulated by polycomb-group proteins (PRC2) in
trans.

19.6 Arabidopsis thaliana: Homeotic Gene

Homeotic genes comprise a highly conserved gene set responsible for regulating the
anatomical structure development throughout the evolution tree. Mutations in home-
otic genes result in homeosis, ectopic placement of body parts which are usually
lethal. Flower formation is initiated when vegetative meristem is converted to floral
meristem due to the activity of heterochrony or flowering time genes. The flower
1004 D. Vimal and K. Banu

Fig. 19.23 The ABCE model of floral organ identity. According to this model, the flower
meristem is divided into three regions, A, B, and C, with overlapping gene activity that altogether
defines two adjacent whorls of a flower. Region A determines sepals, while region C specifies
carpels. However, the joint activity of regions A and B determines petals, and activity of regions B
and C determines stamens. Different genes are required for this specification process such as
APETALA1(AP1) and APETALA2(AP2) are necessary for region A, while two genes,
APETALA3(AP3) and PISTILLATA(PI), are required for region B and AGAMOUS(AG) gene
is required for region C. (Adapted from Chanderbali et al. 2010)

meristem is then converted into a flower and is regulated by flower meristem identity
genes. Once the flowering starts in plants, the whorl (sepal, petal, stamens, and
carpel) formation, development, pattern, and structure are regulated by two sets of
genes called cadastral genes and homeotic genes in the same sequence (Fig. 19.23).
The floral development was studied in detail by George Haughn and Chris
Somerville in 1988 in two plant species, Arabidopsis thaliana and Antirrhinum
majus, and ABC model of floral development was given. According to ABC
model, five homeotic genes, A, B, C, D, and E, regulate the formation and develop-
ment of floral organs. All the homeotic genes have been studied in detail through
single gene mutation studies, and it was found that the sepal whorl is regulated by the
action of gene A, while both A and B genes are responsible for petal whorl. The
stamen and carpel whorl are regulated by the co-expression of the B and C and C
genes, respectively. Genes A and C work in an antagonistic manner. Gene D has
been known to regulate the ovule formation and development, whereas gene E is
responsible for the regulation of normal physiological functioning of whole flower.
19 Developmental Genetics 1005

19.7 Caenorhabditis elegans: Development

C. elegans is a free-living transparent soil nematode of ~1 mm in length and shows

bilateral body symmetry. It derives its name from two Greek words: caeno which
means recent and rhabditis which means rod-like. It is a sexually dimorphic organ-
ism with most of the population being hermaphrodites (XX) producing both eggs
and sperm while only 0.001 percent population being males. Hermaphrodites may
self-fertilize or cross-fertilize with the males. C. elegans genome consists of
5 autosomes and X chromosome comprising of ~97,000 kb per haploid genome
which encodes for ~18,000 proteins. C. elegans genome was the first multicellular
genome to be sequenced and served as the basis for human genome project frame-
work. The basic anatomy of C. elegans includes a mouth, pharynx, intestine, gonad,
and collagenous cuticle without any respiratory or circulatory system (Fig. 19.24).
The outer body wall consists of an outer protective epidermis and underlying
muscular layer. There are two tubes in the body of C. elegans: one that is made of
endodermal cells forms the intestine, and the other present between the intestine and

Fig. 19.24 Lateral view of adult hermaphrodite. In the hermaphrodite nematode, sperms are stored
in the storage organ, spermatheca, such that eggs passing through them get fertilized before
reaching the vulva. (Adapted from Gilbert 2000)
1006 D. Vimal and K. Banu

body wall made of somatic cells constitutes the gonad. The adult has 945 cells with
959 somatic nuclei and 302 neuronal cells derived from 407 neural precursor cells.
Sperm enters the oocyte in spermatheca followed by fertilization and is also
responsible for the establishment of A-P axis. Fertilized zygote then enters the uterus
where it undergoes rotational holoblastic cleavage. The microtubule organizing
center of the sperm directs the movement of the sperm pronucleus to the future
posterior pole of the embryo also inducing the movement of PAR proteins in the
embryo which results in asymmetric and asynchronous first cleavage divisions. Each
asymmetrical division produces one founder cell (denoted AB, MS, E, C, and D)
which further produces different cell types and one stem cell (P1-P4 lineage). All the
558 cells of the small worm inside the egg shell are generated by the divisions in the
descendants of each founder cell at specific times and are named according to their
positions relative to their sister cells. The first cell division results in the formation of
cleavage furrow which is located asymmetrically along the A-P axis of the egg
resulting in the formation of a larger blastomere founder cell (AB) and a smaller
blastomere stem cell (P1). At the four-cell stage, the second cell division results in
the formation of ABp and ABa cells due to equatorial division of anterior founder
cell (AB) and a posterior stem cell (P2) and EMS due to meridionally (transversely)
dividing P1 cell. Before the egg is laid, mRNAs of the maternal genes are
accumulated in the egg which interacts with one another directing the early devel-
opment; these genes are called maternal effect genes.
The A-P axis is decided on the basis of the position of sperm pronucleus. When
sperm enters the oocyte, sperm pronucleus is pushed to the nearest end of the oblong
oocyte by the centriole due to cytoplasmic movements; this end becomes the
posterior pole. Another determinant of the A-P axis is the migration of the P
granules, ribonucleoprotein (RNP) complexes that function in specifying the germ
cells. P granules are initially distributed uniformly in oocyte but associate at poste-
rior pole just before the first cleavage so that they only enter the P1 blastomere. The P
granules from the P1 cell are passed to the P2, P3, and eventually to P4 cell,
descendants of which become the sperm and eggs of the adult. The partitioning of
the P granules is thought to be regulated by the par (partition-defective) genes. Par
(partitioning-defective) gene family is a maternal effect gene family containing six
genes that are responsible for organizing cell asymmetry and polarization of the
cytoskeleton in nematode egg. Par genes have homologs in vertebrates. Par proteins
orient the mitotic spindle in such a way that P granules are segregated to only the
posterior daughter cell and not to the anterior daughter cells. In this way, at the
16-cell stage, there is just one cell that contains the P granules which gives rise to the
germline.
The dorsal-ventral axis of the nematode is defined through Wnt and Notch
signaling, by the second division of the AB and P1 cells with their descendant
cells. P2 cell expresses a homolog of the Notch ligand, Delta, while the ABa and
ABp cells express the corresponding transmembrane receptor, homolog of Notch.
Due to the elongated shape of the nematode egg, ABa acquires the anterior end,
while ABp and EMS acquire leaving only ABp and EMS cells exposed to the signal
from P2. This signal acts on ABp, making it different from ABa and defining the
19 Developmental Genetics 1007

Fig. 19.25 Cell lineage chart. The germline segregates into the posterior portion of the most
posterior (P) cell. Three cell lineages, AB, C, and MS, are produced from the primary cell divisions.
The newly hatched larva contains a total of 558 cells, with different numbers of cells (in brackets)
belonging to different tissue types, while more divisions further produce the 959 somatic cells of the
adult. (Adapted from Gilbert 2000)

future dorsal-ventral axis of the worm (Fig. 19.25). The ABp cell defines the future
dorsal side of the embryo, while the EMS cell, the precursor of the muscle and gut
cells, marks to future ventral surface of the embryo. In addition, P2 also expresses
Wnt protein, which acts on the Frizzled protein (Wnt receptor) present on the
membrane of the EMS cell resulting in the orientation of the mitotic spindle. As a
result of the Wnt signal from P2, EMS gives rise to two daughter cells, one MS cell,
which give rise to muscles and various other body parts, and an E cell, which is the
precursor for all the cells of the gut. The left-right axis is specified at the 12-cell stage
where the descendant of EMS cell, MS blastomere, contacts the descendant cells of
the ABa cell, distinguishing the right side of the body from the left side.
The molecular mechanism involved in the developmental potential of the indi-
vidual cells in the embryo can be determined by various methods which includes
laser microbeam microsurgery and genetic screens. Laser microbeam microsurgery
can be used to alter the cell’s environment by killing the neighboring cells or
rearranging the cells inside the eggshell. Such studies revealed that if the relative
positions of ABa and ABp are flipped at the four-cell stage of development, Aba
acquires the fate of the ABp cell and vice versa showing that the two cells initially
have the same developmental potential and depend on the signals from their
neighbors to make them different. Genetic screens with different mutant worm
strains are used to study the cell-cell interaction. Two mutants, one in which no
gut cells are induced (mom mutants) and another in which extra gut cells are induced
(pop mutants), were used in order to understand the P2-EMS cell interaction.
Genetic screening of the mom genes revealed that these genes encode for the Wnt
1008 D. Vimal and K. Banu

signal protein that is expressed in the P2 cell, as well as for the Frizzled protein
(a Wnt receptor) that is expressed in the EMS cell. In the absence of this signaling
between P2 and EMS in mutant worms, both the daughter cells of the EMS cell fail
to induce resulting in more mesoderm. On the other hand, in the absence of pop-1
gene activity both daughter cells of the EMS receive the Wnt signal from P2
resulting in plenty of pharynx due to extra gut.

19.8 Caenorhabditis elegans: Vulva Formation

The vulva is a hermaphrodite female-specific organ of utmost importance in

C. elegans as it connects the hermaphrodite uterus to the outer environment. It is
required for egg laying after internal fertilization and for sperm injection during
copulation with male. Vulval development is extensively studied in C. elegans as it
provides an excellent method for studying the process of morphogenesis and tissue
remodeling (Ririe et al. 2008). Another advantage in studying vulva formation is the
ease of genetic analyses of signal transductions and regulatory pathways that work as
the bridge between network pathways and organogenesis. Vulval development
occurs during the L3-L4 larval development stages, but the opening can only be
seen after L4-adult molting (Fig. 19.26). Vulval development includes some major
steps:

1. Generation of vulval precursor cells (VPCs): After hatching, L1 larva of

C. elegans consists of a set of 12 ventral epidermal blast cells called the Pn.
p cells from P1.p to P12.p. Anterior Pn.p cells give rise to neuronal daughter cells,
while posterior Pn.p cells acquire different fates, viz., primary, secondary, or
tertiary. During larval development six epidermal blast cells named P3.p–P8.p on
the ventral surface are induced by a somatic gonadal cell, anchor cell, to become
the vulva. Through LIN-3/epidermal growth factor (EGF) and LIN-12/Notch
pathways, these epidermal cells are induced to become the VPCs. P6.p acquires
the primary fate to become the 1
vulval precursor cell (VPC), while P5.p and P7.
p are induced to acquire secondary fates and become 2
VPCs. P1.p–P4.p and P8.
p–P11.p acquire the tertiary fate where they fuse either directly or after a single
cell division with the syncytial hypodermis hyp7. P12.p gives rise to two daugh-
ter cells of which the posterior progeny cell, P12.pp, undergoes apoptosis, while
the anterior daughter cell, P12.pa, forms anal hypodermis called hyp12
2. In order to understand the signals that provoke different Pn.p cells to assume and
acquire a particular developmental fate, different experimental methods like laser
ablation and mutation studies are done. In the absence of induction by anchor cell
both due to ablation of anchor cell or signal cascade, all the VPCs acquire the
tertiary fate and fuse with hyp7, and no vulva formation takes place. The
phenotype thus produced is called Vul for vulvaless. In case of removal or
incompetence of any Pn.p cells, the remaining ones can acquire and assume its
position and function depending upon the time of ablation. Thus all the Pn.p cells
from P3.p to P8.p form the vulval equivalence group where all the cells are
19 Developmental Genetics 1009

Fig. 19.26 Morphogenesis of the vulva in C. elegans. In the L3 stage larvae, a somatic gonadal
cell, anchor cell (AC), induces three precursor cells, P5.p, P6.p, and P7.p, out of the total six (P3.p–
P8.p) vulval precursor cells (VPCs) to adopt the vulval fates. The non-vulval cells, P3.p, P4.p, and
P8.p, undergo a single proliferation step and fuse with the underlying syncytial cell. After the
formation of vulva primordium, the cells from the outer regions carry out short-range migrations in
a symmetrical fashion toward the central midline fusing with specific partners. A seven-toroid (vulA
to vulF) stack is formed in the center where component cells from each ring fuse in a specific
manner resulting in the formation of epithelial tube connecting the uterus to the outside. (Adapted
from Sharma-Kishore et al. 1999)

competent enough to acquire each other’s role ensuring normal vulva formation.
For example, in case of P6.p ablation, the surrounding Pn.p cells, P5.p or P7.p,
change their fates and adopt the primary fate, and P4.p or P8.p adopts the
secondary fate resulting in the normal vulva formation. Ablation of gonads or
anchor cells results in all VPCs acquiring the 3
fate. Multiple genes affect the
VPC generation and induction, including let-23, lin-12, lin-39, and sem-4. In the
absence of lin-39 expression, VPC generation is terminated due to the fusion of
Pn.p cells with hyp7 epidermis. Sem-4 encodes a zinc finger protein and is
required for the expression of the lin-39, which can also directly affect the VPC
generation
3. Division and patterning of VPCs to produce different progeny cells: After the
VPCs have acquired their sublineage fate, they start to divide and form their
progeny cells. During L3 larval development stage, VPCs is induced by anchor
cell and regulated through let-23 and lin-12 signaling pathways. Three cell
divisions occur in both 1
(P6.p) and 2
VPCs (P5.p and P7.p) to produce eight
cells and seven cells each respectively to form a vulval primordium consisting of
1010 D. Vimal and K. Banu

22 progeny cells. Both 1
and 2
VPCs undergo different morphogenesis regime:

1
VPC produces progeny cells vulE and vulF, while 2
VPCs produce progeny
cells of type vulA, vulB1, vulB2, vulC, and vulD. The division pattern in
secondary fate is asymmetric, and all the progeny cells thus formed result in
vulval structure formation.
4. Anchor cell invasion: After the cell division, all the VPCs are arranged in a linear
fashion with anchor cell in the middle. During the L3-L4 molt, the 1
VPC starts
to move dorsally such that the homotypic cells on both sides of the anchor cell are
migrated toward the center facing each other. As a result a lumen is formed
surrounded by VPC’s progeny cells in early L4 stage. Ring formation and
invagination of VPCs continue resulting in a stack of cells in a dorsal-ventral
manner. The structure thus formed is called toroid with a central hole surrounded
by circular group of cells. Vulval invagination is regulated by eight sqv genes
(sqv-1 to sqv-8). sqv genes encode enzyme for glycosaminoglycan synthesis that
is required to maintain the shape of forming lumen. During toroid formation the
similar cells on both sides of the midline starts to fuse together except vulB1 and
vulB2 generating a multinucleate syncytial structure. In the uterine lumen that
formed, the uterine and vulval epithelial are separated by a thin syncytium utse
membrane (Fig. 19.27).

After the formation of lumen, a connection is established between lumen and

uterus. Uterine anchor cell invades the basement membrane reaching the central 1

VPCs. On the dorsal side, vulF cells contact the uv1 cells on the ventral side of
uterus. The vulF and uv1 cells are covered by a syncytial utse membrane formed by
the fusion of AC cell with uterine cells. The utse membrane separates the uterine and
vulval lumens. The vulE cell extends laterally and contacts with the hypodermal
seam cells that stabilize the vulval structure. The opening and closing of the vulva is
controlled by eight muscle cells, four vm1 and four vm2 that are in turn controlled by
motor neurons. vm1 cells connects the vulva between vulC and vulD toroids, and
vm2 connects vulF and the uterus. On the ventral side, vulA cells contact the hyp7
hypodermal syncytium. During hermaphrodite to adult molt in, finally, the vulva
turns inside out resulting in an everted vulva with a slit for egg laying and copulation.

19.9 Cell Signaling Network in Development

During development, the ability of cells to respond to their microenvironment is

dependent upon the communication process through a numerous number of signal-
ing pathways. This communication occurs between different components within a
cell, adjacent cell, as well as environment that governs basic activities of cells and
coordinates multiple-cell actions to bring about development, tissue repair, and
immunity, as well as normal tissue homeostasis (Fig. 19.28). The signaling network
comprising of binding of a ligand to their specific receptors leading to the activation
of intracellular machinery is a highly conserved mechanism. This general signaling
19 Developmental Genetics 1011

Fig. 19.27 Transverse section of late L4 stage worm showing vulval region and uterus. Vm1s and
vm2s are the vulvar muscles present in two sets of four, where vm1s connects body wall and vulva
while vm2 muscles connect ventral body wall and vulF. (Adapted from Sharma-Kishore et al. 1999)

network comprises of various interconnected signaling pathways as the same signal-

ing component is capable of receiving signals from multiple inputs.
Cell signaling can be of two types depending upon the type of signal: mechanical
where cell sense and response to the forces exerted on the cell or biochemical in
which biomolecules like lipids, proteins, ions, and gases acts as signal. Signals can
be categorized as intracrine, paracrine, or endocrine depending upon the distance
between the secretory cell and its target. Intracrine signals are produced by the target
cell and act inside a cell, regulating intracellular events. Examples of intracrine
signaling include several protein or peptide hormones like renin-angiotensin system:
angiotensin II and angiotensin, fibroblast growth factor 2, and parathyroid hormone-
related protein. Similar to intracrine signaling, autocrine signals can function on the
same cell, but they can also act on nearby cells if they are of same type. Autocrine
signals, hormone or chemical messenger, are produced by the target cell, secreted,
and bind to the same cell through receptors. Examples of autocrine signaling include
immune cells like T-cell lymphocytes and cytokine interleukin-1 in monocytes.
Juxtacrine signaling is also called contact-dependent signaling as it targets the
adjacent cells. A juxtacrine signal can be the ligand from a membrane or a combina-
tion of two membrane proteins from neighboring cells. An intracellular protein or
1012 D. Vimal and K. Banu

Fig. 19.28 Signal transduction. Mechanism of signal transduction includes three steps, reception,
transduction, and response. Ligand binds on specific receptors that produce secondary messengers
which carry the message to the nucleus or other organelles which in turn respond to this message by
carrying out different metabolic reactions. (Adapted from https://biologydictionary.net/signal-
transduction)

compartments of single cell or combination of two adjacent cells can also function as
juxtacrine signal. These signals may function to carry out communication either
between two cells or a cell and its extracellular matrix. In cell to cell signaling, a cell
expresses a specific ligand on the surface of its membrane which binds to the
appropriate cell surface receptor or cell adhesion molecule present on the adjacent
cell. This type of communication can be seen in the Notch signaling pathway
involved in neural development. In one type of juxtacrine signaling, two adjacent
cells can construct communicating channel between their intracellular
compartments, for example, gap junctions in animals and plasmodesmata in plants.
Cell to extracellular matrix signaling can be seen during cell cycle and cellular
differentiation where the cells interact with the glycoproteins secreted by extracellu-
lar matrix through a receptor integrin. Another type of cell-to-cell communication is
paracrine signaling where cell produces a signal into the immediate extracellular
environment that diffuses over a relatively short distance to induce changes in
nearby cells altering its behavior. The highly conserved receptors and pathways of
the paracrine signaling can be organized into four major families based on similar
structures: fibroblast growth factor (FGF) family, Hedgehog family, Wnt family, and
TGF-β superfamily. Endocrine signaling targets distant cells through hormones
produced by endocrine cells which travel through the blood to reach all parts of
19 Developmental Genetics 1013

the body. A number of endocrine glands signal each other in sequence and regulated
by feedback loops are usually referred to as an axis, for example, the hypothalamic-
pituitary-adrenal axis. In comparison, exocrine glands secrete hormones to the
outside of the body utilizing ducts to distribute them throughout the body; common
examples include sweat glands, gastrointestinal tract glands, and salivary glands.
Major endocrine systems include TRH-TSH-T3/T4, GnRH-LH/FSH sex hormones,
CRH-ACTH-cortisol, renin-angiotensin-aldosterone, and leptin-insulin systems.
Hormones secreted by endocrine systems can be categorized into proteins, steroids,
and eicosanoids. All the cellular, physiological, and behavioral function of any
organism is dependent upon the intricate crosstalk between these hormones and
different tissues and organs.

19.9.1 Wnt Pathway

Wnt pathway is one of the most conserved signaling pathways throughout the animal
kingdom that regulates multiple cellular processes (Komiya and Habas 2008). Wnt
signaling plays a critical role in embryonic development, axis patterning, cell fate
specification, cell proliferation, cell migration, and insulin sensitivity (Cadigan and
Liu 2006). The name Wnt is derived from Drosophila segment polarity gene
wingless (Wg) and its vertebrate homolog integrated or int-1. Wnt signaling pathway
was first identified in retroviruses for its role in carcinogenesis. Further, it was found
that int-1 is actually a homologous gene of Wg in Drosophila extensively involved
in embryonic development. Wnt pathway is known to function in at least two
major ways: canonical or Wnt/β-catenin pathway, non-canonical planar cell polarity,
or non-canonical Wnt/Ca2+. Wnt-protein ligand binds to the receptors of Frizzled
family, thereby transmitting the signal intracellularly to the Dishevelled protein.
Wnt ligands are the secreted glycoproteins of ~40 kDa containing several
conserved cysteine residues and are extensively palmitoylated at a conserved serine
residue. The lipid modification on Wnts is required for efficient signaling, binding of
Wnt to its secretion carrier protein, Wntless (WLS), so it can be transported to the
plasma membrane for secretion and for binding to its receptor Frizzled. Similar to
many secretory proteins, Wnt ligands undergo glycosylation in endoplasmic reticu-
lum and are then secreted into the extracellular matrix. The palmitoylation of the
Wnt proteins is regulated by porcupine protein which in turn is regulated by wntless
or evenness interrupted proteins and the retromer complex (Fig. 19.29).
In addition to the receptor, some molecules like low-density lipoprotein-related
protein 5/6 (LRP5/6), receptor tyrosine kinase (RTK), and ROR2 work as the
co-receptor for the signaling. In contrast, some molecules like Dickkopf (Dkk)
proteins, Wnt Inhibitory Factor-1 (WIF-1), and secreted Frizzled-Related Proteins
(sFRPs) work as antagonists to Wnt signaling by binding either to Wnt ligand or to
its receptor.
A basic mechanism of Wnt signaling includes the binding of Wnt ligand to the
extracellular N-terminal cysteine-rich domain of a Frizzled (Fz) family receptor.
Frizzled (Fz) receptor is a transmembrane receptor spanning the plasma membrane
1014 D. Vimal and K. Banu

Fig. 19.29 Wnt biogenesis and secretion. Wnt proteins are highly modified before becoming
mature as a ligand. Wnt proteins are first glycosylated and then lipid modified in the endoplasmic
reticulum which is regulated by porcupine. After the maturation these proteins are transported from
Golgi body to the plasma membrane for secretion by wntless. (Adapted from Macdonald et al.
2009)

seven times and constitutes a distinct family of G protein-coupled receptors

(GPCRs). After activation Fz directly interacts with the intracellular phosphoprotein
Dishevelled (Dsh) and transmits the signal. Dsh is a highly conserved Wnt signaling
molecule present in all organisms and contains conserved domains: an amino-
terminal DIX domain, a central PDZ domain, and a carboxy-terminal DEP domain.
Dsh protein activation might then branch Wnt pathway into various directions
(Fig. 19.30).
Canonical Wnt pathway: Canonical pathway is also called Wnt/β-catenin
pathway as it is dependent on the protein β-catenin. The hallmark of the canonical
pathway is the accumulation of the protein β-catenin into the cytoplasm which is
19 Developmental Genetics 1015

Fig. 19.30 Overview of Wnt/β-catenin signaling. In the absence of Wnt, cytoplasmic β-catenin
levels are very low due to the proteasome-mediated degradation which is regulated through GSK-3/
APC/Axin complex. However, in the presence of Wnt, this complex is inactivated leading to an
increase in the levels of β-catenin in cytoplasm as well as nucleus which in turn interacts with
transcription factors inducing different target genes. (Adapted from MacDonald et al. 2009)

subsequently translocated to the nucleus. In the absence of inducing Wnt signal,

β-catenin in the cytoplasm is destined to degradation by a cytoplasmic destruction
complex that comprises of Axin, adenomatosis polyposis coli (APC), protein phos-
phatase 2A (PP2A), glycogen synthase kinase 3 (GSK3), and casein kinase 1α
(CK1α). β-catenin is targeted for its ubiquitination by proteasomal machinery after
its phosphorylation. In contrast, when Wnt binds to the Fz and LRP5/6 receptor, it
results in the translocation of the Axin to the plasma membrane inactivating the
β-catenin destruction complex. CK1γ or GSK3 phosphorylates LRP5/6 which in
turn dephosphorylates Axin resulting in the reduction of Axin stability and concen-
tration. Further, Dsh becomes phosphorylated and activated resulting in inhibition of
GSK3 of destruction complex by DIX and PDZ domains of Dsh protein. Upon
inactivation of destruction, complex β-catenin starts to accumulate in the nucleus and
induces target genes with TCF/LEF (T-cell factor/lymphoid enhancing factor) tran-
scription factors.
Non-canonical Wnt pathway: Non-canonical pathway can further branch into
two pathways, viz., planar cell polarity (PCP) pathway and Wnt/calcium pathway.
Both of these pathways work in a β-catenin independent fashion. In non-canonical
pathway, receptor Fz uses NRH1, Ryk, PTK7, or ROR2 as a co-receptor for signal
transduction instead of LRP-5/6. Using one of these co-receptors, the signal is then
transmitted to the Dsh protein activating its PDZ and DIX domains which in turn
recruits Dishevelled-associated activator of morphogenesis 1 (DAAM1) protein to
form a complex which in turn activates Rho and Rac protein pathways. In the first
pathway, Dsh-Damm1 activates a Rho guanine exchange factor which in turn
1016 D. Vimal and K. Banu

activates the Rho GTPase. Rho activates Rho-associated kinase (ROCK) and
myosin, which further regulates the cytoskeletal rearrangement. In the second
pathway, DEP domain of Dsh activates the Rac GTPase which further stimulates
JNK activity.
The non-canonical Wnt/calcium pathway is also independent of the β-catenin.
This pathway controls the intracellular calcium levels by regulating the release of
calcium from endoplasmic reticulum (ER). Upon activation of Fz, it interacts with
a trimeric G protein leading to the activation of PLC or cGMP-specific PDE
domain of Dsh. Upon activation of PLC, PIP2 present in the plasma membrane is
cleaved into its two components, DAG and IP3. IP3 binds to its receptor on the
ER releasing calcium in the cytoplasm. Increased concentrations of calcium and
DAG activate protein kinase C which further activates Cdc42, calcineurin, and
calcium/calmodulin-dependent kinase II (CaMKII). All these components regulate
ventral patterning, cell adhesion, migration, and tissue separation. Calcineurin can
interfere with TCF/ß-catenin signaling in the canonical Wnt pathway by activating
TGF-β-activated kinase (TAK1) and Nemo-like kinase (NLK). Activation of PDE
domain of Dsh leads to the inhibition of PKG which in turn impedes the calcium
release from the ER.

19.9.2 Hedgehog Pathway

The Hedgehog (Hh) signaling pathway is one of the key conserved pathways in a
wide range of organisms regulating many developmental processes (Jia et al. 2015).
The Hedgehog (Hh) signaling pathway was first identified in the Drosophila as one
of the genes required for establishing anterior-posterior body axis of the fly.
Christiane Nüsslein-Volhard and Eric Wieschaus performed genetic screens in
Drosophila embryo in order to understand the body segmentation. The protein
derived its name from the appearance of short and spiked phenotype of the cuticle
in Hh mutant embryos which resembles the spikes of a hedgehog.
Drosophila contains a single Hh gene as compared to three homologs in verte-
brate with different spatial and temporal distribution patterns: Sonic Hedgehog
(Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh). Hh undergoes several
post-translational modifications in order to become fully functional. A precursor Hh
protein consist of a signaling N-terminal called “Hedge” domain and a protease
C-terminal called “Hog” domain. The Hog domain can further be divided into a Hint
domain at N-terminal and sterol-recognition region (SRR) at the C-terminal. At the
C-terminal hint domain, a cholesterol moiety is added, while at the N-terminus,
palmitoyl acyltransferase adds a palmitoyl moiety. After autocatalytic cleavage of
the precursor Hh molecule, a dually lipidated active signaling molecule called HhNp
is released from the secreting cell by a transmembrane transporter protein called
Dispatched (Disp). In addition, Scube2, cell surface protein LRP2, and the Glypican
family of heparan sulfate proteoglycans (GPC1–6) also help in trafficking over
19 Developmental Genetics 1017

multiple cells. Functional Hh proteins initiate signaling at the plasma membrane by

binding to transmembrane receptor Patched (PTCH1) facilitated by co-receptors
GAS1, CDON, and BOC.
Hh signaling is regulated by a Hh signaling complex, HSC, comprising of the
transcription factor Cubitus interruptus (Ci), serine/threonine kinase Fused (Fu),
kinesin-like molecule Costal 2 (Cos2), and suppressor of fused (Sufu). In the
absence of Hh, this complex remains bonded to the microtubule in the cytoplasm.
In the absence of Hh signaling, the Ci- Cos2 complex present in the cytoplasm is
targeted by a degradation complex which contains protein Slimb in Drosophila. This
degradation complex targets the Ci protein for proteasome-dependent cleavage
resulting in the generation of protein fragment called CiR. CiR diffuses into the
nucleus from the cytoplasm where it acts as a co-repressor for Hedgehog (Hh) target
genes. In contrast, upon activation by Hh, the complex dissociates and the Cos2-Fu-
Ci complex interacts with the C-tail of Smo domains regulated by Cos2 phosphory-
lation leading to formation of active Ci and consequent pathway activation. In
addition, Hh binds and inhibits a cell surface transmembrane protein called Patched
(PTCH) which in turn results in the accumulation of Smoothened (SMO) inhibiting
the cleavage of Ci. Thus Ci and CiR levels increase and decrease respectively in the
cell allowing Ci to act as transcription factor for many genes in nucleus.

19.9.3 TGF-b Pathway

It is an evolutionary conserved pathway regulating a wide number of cellular

processes including cell growth, differentiation, and development in a wide range
of organisms. TGF superfamily includes various ligands like growth and differenti-
ation factors (GDFs), bone morphogenetic proteins (BMPs), anti-Müllerian hormone
(AMH), Activin, Nodal, and TGF-βs. The result of TGF-β signaling pathway
depends upon the control of the spatial and temporal expression of more than
500 genes which in turn is dependent on the functions of the SMAD protein and
cofactors. There are three major classes of SMAD protein family, R-SMAD (SMAD,
SMAD1–3, and SMAD5), co-Smad (SMAD 4), and I-SMAD (SMAD6–7). An
SSXS motif is present at the C terminal of the receptor-regulated Smads
(R-SMAD) which is phosphorylated by the type I receptors which results in the
formation of phosphorylated R-Smad (p-R- SMAD) which in turn associates with
SMAD4. TGF-β signaling regulates the transcription of more than 500 genes which
contains one or more SMAD binding elements (SBEs) in their promoter region. In
corroboration to SMADs, several SMAD interacting transcription factors modulate
specific target gene expression depending on the cellular type in both physiological
and pathological conditions.
TGF signaling starts when TGF-β ligand binds to the serine/threonine type II
receptor kinase dimer which catalyzes the phosphorylation of the Type I receptor
dimer resulting in the formation of hetero-tetrameric complex. Ligand binding also
1018 D. Vimal and K. Banu

Fig. 19.31 TGF-β Pathway. TGF-β ligand binding results in the assembly of type I and type II
receptors which further transmits the signal by phosphorylation of R-Smad proteins. The activated
R-Smad proteins along with co-Smads translocate to the nucleus, thereby regulating the transcrip-
tion of target genes. (Adapted from Tecalco-Cruz et al. 2018)

phosphorylates cytoplasmic signaling molecules, Mothers against decapentaplegic

homolog (SMAD) family members, Smad1/2/3/5/9, called R-SMADs. The TGF-β/
activin pathway is induced by Smad2 and Smad3, while BMP pathway is induced by
Smad1/5/9. Phosphorylation at the C-terminal of the Smads allows the results in the
activation of co-SMAD (Smad4) forming a RSMAD/coSMAD complex which
translocate to the nucleus resulting in the transcription of target genes through
different transcription factors (Fig. 19.31). Smad6 and Smad7 function in the
antagonistic manner inhibiting the R-Smad activation; therefore both of these
Smads are used in negative feedback loop in activin/TGF-β and BMP signaling
pathway. In addition, two SMAD interacting proteins, Ski (Sloan-Kettering
19 Developmental Genetics 1019

Institute) and SnoN (Ski novel), disrupt the formation of R-Smad/Smad4 complex as
well as inhibit the SMAD association with the p300/CBP coactivators resulting in
the negative regulation of the TGF-β signaling pathway. Ski and SnoN indirectly
bind to the consensus sequence called SBE (50 -GTCTAGAC-30 ), thereby acting as
SMAD corepressors.

19.9.4 Receptor Tyrosine Kinase Pathway

Receptor tyrosine kinases (RTKs) comprise of the family of receptors that in

addition to working as receptors for ligand binding also function as enzymes leading
to intracellular phosphorylation event. RTKs are single-pass, type I receptors embed-
ded in the plasma membrane. Each receptor of RTK is comprised of two domains,
an extracellular domain present at the N terminal region and an intracellular
domain present at the C terminal region. Both the domains together comprise a
single hydrophobic transmembrane-spanning domain. Binding of a ligand (growth
factor or hormone) to the extracellular N terminal domain results in the oligomeriza-
tion generally dimerization of the RTKs called cross-phosphorylation. In
cross-phosphorylation, neighboring RTKs associate with each other resulting in
autophosphorylation and thus activation of the tyrosine kinases present in the
intracellular C domain of RTK (Fig. 19.32). The cross-phosphorylation of tyrosine
kinases produces binding sites for a variety of intracellular protein that further
propagate signal transduction. The proteins bind to the tyrosine kinases through
special domains called Src homology 2 (SH2) domain- and phosphotyrosine-binding
(PTB) domain (Hubbard and Miller 2007). The most common downstream signaling

Fig. 19.32 Receptor tyrosine kinase activation. Upon binding of ligand to the inactivated RTK
receptor (left), the receptors dimerize together and recruit various different proteins that in turn
activate each other through phosphorylation. These phosphorylated proteins in turn alter the gene
expression of target genes, thereby completing the signaling pathway. (Adapted from https://www.
nature.com/scitable/topicpage/rtk-14050230/)
1020 D. Vimal and K. Banu

pathways activated by RTKs are proto-oncogene tyrosine-protein kinase,

phosphoinositide 3-kinase, mitogen-activated protein kinase, janus kinases (JAKs),
signal transducer and activator of transcription proteins, etc.
RTKs target various intracellular signaling pathways; however the commonly
employed pathway is mitogen-activated protein (MAP) kinase cascade regulating
cell proliferation, cell death, differentiation, migration, and angiogenesis. When
SH2-containing proteins bind the activated RTKs, they cause Ras to bind GTP in
place of GDP and become active which in turn activates MAP kinase leading to the
amplification of original signal. The final step in this cascade is the phosphorylation
of transcription regulators, leading to a change in gene transcription.

19.9.5 Notch Pathway

It is a highly conserved signaling pathway evolutionarily present in most animals

regulating cell fate determination and tissue homeostasis during development in
adult. Notch was discovered in the Drosophila due to the appearance of a notch in
the wings of mutant flies. Most of the knowledge on the notch signaling pathway
comes from the studies on Drosophila and C. elegans mutants. Notch receptor is
composed of two domains, an extracellular (NECD) domain and an intracellular
(NICD) domain, both of which together form a single-pass transmembrane protein.
The NECD consists of multiple epidermal growth factor (EGF)-like repeats and
interacts with the Delta/Serrate/LAG-2 (DSL) family ligands mediating juxtacrine
signaling. The NICD of the Notch receptor consists of a transcriptional activation
domain (TAD) which acts as a transcriptional activator in association with CBF1/
Suppressor of Hairless/LAG-1 (CSL) family transcription factors. Notch receptors
are further processed in the endoplasmic reticulum and Golgi body through S1
cleavage and glycosylation resulting in a heterodimer consisting of NECD
non-covalently attached to the TM-NICD. This processed receptor is then
transported by endosome to the plasma membrane and gets embedded in it. In
mammals, Delta-like and Jagged family proteins serve as ligands for Notch signaling
receptors, while in Drosophila Delta and Serrate work as ligands.
Notch signaling is initiated when an adjacent cell expressing notch ligand binds
with notch receptor on another cell; this in result promotes a proteolytic processing
event. An enzyme TACE (TNF-α ADAM metalloprotease converting enzyme)
carries out the S2 cleavage resulting in the release of NECD bound to ligand
which is then endocytosed/recycled by the ligand-expressing cell. The remaining
receptor is then further cleaved by γ-secretase enzyme (S3 cleavage) releasing the
NICD which then translocates to the nucleus resulting in the activation of the target
genes: Myc, p21, and the HES-family members through CSL transcription factors
(Fig. 19.33).
19 Developmental Genetics 1021

Fig. 19.33 Notch pathway. Notch ligand is a cell-surface receptor that communicates with other
transmembrane ligands such as Delta (termed Delta-like in humans) and Serrate (termed Jagged in
humans) on adjacent cells, thereby transmitting short-range signals. Upon ligand binding the Notch
intracellular domain (NICD) gets cleaved and is released into the cytoplasm from where it
translocates to the nucleus and regulates transcriptional activity of target genes. (Adapted from
Kopan 2012)
1022 D. Vimal and K. Banu

Box 19.1: Scientific Concept: Variation and Constraint in Hox Gene

Evolution (Heffer et al. 2013)
The genes that regulate the body plan during embryonic development are
highly conserved throughout the animal kingdom despite the diversification
in body plan symmetry. However some of these conserved genes acquired new
function during the course of evolution which is thought to occur due to the
redundancy or duplication of genes. One such class of genes is Hox family
genes that are highly regulated and play important roles in body plan pattern-
ing during embryonic development. This opens up a new field of investigation
as how these genes acquired new roles due to redundancy and why did not get
lost during the course of evolution. In addition was this acquisition a single
step process or took several stages to convert into a gene with new functions.
The fushi tarazu (ftz) gene in Drosophila is a Hox gene and arose due to the
duplication of an Antennapedia (Antp)-like Hox gene. Due to the presence of
nearby Hox genes like Antp and/or Sex-combs reduced (Scr) which are
responsible for determining the segment identity during embryonic develop-
ment, it acquired a new function of segmentation in higher insects. During
early embryonic development in Drosophila, ftz and even-skipped (eve) are
expressed in alternating 7 vertical stripes constituting a total of 14 stripes
which in the future become the body segments of fly. The expression of ftz in
the seven stripes as compared to the single Hox-like domain requires a
dynamic shift in gene expression. In addition, ftz also plays important roles
during the central nervous system (CNS) development. Midline precursor-
2 cells show high ftz expression in segmentally repeating pattern during 5–6 h
of development followed by expression in other neuronal precursors which
reaches a second peak of abundance at 9 h of development. Moreover, ftz is
also expressed in the hindgut between 11 and 15 h of development. The
functioning of ftz requires a cofactor called Ftz-F1, an orphan nuclear receptor,
which facilitates the binding of ftz to DNA. Ftz-F1 binds the sequence element
50 -YCYYGGYCR-30 in the zebra element of ftz; this interaction is mediated
by a LXXLL motif.
Despite of the dynamic changes in sequence and expression of ftz, it is
found in all arthropod genomes examined to date. Tracking the ftz expression
and Ftz protein domains in different arthropod phylogeny revealed that in
myriapods and chelicerates, ftz retains the Hox-like expression unlike in
crustaceans, basal insect Thermobia, and several holometabolous insects
where striped expression can be seen. Ftz expression as a segmentation gene
or Hox gene in addition with loss or gain of cofactor interaction motifs is
highly variable throughout arthropods. Despite the variations in the ftz expres-
sion as segmentation or Hox gene, its expression is well conserved in the
developing CNS. Interestingly, it was found that the Antp homeodomain can
substitute for the Ftz homeodomain in the core function of ftz in CNS by

(continued)
19 Developmental Genetics 1023

Box 19.1 (continued)

acquiring a neurogenic ftz cis-regulatory elements (CRE) leading to its expres-
sion in a unique group of cells, thereby differentiating its role from neighbor-
ing Hox genes. It was found that homeodomain present in all Ftz proteins is
indispensable for its function unlike YPWM or LXXLL motifs which were
variably present in some organisms. Ftz acquired the neurogenic element
(NE) in the cis-regulatory region which regulates the ftz expression in specific
cells of the developing CNS. Since the function of Antp is also conserved in
the CNS, the question arises that Antp could have taken up the function of ftz
by acquiring the NE; however, this is not the case. This is evident by the
inability of entire Antp coding region under the control of ftz NE to rescue the
eve expression in neurons. Moreover, degeneration of CREs results in the
further change in the ftz expression in the stripped fashion, and change in the
sequences resulted in a switch in cofactor interaction. Ftz requires the
homeodomain for its function in developing CNS, while LXXLL motif is
indispensable for the segmentation function. Based upon all the above obser-
vation, it was proposed that functional loss and acquisition of new function
occur in the stepwise manner during evolution.
In conclusion, the requirement of gene function along with its protein
domain in a specific tissue applies a constraint on the gene, thereby leading
to its retention during evolution as seen in ftz, where CNS activity requires the
homeodomain. In comparison, other domains which are not essential for the
core function underwent extensive diversification leading to variations in the
gene. In this way the balance between constraint and variation allows func-
tional diversification as well as long-term retention during animal evolution
(Fig. 19.34).

19.10 Summary

• Developmental biology explains how different cellular processes interact with

each other at the molecular level to produce an anatomically as well as morpho-
logically complex organism starting from an embryo to adult.
• Different intrinsic as well as extrinsic signals direct the cell to acquire their fate
during development. Intrinsic signals are also called lineage information and are
inherited from the mother cell, while extrinsic signals, also called as positional
information, are received from the cell’s surroundings.
• During evolution the basic developmental pathways have been conserved ranging
from unicellular to higher organisms. This conservation has allowed the
researchers to dissect these processes and gain insights into it using various
model organisms.
1024 D. Vimal and K. Banu

Fig. 19.34 Ftz neurogenic element (NE). Duplication of Hox gene produced ftz which acquired
NE in the homeodomain which allowed it to be retained in almost all the organisms and is essential
for the CNS development. While new domains like LXXLL were acquired, other domains like
YPWM are degenerated. (Adapted from Heffer et al. 2013)

• Model organisms are animals that have certain characteristic features that allow
researchers to study them with ease like large cultures in confined laboratory
space, short generation time, large number of progeny, easy manipulation at the
molecular level, etc. The most widely used model organisms include Saccharo-
myces cerevisiae, Xenopus, Drosophila melanogaster, Mus musculus,
Caenorhabditis elegans, Arabidopsis thaliana, Danio rerio, and Escherichia coli.
19 Developmental Genetics 1025

• Various genetic approaches are extensively applied in order to study the devel-
opmental processes and understand the mechanisms behind it. These approaches
can be divided into two classes, forward and reverse genetics; the goal of both
processes is to associate a gene with its biological function.
• Forward genetics starts with the discovery of a mutant organism with distinct
phenotype followed by the identification of gene responsible. In contrast, reverse
genetics deals with the known candidate gene and then elucidating its function.
• With the identification of a sufficient number of genes and proteins that are
involved, researchers can acquire insights on the underlying molecular processes.
Despite the basic knowledge on these processes, they cannot be used to elucidate
the exact mechanism as behavior of any organism is the culmination of the very
intricate network of molecular processes.
• Therefore, various genetic tools are now being employed to gain insight on the
underlying complex but basic cellular processes.
• There exists a functional homology in developmental genetics, for example, the
Hox genes are conserved among animal kingdom and play important role in axial
patterning. The mutation in Hox genes is known to cause various developmental
defects in different organisms.
• The development of any organisms is based on the interplay and intercommuni-
cation between the underlying signaling networks. A great deal of research is
focused on unraveling the signaling cascade with upstream and downstream
interactions which regulate and relay the information carried on the specific
binding of ligands to their receptors thereby by a ligand to cell surface receptors
and finally to the cellular effectors such as metabolic enzymes, channels, or
transcription factors.
• This cascade is not essentially linear in its function; rather it is highly branched
leading to the interaction between the components of different pathways which
help to regulate multiple functions in a context-dependent manner.
• Recent studies indicate that there is a conserved set of signaling components as
well as pathways that receives signals from cell type-specific inputs and engages
cell type-specific machinery.
• The interconnection between different pathways may be on two levels: first at a
junction which functions as a signal integrator and second, nodes which split the
signal and route them to multiple outputs; this signaling can be both positive and
negative.
• Studies deciphering the signaling mechanisms involved in various human
diseases help to understand the deregulations in human disorders by considering
a broader range of molecular process types.
• In order to understand the larger scheme of cellular networks where different
signaling and metabolic networks function in an integrated fashion, there is a
need to find the common regulatory components of these pathways responsible
for the interconnection.
• By integrating the knowledge obtained in this way, it is possible to elucidate the
crosstalk between metabolic and signaling networks and to find the potential
targets to cure the said disease.
1026 D. Vimal and K. Banu

• During evolution, major pathways and key genes involved are both preserved and
modified to take on more specialized roles, for example, in vertebrates like
humans and mice, Hox genes have been duplicated over evolutionary history
and now exist as four similar gene clusters.

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Quantitative Genetics
20
Anindo Chatterjee

20.1 Introduction to Quantitative Genetics

An overwhelming amount of genetics that is taught in classrooms is derived from a

common assumption, which is that a given phenotype is the output of a given gene.
This one gene-one phenotype assumption actually does not hold true for the majority
of phenotypic traits. Most of the traits that you would have studied till now can be
classified as qualitative traits. They encompass different types or kinds of a trait. For
example, curly hair, wavy hair and straight hair are different kinds of qualities of
hair. Similarly, red or white eye colour in Drosophila refers to a qualitative trait, the
quality in question being the colour. Other examples include human blood-group
types (A, B, AB and O) and the shape of the fruit in squash (spherical, disc-shaped,
elongated, etc.). However, careful observation of the world around us informs us that
a given trait can be given a numerical value (or a unique phenotypic class), and these
numerical values tend to be part of a range of possible degrees and intensity of the
trait in question. For example, the protein content in a seed will have a numerical
value, and these values are going to vary even for seeds of plants of the same species.
Thus, protein content of a seed is a typical quantitative trait. Such traits are also
called continuous traits to distinguish them from traits that can be categorized in a
few phenotypic classes. The latter traits are known as discontinuous traits. The tall
and dwarf pea plants of Mendel are considered to be discontinuous traits. However,
we can also study the range of tallness or dwarfness of these plants and analyse it
quantitatively. Therefore, a given qualitative trait can sometimes be further studied
quantitatively. Human IQ scores are also considered to be a quantitative trait.
Quantitative traits are also called as polygenic traits. This is because the pheno-
typic expression of most quantitative traits is regulated by more than one gene. The
polygenic control of a trait was demonstrated by James Crow in an experiment

A. Chatterjee (*)
Jain University, Bangalore, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 1029
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_20
1030 A. Chatterjee

which showed that the resistance to DDT in a population of DDT-resistant Drosoph-

ila was conferred by genes present on chromosomes 2 and 3 and the X-chromosome.
Quantitative traits are also called multifactorial traits because the range of
phenotypes exhibited for a given trait is influenced by more than one factor. The
expression of quantitative traits is regulated by both genetic and environmental
factors. This was convincingly proven by Wilhelm Johannsen through his work on
Phaseolus vulgaris (broad bean) wherein he showed that both genetic and environ-
mental factors contribute to the 150 mg–900 mg range of seed weight observed in
this plant.
An important factor which is usually ignored in the study of classical Mendelian
genetics is the environment the organism grows in. The same genotype has the
potential to exhibit a range of overlapping phenotypes due to differing environmen-
tal factors such as temperature, pH, moisture content, etc. The phenotypic range
(or variation) shown by a given genotype due to variation in environmental factors is
called the norm of the reaction. Similarly, multiple forms of genetic interactions such
as pleiotropy and epistasis also result in phenotypic ranges for a given trait.
The study of quantitative genetics is considered very significant both from the
purview of basic sciences and its application potential. Selection pressures which
have modified multiple traits over an evolutionary scale of time have invariably
acted upon mutations in polygenic and/or multifactorial traits. An understanding of
quantitative genetics also enables us to predict the possible phenotype of the
offspring if the parental phenotypes are known. This is invaluable in artificial
selection and breeding, done to improve the yields of commercially important
plant and animal products.

20.1.1 Types of Quantitative Traits

Quantitative traits can be broadly classified into three types:

1. Meristic traits are traits that can be quantified and exhibit a phenotypic range, but
the numerical value associated with each phenotypic class is a whole number. For
example, the number of children borne by a female of any species, or the number
of seeds in a pod, can only be whole numbers. The number of children borne
cannot be 4.6 or 5.3. Similarly, the number of seeds in a pod cannot be a fraction.
Other examples include the number of eggs laid by a hen and the number of
bristles on the thorax of a fruit fly.
2. Threshold traits resemble qualitative traits superficially. The phenotype is either
present or absent for such traits. However, the underlying mechanism determin-
ing whether a phenotype will be expressed or not is polygenic and/or multifacto-
rial. For example, the symptoms of a given disease may only be expressed if a
certain minimum number of mutant alleles are present in an individual
(Fig. 20.1). The minimum number of mutant alleles required is the threshold;
however, the susceptibility to the disease increases progressively with the
increase in the number of mutant alleles present. Sometimes, the susceptibility
20 Quantitative Genetics 1031

Fig. 20.1 Diabetes as a threshold trait: The phenotype of the disease is not expressed until a
threshold number of predisposing alleles is not reached. The Y-axis plots the frequency or number of
individuals who carry these predisposing alleles in the human population

to a disease might be the output of an interplay between genetic and environmen-

tal factors; however, till this output does not reach a threshold value, the disease
phenotype will not be expressed. Such a mechanism is generally agreed to
regulate the phenotypic expression of type II diabetes.
3. Continuous variations are characterized by a potentially infinite number of
numerical values that can be assigned to the trait of interest. For example, the
variation in height in a population of students can range from a lower extreme to
an upper extreme value. Within this range of extreme values, one can observe an
infinite number of variations in height. Two people having the same height up to
1 decimal point may vary in height if it is calculated up to 2 decimal points, hence
the expression of infinite variation.

20.1.2 Multiple Gene Hypothesis (or the Polygene Hypothesis)

The study of quantitative traits is a pre-Mendelian effort. Famous statisticians

F. Galton and K. Pearson had already shown that quantitative traits like height and
intelligence are heritable. Due to the pre-Mendelian nature of that time, they were at
a loss to explain how the underlying system of inheritance functioned. The redis-
covery of Mendel’s work resulted in a heated debate over the application of
Mendelian principles to explain the inheritance of quantitative traits and the pheno-
typic variation seen in them. Biometricians used statistical techniques to study
quantitative traits and staunchly opposed the idea that single genes (of the nature
controlling Mendelian traits) can explain the inheritance of quantitative traits. On the
other hand, the Mendelians strongly believed that genes ultimately regulate the
inheritance and expression of quantitative traits. Experiments conducted by Wilhelm
1032 A. Chatterjee

Johannsen on the phenotypic variation in the weight of beans (Phaseolus vulgaris)

indicated that genetic components do contribute to quantitative trait expression. The
mathematician George Uduy Yule in 1906 demonstrated that phenotypic variation in
a quantifiable trait can be derived from a system of multiple genes interacting
together. Finally, Ronald Fisher in 1918 showed that variation in quantitative traits
is an end product of the cumulative effect of multiple genes. It is now broadly agreed
upon that multiple genes regulate the expression of quantitative traits. This is also
known as the polygene hypothesis or the multiple gene hypothesis for quantitative
inheritance. These multiple genes can occupy multiple loci on different
chromosomes. It is their collective output in a population of individuals that is
reflected as the phenotypic variation of that trait in that population. The collective
output can be an aggregate of the additive effects of different alleles belonging to
different genes. It can also be a result of dominance interactions between alleles of a
given gene and epistatic interactions between genes. The additive effects of genes
are of great value as it makes the trait manipulable and therefore amenable to
artificial selection. We will study about these phenomena in the coming sections.

20.1.3 Variation in a Quantitative Trait and the Number of Genetic

Loci Involved

Variation in the phenotype of a trait can be caused either due to genetic or environ-
mental factors. One of the main genetic factors contributing to phenotypic variation
is additive. It simply means that for a polygenic quantitative trait, the alleles present
in each of the participating loci contribute to a certain definite amount to the final
phenotype. In the case of a coloured trait, each allele might contribute to a certain
degree of pigmentation. Different F2 phenotypic ratios can be obtained based on
how many number of genetic loci regulate a trait and by the nature of the alleles in
each of those genetic loci.
Trait controlled by a single genetic locus: To understand quantitative variation
controlled by a single gene, let us consider the hypothetical example of the number
of heads on a sea monster. Let us assume that there are two varieties of sea monsters:
one having eight heads and the other having two heads. Let the eight-headed sea
monster be assigned the genotype AA, wherein each allele “A” contributes to the
formation of four heads. The two-headed monster has the genotype aa, wherein each
allele “a” contributes to the formation of one head. All the F1 have the genotype Aa
and hence have five heads (4 + 1). Selfing of the F1 generates a phenotypic ratio of 1:
2:1 of an eight-headed, five-headed and two-headed sea monster, respectively
(Fig. 20.2a). Interestingly, the phenotypic class with the maximum representation
in the F2 population (two sea monsters with five heads in this case) has the same
phenotype as all the F1. However, unlike the F1, the F2 also displays a variation
around this mean phenotype.
Trait controlled by two genetic loci: Let us take the hypothetical example of the
number of dragon wings to understand phenotypic variation when a trait is con-
trolled by two genes. Let us assume that the alleles “A” and “B” contribute to one
20 Quantitative Genetics 1033

Fig. 20.2 The number of phenotypic classes in relation to the number of underlying genes
governing that trait additively: (a) Three distinct phenotypic classes are observed when a quantita-
tive trait is regulated additively by two alleles of one genetic locus. (b) Five distinct phenotypic
classes are observed when a quantitative trait is regulated additively by four alleles of two genes. (c)
Up to seven distinct phenotypic classes are observed when a quantitative trait is regulated additively
by six alleles spread across three genetic loci

wing each, while the alleles “a” and “b” do not contribute to the development of a
wing at all. One parent, having the genotype AABB, has four wings and the other
parent having the genotype aabb is wingless. Their progeny (F1) will have the
genotype AaBb, and hence all of them will have two wings. Selfing of F1 generates
five distinct phenotypic classes in the F2 population, with number of wings ranging
from 4 to 0. The F2 generation exhibits a phenotypic ratio of 1:4:6:4:1 (Fig. 20.2b).
1034 A. Chatterjee

Maximum number of dragons in the F2 generation have two wings (six dragons)
which is equivalent to the phenotype shown by all the F1 dragons. However, there
was no phenotypic variation associated with the F1 trait (if we disregard environ-
mental variation). In contrast, the F2 represents a whole range of phenotypes with
respect to the number of wings on the dragons.
Trait controlled by three genetic loci: Let us assume that the number of snakes on
Medusa’s (Greek mythological character) head is a polygenic quantitative trait
controlled by three genetic loci. The alleles “A”, “B”, and “C” code for one snake
each, while the alleles “a”, “b”, and “c” do not code for any snakes. The mating of a
Medusa having the genotype AABBCC (having six snakes on its head) with another
Medusa having the genotype aabbcc (snakeless head) results in an F1 progeny
having the genotype AaBbCc (three snakes on their head). Selfing of the F1 progeny
will result in an F2 progeny consisting of 6, 5, 4, 3, 2, 1 and 0 snakes on their heads
in the ratio 1:6:15:20:15:6:1 (Fig. 20.2c). As in the previous two examples, the
phenotypic class with the maximum representation in F2 (three snakes in the head) is
also the phenotype of all the F1 progeny. However, the former demonstrates a range
of phenotypes, while the latter does not (assuming no environmental variation).
Therefore, one can extrapolate principles of classical Mendelian genetics to derive
phenotypic ratios of polygenic quantitative traits.
One can estimate the number of genes (n) involved in regulating the variation of a
phenotypic trait by using the formula:

1
= Ratio of F2 progeny which phenotypically resemble either of the two parents
4n
In the example concerning the number of wings a dragon has, 1 out of 16 F2
progeny was either wingless or had 4 wings. Therefore the number of genes
regulating the variation in this trait is equal to 41n ¼ 16
1
; therefore, n ¼ 2. Similarly,
in the example concerning the number of snakes on Medusa’s head, the number of
F2 progeny which phenotypically resembled either of the two original parents was
1 in 64. The number of genes regulating the variation in this trait is equal to 41n ¼ 641
;
therefore, n ¼ 3. It is important to bear in mind that this formula assumes that the
parental phenotypes represent the opposite extremes of the phenotypic range for the
trait under consideration.
The number of phenotypic classes that are generated in the F2 generation by a
trait controlled by n number of genes is provided by the formula 2n + 1. This means
that a trait controlled by two genes will generate five [¼ (2  2) +1] distinctly
observable phenotypic classes in the F2 generation. Similarly, a trait controlled by
three genes will generate seven [¼ (2  3) +1] distinct phenotypic classes in the F2
generation. This is also what we observe in the examples given above.
20 Quantitative Genetics 1035

20.1.4 Examples of Polygenic Inheritance

1. Kernel colour in wheat: In 1909, Nielsen and Ehle provided one of the earliest
proofs of the polygene hypothesis for a quantitative trait. They studied the colour
of kernels (seeds) in wheat. They selected a variety which produced purple (deep
dark-red) kernels and crossed it with a variety of wheat which produced white
kernels. The F1 produced kernels of an intermediate phenotype of red colour.
Selfing of the F1 generation generated five distinct phenotypic classes of colours
in F2 wheat kernels: purple (deep dark-red), dark red, red, light red and white in
the phenotypic ratio 1:4:6:4:1. This phenotypic ratio can be explained if we
assume that the trait is regulated by two genetic loci, each having two alleles.
One allele at each locus, “A” and “B”, contributes to red pigment formation,
whereas the other two alleles, “a” and “b”, do not synthesize any pigment.
Applying the principle of additive genetic contribution, we can derive all the
observed phenotypic classes in the experiment. It was later found that kernel
colour in wheat is actually controlled by three genetic loci. The third loci had had
the same set of alleles (CC or cc) in both the varieties in the previous experiment;
therefore, its contribution went unnoticed. Crosses carried out between two wheat
strains that differ in kernel colour due to differences in allele identity at all the
three loci resulted in a F2 phenotypic ratio of 1:6:15:20:15:6:1 (Fig. 20.3). This
represented seven phenotypic classes of kernel colour ranging from purple (deep
dark-red) to successively lighter shades of red and finally white. This experiment
also demonstrated how simple Mendelian nature of genetic characteristics can
successfully explain polygenic quantitative inheritance.
2. Ear length in maize: In 1913, while working on the ear length in maize (Zea
mays), Emerson and East provided an example of continuous variation. They
generated two parental strains, each exhibiting an extreme phenotype of ear
length. The Black Mexican variety of maize had a mean ear length of 16.8 cm,
while the Tom Thumb popcorn variety had a mean length of only 6.6 cm. The two
varieties were obtained after generations of inbreeding. In crossing the above two
parental varieties, F1 plants were obtained exhibiting a relatively intermediate ear
length of 12.1 cm. The F1 were considered to be all homogenously heterozygous
at the gene loci controlling ear length as the parents used were purebreds and
hence assumed to be homogeneously homozygous at all the genetic loci. The F2
plants produced as a result of selfing F1 exhibited a mean ear length of 12.9 cm
and a large phenotypic variation. Even amongst 646 plants analysed, the
extremely long or short ear length parental phenotype was not observed. This
suggests that ear length in maize is controlled by five or more genes.
3. Skin colour in humans: Skin colour in humans is considered to be a polygenic
trait exhibiting continuous variation. Skin colour depends on the deepness of
pigmentation provided by the pigment melanin and is a function of both genetic
components and environmental factors.

Studies have implicated two to six loci contributing to this trait; however, it is
generally accepted that three to four loci controlling this trait adequately explains
1036 A. Chatterjee

Fig. 20.3 Variation observed in kernel colour in wheat: Assuming three genes are involved in
determination of kernel colour in wheat, each of the alleles contributing to the red pigmentation of
the kernel has been denoted as an upper-case letter (A, B, C). The alleles not contributing to kernel
pigmentation have been denoted in lower-case letters (a, b, c). A gradation in kernel colour is
observed in the F2 generation in the ratio 1:6:15:20:15:6:1, as can be seen on the Y-axis

the observed phenotypic variation in skin colour. Assuming that three pairs of genes
(A/a, B/b, C/c) control the degree of skin pigmentation, with the dominant alleles
“A”, “B”, and “C” contributing to melanin synthesis and the alleles “a”, “b”, and “c”
not coding for the pigment, we can arrive at a situation where we observe seven
different F2 phenotypic classes in the ratio 1:6:15:20:15:6:1, wherein 1/64 of the F2
progeny will be black (or white) and the remaining phenotypes will exhibit skin
20 Quantitative Genetics 1037

colour darker than white but lighter than black. The parental genotypes for the above
cross were AABBCC (black) and aabbcc (white), and the F1 were brown with the
genotype AbBbCc (intermediate phenotype). The F2 generation will exhibit a fine
gradation of skin tones, wherein the larger the number of dominant alleles the child
inherits, the darker he/she will be. The F2 generation will show continuous variation,
and it will be tough to demarcate distinct phenotypic classes of skin colour amongst
them. From an evolutionary perspective, populations living close to the equator
synthesize more melanin than those in the temperate regions in order to absorb the
excess UV radiation in such areas and hence prevent its harmful effects (such as
cancer and excess vitamin D synthesis which is toxic for the body).

20.1.5 Polygenic Traits and Oligogenic Traits

Polygenic traits are phenotypic traits that are regulated by multiple genes in the
genome of the individual. Polygenic traits are quantitative traits that possess a
magnitude and a range and display a number of overlapping phenotypic classes.
Oligogenic traits are phenotypic traits that are controlled by one to a few genes and
are usually qualitative in nature. Mendelian traits are considered to be oligogenic.
There are multiple features which differentiate polygenic and oligogenic traits:

1. Discontinuous variation is seen in case of qualitative traits, while continuous

variation is observed in quantitative traits. Distinct phenotypic classes are often
hard to discern in case of quantitative characteristics due to overlap of phenotypic
expression.
2. Most quantitative traits are polygenic with each gene (and the alleles within those
genes) having a small effect on the overall phenotype. Therefore, they are known
as minor genes. Qualitative traits on the other hand are regulated by one or a few
genes, each having a large and easily detectable contribution to the phenotypic
trait. Therefore, they are known as major genes.
3. Quantitative characters are usually the output of additive gene action and in some
cases are regulated by dominance and epistatic gene interactions. Qualitative
traits are usually governed by non-additive gene interactions such as
dominance-recessive relations and epistasis.
4. Environmental factors can contribute heavily to the phenotypic variation seen in
quantitative traits. The effect of environmental factors on qualitative traits is
considered to be minimal. Because quantitative traits are susceptible to environ-
mental factors, they are relatively less stable than qualitative traits.
5. Numerical values can usually be assigned to the magnitude or intensity of
quantitative traits. Qualitative traits are usually studied in regard to features
such as overall colour and shape of the trait.
6. Transgression is the appearance of a phenotype which is higher or lower than
either of the two parental phenotypes. This happens when the parental phenotypes
to begin with were intermediates within a range of possible phenotypic classes.
Transgression is not seen for qualitative traits.
1038 A. Chatterjee

7. As quantitative traits are susceptible to environmental influence, their heritability

is much lesser than qualitative traits, which are predominantly dependent on the
genotype of the individual.
8. Qualitative traits are mostly analysed using segregation ratios and applying
chi-square tests to determine the statistical significance between observed and
expected values. Analysis of quantitative values utilizes the statistical concepts of
variance, standard deviation, correlation and regression. These concepts are used
over and above the calculation of segregation ratios for quantitative traits in a
population.

There are certain features which are common to oligogenic (qualitative) traits and
polygenic (quantitative) traits:

1. Both these kinds of traits are regulated by genes in chromosomes.

2. Definite F2 generation segregation ratios are observed for both qualitative and
quantitative traits.
3. Qualitative characters also show variation due to environmental factors, albeit to
a much lesser degree than quantitative traits.
4. Dominance and epistatic genetic interactions contribute to both qualitative and
quantitative trait manifestation.
5. Polygenes also exhibit linkage like oligogenes. Linkage can also be observed
between a polygene and an oligogene.

20.2 Statistical Analysis of Quantitative Traits

Quantitative traits exhibit continuous variation. Analysis of these kinds of traits

requires statistical descriptions of the phenotype in a population. This differs from
the simple segregation ratio analysis of classical Mendelian traits.

20.2.1 Frequency Distributions, Samples and Population

Individuals in a group display a phenotypic range for a given trait. The magnitude or
intensity of the phenotype in different individuals tends to vary. In order to summa-
rize such a trend, geneticists often construct frequency distribution graphs. Such
graphs depict the range of magnitudes in an increasing (or decreasing) order on the
X-axis and the number of individuals exhibiting a given magnitude of the trait on the
Y-axis. The latter is also known as the frequency of individuals exhibiting a particu-
lar trait. Connecting all the points on a frequency distribution graphs forms a curve.
For most quant traits, this curve is bell-shaped and is called a normal distribution
(Fig. 20.4). The curve might even have two peaks (instead of the characteristic single
peak of a normal distribution), in which case the curve represents a bimodal
distribution. Different kinds of curves can be generated depending on the nature of
the acquired data; however, the normal distribution is the most common.
20 Quantitative Genetics 1039

Fig. 20.4 A representative

example of a normal
distribution: The frequency
distribution graph depicts the
frequency (or number) of
beans over a range of weights.
A normal curve fits the
frequency histogram

Data is acquired from the individuals exhibiting the phenotype of interest. The
population consists of all the individuals who exhibit the phenotype. It is, however,
impractical and tedious to acquire data from an entire population. For most purposes,
data is acquired from a subsection of the population, called the sample. In order to
faithfully depict the phenotypic range, a sample must be accurately representative of
the entire population. To achieve this, a sample must be chosen at random from the
population and must be large enough to incorporate the phenotypic range usually
exhibited by the population. For example, if we wish to study the range of tusk
lengths in Indian elephants, then collecting data only from the first five elephants that
we come across might not reflect the true phenotypic range. Furthermore, consider-
ation of elephants from only one region of the country might again not be represen-
tative of the elephant population of the entire country. Values of traits extracted from
the population are called parameters, and values derived from representative samples
are called statistics.

20.2.2 Mean, Median, Mode: Measures of Central Tendency

Once the frequency distribution graph for a given trait had been generated, a number
of essential quantitative characteristics can be extracted from it. Most of the data
points are distributed around a central value, which in a normal distribution tends to
have the highest frequency. The computation of this value is known as measuring the
central tendency of the data. Central tendencies can be summarized via the mean,
median, or the mode of the distribution.
The mode is the value which occurs the maximum number of times in a data set.
For example, the following is a data set of the number of lizards found in nine
random quadrants of a forest:
1040 A. Chatterjee

4, 5, 6, 6, 6, 6, 7, 10, 25.

The mode for the above data set is 6 as it is the value with maximum frequency
amongst all the other numbers.
The median can be described as the middle value. A median value divides a data
set into a higher half and a lower half. Following is the data set of the number of
students who failed their genetics exam in nine different colleges:

2, 5, 6, 10, 12, 15, 21, 21, 22.

12 is the median value in the above data set (it does not matter that 21 figures
twice in the data set). There are equal number of data points above 12 and below
12, in this case 4 data points each. If there are an even number of total data points,
then the average of the two middle data points constitutes the median.
The most useful statistic is the mean of the population. It refers to the arithmetic
average of all the data points in the distribution. For a sample it is denoted by the
symbol x and is computed as:
P
x
x¼
n
wherein

x: the mean of the sample data set

∑x: the summation of all the data values in the data set
n: the number of data points in the data set

In the above example,

x ¼ ð2 þ 5 þ 6 þ 10 þ 12 þ 15 þ 21 þ 21 þ 22Þ=9 ¼ 12:66

20.2.3 Variance and Standard Deviation: Measures of Dispersion

On generating a number of frequency distribution graphs, one soon realizes that two
distributions might have the same mean, but the spread of the data points on either
side of the mean is unique for each distribution (Fig. 20.5). This spread of data
around the mean is referred to as dispersion and is computed using the variance (s2)
and standard deviation (s) of a given data set. The variance is calculated as:
P
ð xi  xÞ 2
s2 ¼
n1
wherein
20 Quantitative Genetics 1041

Fig. 20.5 Variance of a distribution: Three different frequency distributions having the same mean
value but different spread of the values around the mean. The spread is represented by the variance
s2. The smaller is the value of the variance, the lesser is the spread of the data points around the
mean. The larger is the value of the variance, the more is the spread of the data points around
the mean

P
ðxi  x Þ: the summation of the difference between the ith value (a given value) of
x and the mean x of the distribution.

The reason we square the above value is because ðxi  xÞ is always equal to
0. This happens due to the fact that the mean x represents the exact mathematical
average and therefore the sum of deviations higher than x will equal the sum of
deviations below x. The denominator n-1 is used, instead of n. The value n-1 denotes
the degree of freedom. This means that if the values of n-1 data points have been
provided, then the last value can be derived even if it is unknown.
An important feature of variance is that it is additive. Multiple variance values can
be added and/or subtracted normally. This helps in quantitating components of
phenotypic variance as we shall soon see.
The problem with using variance is that its units are squared which can be hard to
interpret. In order to measure the spread (or dispersion) in the units the original data
values were recorded in, we can compute the standard deviation (s) of the sample.
pffiffiffiffi
s¼ s2
1042 A. Chatterjee

Fig. 20.6 Standard deviation and distribution of measurements: A normal frequency distribution
curve showing that 66% of the values in a data set lie within 1 standard deviation around the mean,
95% of the values in a data set lie within 2 standard deviations around the mean, and 99% of the
values lie within 3 standard deviations around the mean

A normal distribution can be completely defined by the (x  standard deviation),

wherein 66% of the observed data lies within x  1 s; 95% of the observed data lies
within x  2 s, and 99% of the observed data lies within x  3 s (Fig. 20.6). This also
means that a data point selected at random from the distribution has a 66% probabil-
ity of lying in the x  1 s range of measurements, 95% probability of lying within the
x  2 s range of measurements and 99% probability of lying within the x  3 s range
of measurements.
Sometimes data is gathered for the same phenotype from multiple samples. The
mean value for each sample can be computed independently. The standard error of
means (SEM) computes the standard deviation within a distribution of sample
means.

s
SEM ¼ pffiffiffi
n

wherein

s: the standard deviation of the data set.

n: the number of data points within the data set.
20 Quantitative Genetics 1043

A note regarding symbols: x is denoted as μ, standard deviation as σ, and variance

as σ2 if the data is collected from a population instead of a sample.

20.2.4 Correlation and Regression: Measures of Relation

Sometimes two or more phenotypic characteristics might vary together. In such

cases we can say that the two characteristics are correlated. A change in
the magnitude of one characteristic is associated with a change in magnitude of
the other. For example, the number of eggs laid by hens is correlated to the size of the
eggs produced. A larger clutch of eggs is associated with smaller size of each
individual egg. Correlation between traits (covxy) is measured using the correlation
coefficient (r). To compute the correlation coefficient, we need to calculate the
covariance between the two traits of interest.
P
ð xi  x Þ ð yi  yÞ
covxy ¼
n1
wherein

xi: the ith value of x in the data set for trait 1.

x: the mean value of x in the data set for trait 1.
yi: the ith value of y in the data set for trait 2.
y: the mean value of y in the data set for trait 2.
n: the number of xy pairs.

Once the covariance is known, we can calculate the correlation coefficient using
the following formula:

covxy
r¼
sx sy

wherein

covxy: the covariance of x and y.

sxsy: the product of the standard deviation of the data sets for trait 1 and trait 2.

In the above example, the two traits are clutch size and individual egg size.
The correlation coefficient (r) can range from 1 to +1. A positive value of
r denotes that an increase in the magnitude of one trait is associated with a
concomitant increase in the value of the other. A negative value of r denotes that
an increase in the magnitude of one trait is associated with a decrease in the
magnitude of the other. The absolute value of r denotes the strength of the associa-
tion. A correlation coefficient nearing either +1 or 1 means that a change in the
magnitude of one trait is nearly always associated with a change in the magnitude of
1044 A. Chatterjee

Fig. 20.7 Data points plotted to show correlation between x and y variables: The leftmost graph
shows a random scattering of points with a r ¼ 0; therefore, variations in magnitudes of the two
variables are not associated with each other. A r ¼ 0.7 is a strong positive correlation wherein an
increase in magnitude of the x variable is associated with an increase of magnitude of the y variable.
The opposite to the previous trend is a r ¼ 0.7 wherein an increase in magnitude of an x variable is
accompanied by a decrease in magnitude of the y variable

the other trait. A correlation coefficient near 0 means that either the association
between the two traits is very weak or there is no association between the change in
magnitudes of the two traits under consideration (Fig. 20.7). It is important to
remember that an association does not automatically imply a cause-effect relation
between the changes. It only means that a change in one trait is associated with a
change in the other.
Regression is a type of statistical prediction wherein the value of the magnitude of
one trait can be computed if the value of the magnitude of the other trait is provided.
This plays an important role in breeding experiments as one can predict the offspring
characteristics from the parental traits.
Regression can be calculated by plotting a graph between values on the X-axis
(reflecting the different magnitudes of trait (1) and values on the Y-axis (reflecting
the corresponding values of trait (2). For example, the values on the X-axis can
represent the average wing length of parents in Drosophila, while values on the Y-
axis represent the wing length of the corresponding offspring (Fig. 20.8). The line
that best fits all the points on the graph is called the regression line and is represented
by the equation:

y ¼ a þ bx

wherein

a: the value of y when x ¼ 0 (the y-intercept of the line).

b: the regression coefficient or the slope of the line. It denotes the change in
magnitude of y per unit change in magnitude of x.

Also,
20 Quantitative Genetics 1045

Fig. 20.8 Plotting of a regression graph: A regression line drawn as the best fit for a data set
depicting the correlation between wing lengths (in mm.) of offspring Drosophila and the
corresponding mid-parent wing length values. Mid-parent refers to the average wing length of
both parents. 1.1 is the y intercept of the line

covxy
b¼
s2x

wherein

covxy: the covariance between values of trait 1 (x) and trait 2 ( y).
s2x : the variance of values of trait 1.

Once the value of b has been calculated, the value of a can be derived using the
following formula:

a ¼ y  bx

wherein

y: the mean value for the data set of trait 2.

x: the mean value for the data set of trait 1.

Once the values of a and b are known, then for any given value of x, the value of
y can be computed.
1046 A. Chatterjee

20.2.5 ANOVA: Measure of Comparison

Analysis of variance (ANOVA) is often used to confirm whether the computed

means between two or more groups is significantly different. For example, the mean
height of basketball players in a college is 6 ft. 4 in., while the mean height of cricket
players is 6 feet. ANOVA computes the probability that the difference in means
between the two samples arises from chance. The analysis might provide a proba-
bility value of less than 0.02 ( p < 0.02), which means that there is less than 2%
probability that the difference in mean heights between the basketball and cricket
players results from random chance. One can then infer that the difference in heights
between the players of the two sports is a consequence of definite factors, which can
be both genetic and environmental.
ANOVA can also be used to calculate what proportion of a phenotypic variance is
genetic and how much of the variance is environmental. In the above example,
ANOVA has been used to determine whether the means of two groups are statisti-
cally significant; however, it can also be used to determine statistical significance
between multiple means derived from multiple groups.

20.3 Components of Phenotypic Variance

Variation in any given phenotype exists in the parental generation, the F1 generation
and the F2 generation. This variation is represented as the standard deviation from
the mean value for the trait. Variance is calculated as the square of this standard
deviation. Fisher in 1918 proceeded to dissect this phenotypic variance and deduce
the various contributing factors to it. The phenotypic variance (Vp) can foremost be
divided into variance (or variation) originating due to genetic differences between
individuals (Vg) in the sample or population and variance (or variation) arising due to
differences in the environment that different individuals in a sample or population are
exposed to (Ve).
Therefore,

Vp ¼ Vg þ Ve

wherein

Vp: the total phenotypic variance.

Vg: the total genotypic variance.
Ve: the total environmental variance.
20 Quantitative Genetics 1047

20.3.1 Genetic Contribution to Phenotypic Variance

The total genetic variance, Vg, can be further divided into three main
sub-categories—additive variance (Va), dominance variance (Vd) and epistatic
variance (Vepi).
Additive genetic variance is observed when different alleles contribute a definite
amount or quanta to the final magnitude of the phenotype. For example, for a given
trait controlled by the alleles “A” and “a” belonging to the same gene, let us assume
that the allele “A” contributes 10 units to the phenotype of interest, while the allele
“a” contributes 2 units to the trait of interest. In such a scenario, the genotype AA
will express 20 units of the trait, Aa will express 12 units of the trait, and aa will
express only 4 units of the trait. The reader will recognize that the examples given so
far in this chapter all follow the additive model of genetic contribution to the
phenotype.
The dominance component of genetic variance is what most of the classical
Mendelian traits display. In such cases the phenotype depends on the identity of
the two alleles that make up the gene. Let us assume that a given hypothetical gene is
composed of two alleles, “A” and “a”. Different dominance relations can be at play
between these two alleles. In a situation where the expression of the allele “A”
completely masks the expression of the allele “a”, the heterozygotes Aa will
completely resemble the homozygotes AA. This is called complete dominance. It
is to be appreciated that the phenotypic variation herein is not caused by the
quantitative contributions of each individual allele. Other forms of dominance
include incomplete dominance and codominance which have been explained in
detail in the previous chapters. Sometimes the heterozygote Aa will have a pheno-
typic value that is larger than the dominant homozygote (AA) or lower than the
recessive homozygote (aa). The former is called overdominance, and the latter is
termed underdominance. Depending on the fitness value of the genotype, the
heterozygous condition may have a higher or lower frequency of occurrence in a
population. For example, in cases of sickle-cell anaemia, the heterozygous condition
is known to confer resistance to malaria; therefore, in regions prone to malaria
outbreaks, heterozygotes will be seen more in number in comparison to regions
where malaria infections are uncommon. Overdominance or underdominance may
also reduce fitness values. If we assume the wingspan in butterflies to be regulated by
dominance relation between alleles, then a larger wingspan in heterozygotes (Aa) as
compared to the dominant homozygotes (AA) would be exemplary of overdomi-
nance. The larger wingspan might be burdensome to the heterozygote butterfly and
might impede flight speed, thereby increasing its chances of being predated upon.
Similarly, a smaller wingspan in heterozygotes (Aa) as compared to the observed
wingspan in recessive homozygotes (aa) would be exemplary of underdominance.
This might again reduce flight efficiency due to the reduced size of the wings and
increase its chances of being captured by a predator. In such populations the
homozygous butterflies (AA and aa) will be higher in number than the heterozygote
butterflies (Aa). Partial dominance is seen when the heterozygote has an
1048 A. Chatterjee

intermediate phenotype, but the phenotype expressed resembles the dominant trait
relatively more than the recessive trait.
Both additive and dominance genetic variance represents variations originating
from within a single genetic locus. However, polygenic quantitative traits are
controlled by multiple genes. This introduces the possibility of the final phenotypic
variance being a result of genic interactions. This is called epistatic variance.
Epistatic variance can be further partitioned into additive x additive, dominance x
dominance and additive x dominance. The additive x additive interaction
summarizes the contribution to phenotypic variance by two genetic loci, each
regulating the trait of interest in an additive manner. These loci are of immense
interest to plant and animal breeders because they allow for easy prediction of
phenotype in the F1 and F2 generations if the parental phenotype is known. The
dominance x dominance interactions summarize the interaction between two genetic
loci which regulate phenotypic variance using dominance-recessive relations
between the alleles. The additive x dominance genetic interactions summarize the
contribution to phenotypic variance by two genetic loci wherein one of the genes is
composed of alleles contributing additively to the trait of interest, while the other
gene expresses itself on the basis of the principles of dominance relation between the
alleles.
By incorporating all the above information into the formula for phenotypic
variance, we can generate a more nuanced formula:

V p ¼ V a þ V d þ V epi þ V e

wherein

Vg ¼ Va + Vd + Vepi.

20.3.2 Environmental Contribution to Phenotypic Variance

Ideally a given genotype should only express a given phenotype. However, general
observation shows that a given fixed genotype can express a range of phenotypes,
that is, it can display phenotypic variation. The source for such variation lies in the
environment the organism is growing in. It is now generally agreed upon that while
the genotype of an individual organism decides the range of possible traits that it can
display, its environment determines where in that range the organism stands. This
specially becomes important when considering the environmental conditions under
which one wants to rear economically important domestic animals or grow food
crops. Under favourable environmental conditions, a given genotype might double
its productivity as compared to its conspecifics being raised in a relatively poor
environment. The entire range of phenotypes that a given genotype can express
when exposed to all possible environments is called the range of reaction or norm of
reaction for that particular genotype.
20 Quantitative Genetics 1049

Most phenotypes are a result of several gene products interacting with each other.
Therefore one can imagine the humming of an entire genetic background engine at
play to express a single phenotype. One can also assume that over the course of
evolution, the optimum expression of a trait has been standardized by that organism.
This means that the permutation and combination of interaction between genes and
gene products in an organism have already been tuned towards the maximization of
the probability of its survival and its reproductive success. The visible output of this
interaction is the population mean for a given trait in a species. Any deviation from
this mean, either due to environmental changes or genetic mutations, is resisted. This
property of resistance is called canalization of development or developmental
homeostasis.
Environmental effects are of two kinds—external environmental effects and
internal environmental effects. The external environment encompasses all the
sources of variation which originate from outside the body of the organism, while
changes in internal environment originate from within the concerned organism.
External environmental factors can either be abiotic (or non-living) or biotic (living).
Temperature, water content and soil properties are some typical abiotic factors at
play, while age and sex constitute an organism’s internal environment.

20.3.2.1 External Environment

Temperature is an important environmental factor due to its ability to control rates of
chemical reactions. Most phenotypes are dependent on chains of chemical reactions
being catalysed by different enzymes coded by different genes. In a mutant of the
Chinese primrose plant Primula sinensis, the colour of the flower depends on the
temperature the plant was exposed to at an early critical time of flower development.
Flowers blooming at a relatively higher temperature of 23  C tend to be red in colour,
while flowers blooming at colder temperatures of below 18  C are usually white in
colour. This is a clear example of a given genotype expressing two different
phenotypes based on its environment. This is also seen in barley wherein colder
temperatures usually cause albinism while warmer climates promote the develop-
ment of the normal green colour in the plants. In virescent maize the seedlings tend
to be yellowish green; however, they turn completely green if grown in higher
temperatures. Certain thermosensitive crops such as wheat and maize depend on
perceiving a certain degree of temperature before flowering.
Phenotypic variance due to temperature has also been observed in animals. Many
genes in Drosophila, called heat shock protein genes (hsp genes), are transcription-
ally activated when flies are exposed to higher temperatures. An interesting example
is the determination of hair colour in the Himalayan strain of rabbits. While the
normal coat colour tends to be white, portions of the body which are susceptible to
heat loss like the tail, nose, paws and ears tend to be black. This is due to inactivation
of an enzyme (tyrosinase) governing melanin content at higher temperatures, leading
to the white coat colour. If one shaves off the fur from a region of the body naturally
consisting of white fur and artificially reduces the temperature locally in that region,
then one will observe the growth of black fur instead of white in that region. The
determination of coat colour in Siamese cats also follows similar principles.
1050 A. Chatterjee

Sometimes the external temperature can be lethal for an organism. The water flea
Daphnia dies if the temperature exceeds 28  C. Interestingly, a mutant within the
species requires higher temperatures for survival and dies at the temperatures
normally required for survival by its conspecifics.
A certain mutation in Drosophila (tetraptera) causes the halteres to develop as
wings. The probability of active expression of the mutant gene is a function of
temperature with higher temperatures associated with a higher tendency for the
expression of the mutant phenotype. Another mutation (Bar) in Drosophila reduces
the number of facets in the eyes of the fly. The number of facets in the eyes of this
mutant decreases as the temperature increases. In another eye mutant (infrabar), the
opposite trend has been observed.
Light is also a crucial factor. The expression of chlorophyll is highly dependent
on exposure to sunlight. Colourless kernels in maize can become bright red on
exposure to sunlight. Certain photoperiod sensitive plants only flower if they receive
light more, or less, than a specific number of hours. This determines the season of
flowering for that plant. Some genes, for example, rbcs (encoding a small RuBISCO
subunit) and cab1 (encoding chlorophyll binding proteins), have upstream light
response elements regulating their transcriptional activity. Freckling in humans is
also controlled by the amount of exposure to light. Identical twins freckle to different
degrees with the twin working outdoors developing more freckles than the twin
staying indoors.
Nutrition affects the phenotype of multiple organisms. Certain mutants of Dro-
sophila can grow to attain giant sizes; however, the final size attained is dependent
on the amount of food resources available. Under conditions of scarce food supply,
these mutants develop into wild-type-sized flies. On the other hand, a sufficient
amount of food supply leads to the attainment of giant sizes amongst these flies.
Yellow fat (y) mutants in rabbits store yellow-coloured subcutaneous fat if their diet
includes xanthophyll-containing green vegetables. In the absence of green
vegetables in their diet, these mutants store white-coloured fat.
Auxotrophic bacteria are nutritional mutants that are unable to synthesize a
common compound required for its survival. However, they grow normally, like
wild-type prototrophs, if the compound they cannot synthesize is added artificially to
their nutrient substrate.
Soil acidity regulates the colour of the flowers in hydrangeas. An acid pH causes
the flowers to be blue in colour, while a relatively basic pH causes the flowers to be
pink or off-white.
Maternal environment provided to the progeny affects its phenotype as well.
Consumption of nicotine, drugs, or alcohol can be deleterious to the developing
foetus. Incompatibility in the blood Rh factor can cause Rh-negative mother to
mount an immune response against their Rh-positive foetus.
Mice homozygous for the allele hair-loss (hl) tend to lose their hl+/hl- heterozy-
gous progeny due to calcium loss. These progeny survive if the mother is hl+/hl + or
hl+/hl-. Therefore the genotype of the mother can establish an external environment
which can affect the phenotype of the progeny. Seed characteristics such as seed size
20 Quantitative Genetics 1051

and protein content in crop plants are also dependent on the genotype of the parent
plant.
Moisture content or humidity affects the morphology of the abdomen in the
abnormal abdomen Drosophila mutants. These mutants develop a distorted abdom-
inal appearance due to irregular chitinous bands. The mutation expresses itself
predominantly under moist culture conditions, but flies show normal abdominal
banding under dry conditions. Also, disease susceptibility as well as the resistance to
lodging in plants is often dictated by moisture content.
Other factors such as the presence of symbionts, immune response to parasites, as
well as the surrounding population density of conspecifics can cause the same
genotypes to be phenotypically different.

20.3.2.2 Internal Environment

Internal factors originate from within the body of the organisms. Many genes are
actively transcribed only after the concerned organism attains a certain age. In plants
the genes determining floral, fruit and seed characteristics become active once the
plant reaches sexual maturity. Similarly, genes regulating milk production in cattle
and egg production in poultry are expressed only after attainment of the appropriate
age. While genes determining the blood group of a person are active even before
his/her birth, genes regulating alkaptonuria, phenylketonuria and rickets are
expressed postnatally. Genes responsible for baldness, diabetes and Huntington’s
disease are predominantly expressed in adults. Therefore age is an important internal
factor.
Phenotypic expression of traits can vary based on the sex of the individual.
Recessive mutant alleles for genes exclusively located on the X-chromosome are
expressed more often in males than females. This is because even a single mutant
copy of the allele is sufficient to express the mutant phenotype in the heterogametic
sex, a phenomenon known as pseudodominance. Sometimes, though a gene is
present in both the male and female of a species, only one of the sexes expresses
the trait. For example, the genes controlling milk production and egg laying are
active in the females of the species only. Such traits are called sex-limited characters.
Sex-influenced characters are expressed in both the sexes of a given species, but the
dominance relation between the alleles of the gene changes, that is, the dominant
allele for a trait in males behaves as the recessive allele in females. Examples of this
include presence of horns in sheep and crown baldness in humans.
The presence of a particular substrate can also decide whether a phenotype will
be expressed. Inclusion of tyrosine and phenylalanine in the diet of patients suffering
from alkaptonuria causes their urine to turn black on exposure to air. This is because
of the build-up of alkapton or homogentisic acid in the urine as a result of the partial
breakdown of tyrosine and phenylalanine. A tyrosine- and phenylalanine-deficient
diet leads to a significant decrease in the symptoms of the disease. Similarly, the
levels of sugar in patients suffering from diabetes can be controlled on consuming a
diet restricted in both fats and carbohydrates. This helps in reducing the intense
symptoms of the disease.
1052 A. Chatterjee

Several genes are only expressed in places where their products are physiologi-
cally required. The human liver, pancreas and lungs will have many different genes
which are expressed exclusively in those organs. For example, the genes for insulin
are expressed in the pancreas. Similarly, genes responsible for protein storage in
seeds are going to be exclusively expressed in seeds. Therefore, the identity of the
tissue is an important factor determining gene expression. Furthermore, a phenotype
is the end result of a number of gene products interacting with each other, and thus
the genetic background of the individual finally decides whether a phenotype will be
expressed or not.
Therefore the formula for phenotypic variance can be further dissected as:

V p ¼ V a þ V d þ V epi þ V eext þ V eint

wherein

Ve ¼ Ve-ext + Ve-int.
Ve-ext: the phenotypic variance caused due to external environmental factors.
Ve-int: the phenotypic variance caused due to internal environmental factors.

20.3.2.3 General and Special Environmental Variance

Environmental variance can also be described as general environmental variance
and special environmental variance. Special environmental variance measures the
intra-individual (or within-individual) variance arising from temporary or localized
circumstances. For example, measuring the milk yield from the same cow during
different months of the year will aid in estimating special environmental variance.
Such effects result in transient changes in the phenotype. General environmental
variance originates from permanent or non-localized environmental factors. Such
factors may originate from differences in the amount of nutrition available during
childhood of an organism which results in permanent effects on the phenotype. Such
factors will remain constant between the months and seasons of a year and therefore
do not contribute to special environmental variance.
Therefore, another equation for phenotypic variance can be written as:

V p ¼ V a þ V d þ V epi þ V egen þ V esp

wherein

Ve ¼ Ve-gen + Ve-sp.
Ve-gen: the phenotypic variance caused due to general environmental factors.
Ve-sp: the phenotypic variance caused due to special environmental factors.
20 Quantitative Genetics 1053

20.3.3 Other Factors Contributing to Phenotypic Variance

Having considered the genetic and environmental contributions to the overall phe-
notypic variance, we must also include variance due to genotype and environmental
interactions (Vg x e). This factor arises due to the reason that different genotypes
might interact uniquely to a given range of environmental conditions. For example,
in Fig. 20.9c, the height of both the plants was different to begin with. An increase in

(a) (b)

G1, G2
Plant height (cm)

Plant height (cm)

G2

G1

15 20 25 30 15 20 25 30
Temperature (°C) Temperature (°C)

(c) (d)

G2
Plant height (cm)

Plant height (cm)

G1
G2

G1

15 20 25 30 15 20 25 30
Temperature (°C) Temperature (°C)

Fig. 20.9 Different sources of phenotypic variation: A hypothetical variation in plant height as a
function of temperature has been shown in the graphs above. G1 (blue) and G2 (red) denote the
genotypes of the two plants. Green represents an overlap of the phenotypic trend in both the plants.
(a) Represents a situation where neither plant is affected by temperature and the height is deter-
mined genetically only. (b) Plant height herein is completely dependent on temperature, and there is
no genetic source of height variance. (c) Temperature and the individual genotypes of the plants
independently and additively determine plant height. (d) Each plant genotype interacts uniquely
with a given temperature value and produces independent trends of plant height. This is a case of
genetic x environment interaction
1054 A. Chatterjee

temperature is correlated with an increase in plant height; however, the difference in

height of the two plants remains constant at any given temperature. In this case the
genetic and environmental factors are not interacting, and each has an independent
effect on the final phenotype. In contrast, in Fig. 20.9d, the height of one plant is
negatively correlated with an increase in temperature, while that of the other is
positively correlated to the same environmental factor. The genotype of each plant in
this case interacts with temperature in a different way. Variances caused due to such
interactions are labelled as genetic-environmental interaction variance (Vg x e).
Figure 20.9a, b represents pure genetic and environmental contribution to pheno-
typic variation, respectively.
Sometimes the underlying reason for phenotypic variance cannot be easily
partitioned into a genetic, environmental, or genetic-environmental interaction.
Variances originating from unknown causes are referred to as intangible variation
(VI). It is hard to discern VI through planned experimental paradigms. These are
usually random and unpredictable fluctuations during development which end up
having permanent phenotypic effects on the individual.
We can now write the equation for phenotypic variance as:

V p ¼ V a þ V d þ V epi þ V egen þ V esp þ V g x e þ V I

Or,

V p ¼ V a þ V d þ V epi þ V eext þ V eint þ V g x e þ V I

wherein

Vg x e: the phenotypic variance caused due to genetic-environmental interactions.

VI: the intangible causes for phenotypic variance.

Some geneticists include another factor while considering the source of pheno-
typic variance. It is the covariance between a genotype and the environment it is
exposed to. For example, for a farmer selling milk, the cows yielding a higher
volume of milk are more important than the ones producing less milk. This may
cause the farmer to give more food (hence more nutrition) to the cows producing
more milk. Also, the farmer might simultaneously give lesser food (hence lesser
nutrition) to the cows producing less milk, especially if the amount of food is limited.
This will result in an even higher yield of milk from cows which were producing
more milk to start with and a decrease in milk production from cows which were
yielding less milk to start with. In this situation the genotype and environment for an
individual covary. This fraction of the contribution to phenotypic variance is called
genetic-environmental covariance.
20 Quantitative Genetics 1055

20.4 Heritability

The partitioning of the phenotypic variance results in the identification of the

multiple contributors to the variation within a given phenotype. For commercial
purposes such as artificial breeding, one is often specifically interested in that
component of the phenotypic variance which is genetically determined. The propor-
tion of the phenotypic variance in a population that is controlled by genetic factors is
termed as heritability. The concept of heritability plays an important role in the
process of artificial selection.

20.4.1 Types of Heritability

Broad-sense heritability (H2): The value of H2 estimates the fraction of phenotypic

variance that is regulated by genetic factors. The superscript 2 is there to remind us
that heritability is a type of variance.

Vg
H2 ¼
Vp

wherein

Vg: the genetic component of phenotypic variance.

Vp: the total phenotypic variance.

Let us try to calculate the H2 of inter-moulting interval time in a hypothetical

snake species. Let us assume that this species has two distinct varieties of snakes,
one with an inter-moulting interval time of 7.6 days  1.2 days and the other
23.3 days  2.7 days. The F1 has an intermediate inter-moulting interval time of
15.7 days  3.1 days. The F2 generation exhibits an inter-moulting interval time of
15.4 days 5.5 days. As expected the standard deviation for the trait has increased
considerably in the F2 generation. It is important to remember that the phenotypic
variance in the parental and the F1 generation is due to environmental factors as the
genotypes do not vary between the individuals in these generations (or at least we
assume so). We can therefore average the variances observed in the parental and F1
generation to obtain a mean value of Ve in this species for this trait. Please keep in
consideration the fact that the variance is the squared value of the standard deviation.
Vp ¼ Vg + Ve
(5.5)2 days2 ¼ Vg + [ {(1.2)2 + (2.7)2 + (3.1)2}/3 ] days2
30.25 days2 ¼ Vg + [ (1.44 + 7.67 + 9.61)/3 ] days2
Therefore, Vg ¼ [30.25 – 6.24 ] days2 ¼ 24.01 days2
Hence, H2 ¼ 24.01 days2 / 30.25 days2 ¼ 0.79
This means that 79% of the variation in inter-moulting interval time in the species
of snakes can be attributed to genetic factors.
1056 A. Chatterjee

Table 20.1 Narrow-sense Trait h2 value

heritability values for some
Stature in human beings 0.65
quantitative traits
Milk yield in dairy cattle 0.35
Litter size in pigs 0.05
Egg production in poultry 0.10
Tail length in mice 0.40
Body size in drosophila 0.40
Adapted from Principles of Genetics by Snustad and Simmons, 6th
edn. (2012)

Genetic contribution to a trait can be due to additive, dominance, or epistatic

relations. Of these the most manipulable and therefore of the most interest to plant
and animal breeders is the additive component of genetic variance. One is usually
not privy to the actual genotype of each individual in a population. Furthermore, for
most polygenic traits, the exact genes regulating the trait of interest have not been
deciphered; therefore, manipulating dominance and epistatic genetic interactions is
very tough. Under such circumstances one relies on the observable phenotype of a
trait in the parents and tries to predict the possible phenotypic range of the trait in the
offspring. This can be done most faithfully by calculating the additive components
of genetic variance for the trait of interest. The fraction of the total phenotypic
variance that is regulated by additive components of genetic variance is called as the
narrow-sense heritability (h2).

Va
h2 ¼
Vp

wherein

Va: the additive component of the genetic contribution to phenotypic variance.

Vp: the total phenotypic variance.

The values of H2 and h2 range from 0 to 1. A value of H2 close to 0 means that the
genetic contribution to phenotypic variance is nearly non-existent. A H2 value close
to 1 means that the genetic contribution to the phenotypic variance for the given trait
is very high. Similarly, a value of h2 close to 0 means that the additive components of
genetic variance do not regulate the phenotypic variance, while a value close to
1 means that the additive components of genetic variance can predominantly account
for the variance in the phenotype. The h2 values for some quantitative traits have
been presented in Table 20.1.
Before we proceed further, it is important that we correctly understand how to
interpret the values of heritability. The need for this arises from the fact genetic
studies in this field have been historically politicized and used to unfairly target
certain sections of the population. Please keep the following points in mind while
interpreting heritability values:
20 Quantitative Genetics 1057

1. The heritability value calculated does not hold true for an individual. It is an
estimate of the genetic contribution to phenotypic variance for a population. For
example, a H2 value of 0.67 for plant height in a population (or sample) of
sunflowers does not mean that for each individual sunflower, 67% of its height
is regulated by genes. The correct interpretation would be that 67% of the
variance in plant height amongst sunflowers can be attributed to genetic factors.
2. A high value of heritability does not mean that the phenotype is regulated by
genes only. It simply means that in that particular sample or population, the cause
of phenotypic variance was genetic in nature. This might be because all the
individuals in the sample or population were reared in near identical environmen-
tal conditions, thereby eliminating variations due to environmental factors. Simi-
larly, a very low heritability value does not mean that genes do not regulate that
particular phenotype. For example, in a population of lizards that is genetically
inbred (and therefore homogenously homozygous), the genotype will nearly be
the same in all the individuals. This prevents the genotype from contributing to
the phenotypic variance of a given trait like tail length. The variation in tail length
in this population of lizards will be only due to environmental causes. This does
not mean that genes do not play an important role in determining tail length.
3. Heritability values only allow for an estimation of the fraction of the genetic and
environmental contribution to phenotypic variance. It does not tell us anything
about the actual genes or environmental factors controlling the trait.
4. Similarity amongst relatives of a family is not to be confused with heritability.
Members of a family might resemble each other solely due to spatially and
temporally shared environmental factors. Shared resemblance of phenotypic traits
amongst family members is called familiality and is not the same as heritability.
5. Heritability is not fixed for a trait. Heritability estimates for milk yield in an
endemic population of cows in England cannot be extrapolated to breeds of cows
in India. Moreover, this estimate will probably be untrue for other breeds within
England.
6. If two distinct populations exhibit very high heritability values for the same trait,
it does not automatically mean that the trait is predominantly regulated geneti-
cally. For example, the estimation of heritability of human height in a developed
country and in a developing country might yield very high values. One can
assume that individuals in the developed country have access to sufficient food
sources and hence are uniformly nourished. In a poor region of a developing
country, the access to food might be compromised and hence the population may
be uniformly undernourished. In such a situation, the environmental factor of
nutrition exerts an equal effect on individuals belonging to a given population and
thus does not contribute to the phenotypic variance of height within a given
population (one population being from the developed country and the other being
from the developing country). In this case the calculated heritability for height
will only reflect the genetic component to variance in height. It would be incorrect
to deduce from these results that environmental factors like nutrition do not play
an important role in determining human height.
1058 A. Chatterjee

20.4.2 Factors Affecting Heritability

Measures of heritability can be affected by the following factors:

1. Genetic diversity of the parental generation decides the degree of phenotypic

variation that will be observed in the F2 generation. The more is the allelic
variability at the loci amongst the parents, the larger is the number of novel
permutation and combination of genotypes at the F2 level and hence greater is the
phenotypic variability.
2. As it is impractical to study the entire population for a trait of interest, usually
smaller samples are chosen for analysis. However, if the sample is too small, then
the measured genetic variance might not faithfully represent the actual
population-level value. Larger samples are preferred.
3. Random sampling of a population provides genetic variance values (heritability)
closer to the actual population-level values. Biased or non-random sampling
(repeatability) is unable to estimate the true value of the same.
4. Manual errors in measurement of values can lead to a deviation in variance values
from the true value. Such errors can be minimized by increasing the number of
samples chosen for evaluation, increasing the sample size itself, measuring the
variance multiple times from a given sample, and conducting collection of data
from multiple testing sites instead of just one.
5. The type of heritability being calculated matters. Broad-sense heritability (H2)
will usually have a higher value than narrow-sense heritability (h2). This is
because H2 estimates also include dominance and epistatic contributions to
genetic variance, apart from the additive component.
6. Genetic linkage between two loci controlling a quantitative trait can lead to an
overestimation or underestimation of heritability values. Linkage in a
cis-arrangement (AB/ab) leads to an overestimation of the dominance and addi-
tive components of genetic variance. Linkage in a trans-arrangement (Ab/aB)
again results in an overestimation of the dominance component of genetic
variance; however, it leads to an underestimation of the additive component.

20.4.3 Measures of Heritability

There are multiple ways of measuring heritability for a given trait. Here we discuss
three such methods.

1. Elimination of one variance component: The phenotypic variance is a sum of the

genetic and environmental components of variance. If we are successfully able to
prevent one of the factors from affecting phenotypic variance, then any variation
in the trait must have arisen from the other component. For studies in plants, one
can reduce Ve ¼ 0 by growing them in a greenhouse. Greenhouses can efficiently
maintain a uniform temperature and moisture content, thereby precluding any
variation in the phenotype due to different plants being exposed to different
20 Quantitative Genetics 1059

intensities of a given environmental factor. Any variation observed for a given

trait within a given species in such a greenhouse can be attributed to differences in
the genotype between the individual plants. In such a situation, Vp1 ¼ Vg. The
same species can then be grown outside a greenhouse where the environmental
factors are not controlled. The phenotypic variance for the same trait can be
calculated; however, this time the variance in the phenotype (Vp2) originates from
both the differing genotypes and the environment. We can utilize the Vg value
estimated previously and divide it by Vp2 to compute the heritability value for the
chosen trait. Needless to say that we are assuming that the genotypes of the plants
grown inside and outside the greenhouse are the same. This is not always true and
hence can be considered a limitation of the technique.
We can also adopt the alternate approach of reducing Vg ¼ 0. The study of
variation of a given trait in an inbred line will yield Vp1 ¼ Ve for the simple reason
that individuals belonging to an inbred line are assumed to be composed of the
same genotype. Therefore any genetic contribution to phenotypic variance is
precluded, and all the variation observed in the trait of our choice is because of
environmental causes. Vp2 can be calculated by estimating the variance for the
same phenotype in a non-inbred line of the same species. Utilizing the previously
calculated value of Ve, we can compute Vg (¼ Vp2 – Ve). Heritability can then be
estimated by using the formula Vg/Vp2. We do assume that the effects of the
environment on both the inbred and non-inbred line are going to be the same. This
is however not always the case, especially because the inbred and non-inbred
lines are genotypically dissimilar. This is a lacuna in the above technique.
2. Computation of parent-offspring regression: In the earlier sections of this chap-
ter, we have examined how a change in the magnitude of one trait can be
associated with a change in the magnitude of another trait. This is called
correlation. Furthermore we learnt how to calculate the corresponding value on
the Y-axis for a given value on the X-axis. This is called regression. Regression
analysis can be used to estimate heritability values for a given trait if that trait is
shared between the parents and the offspring. For example, we can plot a graph
between the wingspan length of parental Drosophila (trait 1 on the X-axis) and the
corresponding wingspan length of their offspring (trait 2 on the Y-axis). As there
are two parents for any given offspring, geneticists usually plot the mid-parental
value on the X-axis. This value reflects the average of the two parental values for
the trait. This usage of the mid-parental value also introduces the element of
treating the phenotypic variance of the trait as having an additive genetic compo-
nent. This is because the mid-parent value is an intermediate value of the trait and
using this value to compare the trait with the offspring means that we have
assumed that there is an additive genetic component to the trait. Remember that
only in cases of additive genetics did the F1 express a phenotype which was
intermediate with regard to the parental phenotype (this is not true in cases of
genetic components of variance derived from dominance and epistatic relations).
Thus one can compute narrow-sense heritability values using these graphs. The
mathematical derivations for the relation between the slope of a correlation graph
1060 A. Chatterjee

Fig. 20.10 Parent-offspring regression plots: The value of h2 can be estimated from the value of
the slope of the graph having the mean parental value on the X-axis and the mean offspring
phenotype values on the Y-axis. (a) There is no relation between the parental and offspring values
of the phenotype. (b) The phenotype values of the offspring are entirely dependent on the phenotype
values of the parents. (c) The phenotypic range in the offspring is a product of additive genetic,
non-additive genetic, and environmental influences

(b) and h2 are beyond the scope of this chapter; however, it is important to know
the following two results of the said derivations:

h2 ¼ b

wherein
h2: the narrow-sense heritability value for the phenotypic trait.
b: the slope of the regression graph.
If the phenotypic values of only a single parent are available for analysis, then
the mid-parent values cannot be plotted. In such a case, we modify the above
equation.

h2 ¼ 2b

wherein
h2: the narrow-sense heritability value for the phenotypic trait.
2b: twice the value of the slope of the regression graph. This is done to
compensate for the absence of the phenotypic values for the other parent.
If the absolute value of b ¼ 1, then all the genetic variance is derived entirely
from additive components. If the absolute value of the slope is smaller than 1 but
larger than 0, then the genetic contribution to phenotypic variance is a mixture of
additive and non-additive components (Fig. 20.10). If the absolute value of b ¼ 0,
then there is no contribution of additive genetic components to phenotypic
variance; however, this does not rule out dominance and epistatic genetic contri-
bution to the variation observed in the phenotype
3. Comparison of phenotypic variances for the same trait in individuals with
varying degrees of relatedness: Individuals related to each other are expected to
bear a resemblance due to shared genes. The closer the relatedness, the higher is
20 Quantitative Genetics 1061

the proportion of genes shared. That would mean that siblings are genetically
more similar than first cousins, who in turn would be more genetically similar
than second cousins. It is known that siblings have 50% of their genes in common
on an average. Similarly, it is known that half-siblings, who share only one of the
parents, have 25% of their genes in common on an average. The fraction of the
observed correlation coefficient between two relatives to the expected correlation
coefficient between the same relatives for a given phenotypic trait computes the
narrow-sense heritability for that trait.

r obs
h2 ¼
r exp

wherein
h2: the narrow-sense heritability value for the phenotypic trait.
robs: the observed value for correlation coefficient between the relatives for the
phenotypic trait.
rexp: the expected value for correlation coefficient between the relatives for the
phenotypic trait.
The occurrence of monozygotic and dizygotic twins also allows for the
calculation of heritability estimates for a given phenotype. Monozygotic twins,
or identical twins, share a complete set of genes with each other, that is, they are
genetically the same. Dizygotic twins, or fraternal twins, share only 50% of their
genes on an average like any two siblings. An estimate of the broad-sense
heritability value can be obtained on doubling the difference between the corre-
lation coefficients for a given trait calculated for both monozygotic and dizygotic
twins.

H 2 ¼ 2 ðr MZ  r DZ Þ

wherein
H2: the broad-sense value for the phenotypic trait.
rMZ: the correlation coefficient for the phenotypic trait amongst monozygotic
twins.
rDZ: the correlation coefficient for the phenotypic trait amongst dizygotic
twins.

Such estimates, however, should be interpreted with caution as one assumes that
the environment shared by monozygotic twins is no more different to the one shared
by dizygotic twins. That is not always the case. Monozygotic twins might share a far
more similar environment than dizygotic twins as they are often treated very
similarly. The above assumption is a limitation of this technique.
A trait that is expressed by both individuals of a twin-pair is considered to be
concordant if both the twins express it, or neither of them does. A trait is considered
to be discordant if one of the individuals of the twin-pair expresses it and the other
one does not. A comparison of the concordance values for a given trait between
1062 A. Chatterjee

monozygotic and dizygotic twins helps in the estimation of the genetic contribution
to a given phenotypic trait. If two individuals of a monozygotic pair, who have been
reared apart, show very high concordance values for a given trait, then it is quite
certain that there is a heavy genetic contribution to the trait. On the other hand, if a
large concordance value is observed for a given trait in both monozygotic and
dizygotic twins, then there might be environmental factors contributing to the
phenotype as well. This is because dizygotic twins only share 50% of their genes,
and a high concordance value might mean that the similar phenotype is a result of
shared environment too.

20.5 Artificial Selection

Evolution works on the principle of selecting beneficial traits in a population and

increasing the frequency of individuals exhibiting that beneficial trait. This kind of
selection is called natural selection because it happens in nature as a default process.
However, through the process of selective breeding, humans have over the last
10,000 years artificially changed the mean values of the phenotypic traits that are
of interest to him. This includes milk yield from cattle, wool yield from sheep, egg
production, etc. Most of these traits are of commercial value and artificial selection
for these traits has greatly improved standards of living in recent human history. The
parameter to be exploited for artificial selection is the phenotypic variance exhibited
by the original (non-selected) population. Within the range of phenotypes exhibited
by the original population, a few individuals will have relatively superior traits as
compared to others within the same population. Artificial selection works if there is a
large additive genetic component to the phenotypic variance. Dominance, epistatic,
and environmental components of phenotypic variance are not useful for long-term,
permanent changes in trait characteristics. Dominance and epistatic components of
phenotypic variance are hard to artificially control and manipulate while the envi-
ronmental components are not transferrable form one place to another.
To further understand artificial selection, let us assume the mean volume of milk
produced by a cow in a local herd to be 50 L/week (Ῡ). In order to increase the milk
yield, we select breeding pairs which have an average output of 80 L/week (Yp). The
difference between the milk yield in the general population and the selected parents
for breeding is 80–50 ¼ 30 L/week. This difference is known as the selection
differential (S). After breeding, let us assume that the average milk yield of an
offspring is 65 L/week. The difference between the output of the offspring and the
original population is known as the genetic gain, or the response to selection (G). In
this case G ¼ 65–50 ¼ 15 L/week (Yo). Considering that the observed increase in
average milk yield is due to additive components of phenotypic variance, we can
calculate the narrow-sense heritability using the formula:
 
h2 ¼ ðY o  Ῡ Þ= Y p  Ῡ ¼ G=S

wherein
20 Quantitative Genetics 1063

Y Mean yield of original population

P1

Frequnecny

YP Mean yield of
selected parents

Yield (phenotype) YP − Y =
Selection
differential

YO Mean yield of F1
F1

YO − Y = Gain

Fig. 20.11 Change in mean value of a trait due to artificial selection: Realized heritability is the
genetic gain (Yo – Ῡ) divided by the selection differential (Yp – Ῡ). The mean of the phenotypic trait
has shifted to the left in the F1 generation denoting a positive selection for the trait under
consideration

h2: the narrow-sense heritability value for the phenotypic trait.

Ῡ: the mean milk yield by a cow in the original population.
Yp: the mean milk yield by a cow in the population selected for breeding.
Yo: the mean milk yield by a cow in the offspring population.

In this case h2 ¼ 15/30 ¼ 0.5.

The narrow-sense heritability in the above example is called realized heritability
since the value is computed after the breeding is done (Fig. 20.11). It is an estimate of
true heritability which encompasses both narrow-sense and broad-sense heritability.
Once the h2 for a trait is known, then predictions can be made about the change of
magnitude to be expected in the offspring generation. G. Clayton et al. studied the
number of abdominal bristles in Drosophila melanogaster. Using multiple
techniques, they calculated the h2 for the number of abdominal bristles to be 0.52.
They selected parents with mean bristle number of 40.6, while the population mean
was 35.3. This means the selection differential (S) is 5.3 (40.6–35.3). Therefore the
response to selection (or genetic gain, G) ¼ 0.52  5.3 ¼ 2.8. This value can be
1064 A. Chatterjee

interpreted as the expected increase in magnitude of abdominal bristle number in the

offspring generation; 35.3 + 2.8 ¼ 38.1, which turned out to be very close to the
observed value of 37.9.
Every phenotypic trait has a variance associated with it. Therefore we see a range
of phenotypic values for any given trait. It is important to also factor in this variance
for artificial selection. The selection intensity (i) is defined as the selection differen-
tial per unit standard deviation of the phenotypic variance of the original population.
Genetic advance (GA) is defined as the improvement in the mean genotypic value of
a selected trait in the offspring generation over the original parental population.

i ¼ S=σ p ;

GA ¼ i  σ 2 p  H 2 ¼ S  σ p  H 2

wherein

i: selection intensity.
σ 2p: the phenotypic variance of a trait.
σ p: the standard deviation of the phenotypic variance.
S: selection differential.
H2: the broad-sense heritability value of a trait.
GA: genetic advance.

Different populations being reared in different environments can give different

values for h2 for the same trait under study. Therefore, it is prudent to conduct
independent research on heritability of a trait for each individual population.
Artificial selection has been practiced for multiple commercially important traits
such as milk yield, egg number, egg size, and wool yield. Milk yield can be
measured in successive lactations, and several measurements from each individual
can be obtained. Usually both the first and the second lactation are measured for milk
yield. Variance per yield per lactation can be analysed within the same individual
and between individuals. The within individual component is entirely environmental
in origin, while the between individual component is partly environmental and partly
genetic in origin. A positive correlation has been reported for milk yield in the first
and second lactation. The h2 for milk yield has been estimated to be around 0.35
which means that 35% of the variance in milk yield can be explained by additive
genetic variance. Milk yield cannot be measured in males; hence, its breeding value
can only be judged by assessing the performance of its female relatives. Milk yield
has been reported to decrease over time as a result of inbreeding depression. An
increase in milk yield has also been correlated to a decrease in protein and butterfat
content in the milk. Milk yielding varieties of cattle include Dutch Holstein –
Friesian, Finnish Ayrshire and Holstein Dairy. Indian milk yielding cattle include
Sahiwal and Frieswal. Jakhrana is a milk-yielding goat variety in India. Genes
regulating milk yield have been localized to chromosomes 6, 14, 17 and
20 (Arranz et al. 1998 and Zhang et al. 1998). The DGAT1 gene on chromosome
20 Quantitative Genetics 1065

14 has been implicated in regulating milk yield in cattle (Grisart et al. 2002). Genes
implicated in milk yield and composition in dairy sheep have been localized to
chromosomes 1, 2, 3, 20, 23 and 25 (Gutiérrez‐Gil et al. 2009).
The number of eggs laid is known as the clutch size. Egg size and egg number are
both considered to be quantitative traits. It has been studied both in poultry and
Drosophila. The h2 estimate for egg weight in poultry is around 0.50. The h2
estimate for egg number in poultry and Drosophila is 0.10 and 0.20, respectively.
Egg number and egg size variation cannot be studied in males; therefore, their
selection potential is calculated by assessing their female relatives (as was noted
previously for artificial selection concerning milk yield). Both these traits have been
improved over the years using artificial selection. The level of circulating gonado-
tropin hormone has also been implicated in determining the number of eggs laid by
poultry. A positive correlation has been observed between the body weight of the
mother and the weight of the eggs she lays, while a negative correlation has been
reported between the weight of the eggs and the number of eggs laid. Variation in
egg numbers and egg size can be regulated by additive, dominance and epistatic
genetic interactions. Genes on chromosomes 2, 4 and 5 have been implicated in
regulating variation in egg weight and number in poultry (Wolc et al. 2012).
Candidate genes responsible for regulating egg weight include CECR2, MEIS1
and SPRED2 (Liu et al. 2018). Genes implicated in regulating egg number include
GTF2A1 and CLSPN. An SNP mutation on chromosome 5 can cause a phenotypic
difference of nearly seven eggs between two homozygous genotypes (Yuan et al.
2015). The breeds on which such studies have been done include the red junglefowl
and white leghorn.
Approximately 90% of the world’s sheep produce wool. One sheep can produce
anywhere between 1 kg to 13 kg of wool annually. Wool yield is usually measured at
the first and second shearing. The amount of wool a sheep produces is a function of
its breed, genetics, nutrition and shearing interval. The wool length and diameter of
wool fibres predominantly determine wool yield. The sex of the lamb can also affect
wool yield. Wool yield is of immense economic interest and has been subjected to
artificial selection. It is also considered to be a polygenic trait. Breeds of sheep that
have been used in an attempt to increase wool yield include Merino, Romney-Marsh
and Lincoln. Indian breeds of sheep used for wool production include
Muzaffarnagari sheep and Garole sheep. Heritability estimates for wool yield are
quite low – approximately 0.15  0.07 for Muzaffarnagari sheep (Sinha and Singh
1997). Heritability estimates for wool yield range from 0.23 to 0.37 for breeds such
as Rambouillet and Romnolet (Vesely et al. 1970). Genes regulating wool yield have
been localized to chromosomes 3, 4 and 24 of the Merino sheep (Bidinost et al.
2008). Additional genes regulating wool yield have been found on ovine
chromosomes 1 and 11 in later studies (Roldan et al. 2010).
Selection can continue till the time there is observable phenotypic variance in the
trait of choice. But constant selection for a trait finally leads to the appearance of
genotypes which are nearly homozygous for all the genes contributing to the trait. At
this point h2 ¼ 0, and there is not much scope to introduce new variations, and the
selection process comes to an end (Fig. 20.12). Remember that h2 ¼ 0 does not mean
1066 A. Chatterjee

Fig. 20.12 Response to selection of a trait plateaus after a number of generations: In an experiment
to increase the number of abdominal bristles in female fruit flies, the response to selection levelled
off after 20 generations of selection. The trend for both the selected line (the line which was
subjected to selection for the trait) and the control line has been shown

that the phenotypic variance does not have a genetic component. It means that
variation due to genetic reasons cannot be introduced anymore. Also, as homozy-
gosity increases in a selected line, detrimental mutations start expressing themselves
which leads to a general decrease in yield and vigour. This is known as in-breeding
depression. Limitations to selection are also due to phenotypic and genetic
correlations, which is the following topic.

20.6 Phenotypic and Genotypic Correlations

A connection between two or more things is defined as a correlation. The variations

of two phenotypic traits that are correlated influence each other. For example, tall
people also tend to weigh more than shorter people. Herein the quantitative traits of
height and weight are correlated to each other. Such correlations are called pheno-
typic correlations. Another example of phenotypic correlation is the observation that
fair skin, blond hair and blue eyes are often found in the same individual. Phenotypic
correlations can be either due to environmental correlations or genetic correlations.
A non-zero correlation coefficient between two traits is seen if they are phenotypi-
cally correlated.
If two or more traits are affected by the same environmental factor, then the traits
are considered to be environmentally correlated. For example, plants that are grown
in climates affording enough moisture tend to grow taller and produce more number
20 Quantitative Genetics 1067

of seeds in comparison to plants that are made to grow in places of limited water
supply. Similarly, plants growing in soil supplemented with fertilizers grow taller
and bear more number of flowers in comparison to plants which are reared without
any fertilizers. In both cases, a common environmental factor was the cause of
correlation between two traits. In the first case, the height of the plant and the
number of seeds are phenotypically correlated due to the moisture content in their
environment. In the second case, the height of the plant and the number of flowers
are correlated due to the use of fertilizers.
Phenotypic correlations can also be due to genetic factors. When two phenotypes
are correlated due to underlying genetic causes, it is called a genetic correlation.
This might happen due to pleiotropy or genetic linkage. Pleiotropy is the phenome-
non wherein a single gene regulates multiple phenotypes. For example, people who
are taller tend to have bigger hands and vice versa. This is due to the fact that in
general the size of various body parts is dependent on growth hormones, and there
are genes which regulate the amount of growth hormone being secreted by the
pituitary. Therefore, a common group of genes is able to affect the size of multiple
parts of the human body. Furthermore, genetic linkage is observed when two genes
are physically located very close to each other because of which they have a very
high tendency of being inherited together generation after generation. Two such
genes tend to show genetic correlation if they are controlling two different pheno-
typic traits.
Genetic correlations can either be positive or negative. Positive correlation is
seen when the genes that cause an increase in the measure of one quantitative trait
also simultaneously increase the measure of another trait. This would also mean that
a decrease in the magnitude of one trait is accompanied by a decrease in the other.
For example, the genes that control thorax length and wing length in Drosophila are
common; therefore, an increase in thorax length is accompanied by an increase in
wing length. This is also true for the size of a chicken and the mean weight of the
eggs it lays. Negative correlation is seen when the genes contributing to an increase
in the measure of one trait cause a decrease in the measure of another trait. The
amount of milk production in cattle is negatively correlated to the percentage of
butterfat in the milk. Also, the size of the eggs and the number of eggs laid by a
chicken are negatively correlated.
Genetic correlations are important from the standpoint of both natural and artifi-
cial selection. This is because a change in one trait can be accompanied by a change
in magnitude of another trait (Table 20.2). This becomes a problem because many
traits are optimized for the environments we have to survive in. Genetic correlations,
especially negative ones, can lead to the decrease or increase in measure of a trait that
results in the organism to be unfit for its own environment. For example, there is a
negative correlation between body size and fertility in turkeys. Attempts at produc-
ing larger turkeys for commercial purposes led to the decrease in fertility of the
population. This provided an upper limit to artificial selection for size in turkeys.
Selection pressure in the natural world also fine-tunes the effects of genetic correla-
tion. Garter snakes tend to prey on toxic newts which produce the neurotoxin
tetrodotoxin. These snakes also have to be fast in order to escape their own predators.
1068 A. Chatterjee

Table 20.2 Genetic correlations between traits in multiple organisms

Organism Traits Genetic correlation
Humans IgG, IgM 0.07
Cattle Butterfat content, milk yield 0.38
Pigs Weight gain, back-fat thickness 0.13
Chickens Body weight, egg production 0.17
Body weight, egg weight 0.42
Mice Body weight, tail length 0.29
Jewelweed Seed weight, germination time 0.81
Milkweed bugs Wing length, fecundity 0.57
Wood frogs Developmental rate, size at metamorphosis 0.86
Drosophila Early life fecundity, resistance to starvation 0.91
Adapted from iGenetics: A Molecular Approach by Peter J. Russell, 3rd edn. (2014)

However, there is a negative correlation between speed and resistance to tetrodo-

toxin in these snakes. This means that neither trait can be allowed to increase
indefinitely, and therefore only a mutually beneficial range of expression of the
two traits are selected for.

20.7 QTL Mapping

Our analysis and understanding of quantitative traits have predominantly stemmed

from statistical analysis of observable phenotypes in successive generations of
artificially bred plants and animals. However, each of the traits studied must have
an underlying group of genes and alleles which interact to produce the observable
phenotype. In other words, the true physical basis of quantitative traits lies within
segments of DNA which, via multiple modes of intercommunication, finally produce
a phenotype. Unlike classical qualitative Mendelian traits, the search for multiple
genes and alleles responsible for quantitative traits is quite a daunting task. Unlike
simple recombination frequency calculations required to map out the former kind of
genes, different strategies are employed to map the genes contributing to a quantita-
tive trait. These strategies predominantly include the use of different observable or
quantifiable markers and then checking whether any of these markers consistently
get inherited along with the quantifiable trait under study. This kind of consistent
inheritance of the marker with a trait of choice is called co-segregation. When
co-segregation happens, one can assume that the chosen marker is physically close
to a segment of DNA which is contributing to the trait under study. This is because
co-segregation happens when two segments of DNA are very close to each other and
therefore very rarely get separated due to crossing over. In case co-segregation is
seen, then we can say that the marker is close to a Quantitative Trait Loci (QTL). A
QTL is a segment of DNA which may contain a single gene or a group of genes
which regulate a quantitative trait.
20 Quantitative Genetics 1069

A number of markers have been used by multiple research teams. These include
using visible phenotypes, like eye colour, which are distinct from the trait under
study but can be checked for co-segregation. Other markers include using proteins
having different electrophoretic mobilities, for example, isozymes. However, the
most commonly used markers are DNA markers such as single nucleotide
polymorphisms (SNPs), variable number of tandem repeats (VNTRs) and restriction
fragment length polymorphisms (RFLPs). These markers are spaced out throughout
the genome of all species, and their locations on different chromosomes for most
model organisms are already known. One can check whether any of the above
markers co-segregate with the trait under study, and this may tell us the possible
location of a QTL.
The experimental paradigm employed to find QTLs includes using two artificially
bred or naturally found inbred lines which exhibit two extremes of a phenotypically
variable trait, for example, short longevity versus long longevity in Drosophila.
Each of these lines is assumed to be homozygous for the alleles regulating the
quantitative trait, as generations of inbreeding cause the formation of pure-bred lines.
These two lines are used as the parental generation to breed the F1 generation. The
F1 generation usually exhibits a phenotype which is the mean of the quantitative
traits in the parental generation and is assumed to be heterozygous for all the alleles
regulating the quantitative trait. The F1 progeny are selfed to produce the F2
generation. Individuals of the F2 generation exhibit a large phenotypic variance
for the quantitative trait under study. The F2 generation is also known as the QTL
Mapping Population. The large phenotypic variance in F2 is assumed to be the result
of the mixing up of all the alleles contributing to the quantitative trait in various
permutations and combinations in the gametes of the F1 generation. This gives rise
to a large phenotypic variance. Before we execute the above paradigm, we must have
a list of the DNA markers which differ between the parental lines. To begin with we
do not know which of these DNA markers is close to a QTL. After breeding up to the
F2 generation, we can now check which of the original DNA markers have
co-segregated with the quantitative traits of interest (Fig. 20.13).
The above paradigm can be better understood using an example. A study was
conducted by Steven Tanksley and colleagues to find the QTLs responsible for fruit
weight in tomatoes. They used two varieties of tomatoes which differ drastically in
their weight, Lycopersicon esculentum and Lycopersicon pimpinellifolium. While
the tomatoes from the former plant weigh around 500 g, those from the latter weigh
1 g on an average. They positioned 88 RFLPs on the 12 chromosomes of
Lycopersicon and used these RFLPs as DNA markers. The F1 tomatoes produced
weighed on an average 10.5 g (not exactly the mean of the two parental weights but
more than L. pimpinellifolium and less than L. esculentum). The F1 plants were self-
fertilized and the subsequent.
F2 generation exhibited a large range of fruit weights, from less than 5 g up to
45 g. This is due to the segregation of all the genes (and alleles) affecting fruit
weight. Extraction of DNA from each of the F2 plants helped in analysing whether
any of the original parental RFLPs co-segregated with a given fruit weight. Most
RFLPs were not seen to co-segregate with weight; however, there were a few RFLPs
1070 A. Chatterjee

High line Low line

RFLP1 RFLP1 RFLP2 RFLP2

QTL1 QTL1 QTL2 QTL2
P1

RFLP1 RFLP2
QTL1 QTL2
F1

RFLP1 RFLP1 RFLP1 RFLP2 RFLP2 RFLP2

QTL1 QTL1 QTL1 QTL2 QTL2 QTL2

F2

Fig. 20.13 QTL mapping using known RFLP markers in the genome: RFLP1 is a DNA marker for
QTL1, and RFLP2 is a DNA marker for QTL2 in two chromosomes of a hypothetical organism.
RFLP1 is found in a line which exhibits a high magnitude of a given quantitative trait (high line),
and RFLP2 is found in a line which exhibits a low magnitude of the same trait (low line). In the F1
generation, all such loci are heterozygous for the DNA marker. In the F2 generation, co-segregation
of the RFLP markers to individuals exhibiting a certain magnitude of the trait helps in locating the
positions of possible QTLs. In the F2 generation, RFLP1 co-segregates with individuals exhibiting
high magnitude of the trait, while RFLP2 co-segregates with individuals exhibiting lower
magnitudes of the trait. This means that RFLPs 1and 2 are close to the genetic region regulating
this trait

which were always associated with a given fruit weight. These RFLPs were then
considered to mark the position of a possible QTL nearby. Cloning of different
segments of the chromosomal segment housing the possible QTL can finally allow
the identification of the gene responsible for the quantitative trait. Also, candidate
genes in the QTL region having the desired functional annotations can be studied
further. This means that if a known gene having a known function is known to be
located near the co-segregating DNA marker, then that gene becomes a strong
candidate gene for further study. The gene in the above QTL was identified as
ORFX, and it was cloned. Different ORFX alleles have been correlated with plants
producing tomatoes of varied sizes. The product of the ORFX gene seems to inhibit
cell division and when artificially made to express in a plant causes a reduction in the
20 Quantitative Genetics 1071

size of tomatoes produced by the plant. However, the entire range of phenotypic
variance observed in tomato size cannot be explained by the ORFX gene, and there
are certainly multiple genes in other QTLs which contribute to this trait.
Sometimes the observed phenotypic variance is not due to genetic contribution of
multiple genes from several QTLs but due to the existence of multiple alleles all
belonging to the same genetic locus. This was found to be the case for phenotypic
variations in haltere development in fruit flies, as well as acid phosphatase activity in
humans. In both cases, variations in phenotypes were due to the presence of multiple
alleles of a single gene in a given population; however, each of these alleles affected
their phenotypes quantitatively to a definite degree. The cumulative effect of all the
different combinations of these alleles produces a phenotypic variation for the trait in
the population. Furthermore, not all QTLs contribute to protein levels (protein
QTLs). Some are known to regulate phenotypes by altering the RNA transcript
levels of genes (expression QTLs).
Identification of genes in QTLs has advantages. A knowledge of the genes
regulating the quantitative trait of interest allows us to manipulate the genome
more effectively. This allows for larger genetic gains in artificial selection and a
better understanding of genetic predisposition to many medical conditions, espe-
cially those which can be categorized as threshold traits. It also aids in the develop-
ment of more authentic theories to explain evolutionary processes. Recent studies on
QTLs have disproven the assumption of polygenic models that each quantitative
locus contributes nearly equally to a phenotype. It is now widely believed that a few
of the loci, or even one, contribute to a major component of the phenotype; therefore,
mutations in even one important loci can bring about evolutionarily significant
phenotypic changes.
Often closely linked loci do not get separated. Linked QTLs might share a
common DNA marker, and the presumed effect of one QTL might be due to multiple
such loci in that local segment of the chromosome. Sometimes loci with relatively
small effects on the phenotype go unnoticed due to the resolution limit of the
experiment. Importantly, we must remember that the only loci we can detect are
those that were different in the two parental lines to begin with. Genes which are
homozygous in the QTLs of the two parental lines will remain undetected. The
above points are a few reasons why the number of QTLs is usually underestimated.

Box 20.1: Scientific Concept: Genetic Architecture of Natural Variation

in Drosophila melanogaster Aggressive Behaviour – John Shorter
et al. (2015)
Many traits of interest in both plants and animals seem to be controlled by
more than one gene. This kind of polygenic inheritance is often associated
with phenotypes ranging from phenotypic variations in kernel colour in wheat
to the pattern of inheritance of skin colour in humans. The study of polygenic
inheritance influences our understanding of complex traits such as IQ variation

(continued)
1072 A. Chatterjee

Fig. 20.14 Gradation in displayed aggression levels: Aggression levels were measured in
200 available inbred lines of Drosophila (from Drosophila Genetic Reference Panel, or DGRP).
Low and high aggression parental lines were chosen from this population to generate the outbred
lines

Fig. 20.15 Change in gene expression is associated with change in aggression levels: Insertional
mutants of different candidate genes exhibited a decreased magnitude of aggression (except jim,
which showed increased aggression) implicating their role in the polygenic control of the trait. The
two genes on the right were downregulated using RNAi. Decreased aggression levels were
observed in each case

Box 20.1 (continued)

and helps in making economically important decisions concerning the
improvement in quality of domestic animals and cash crops. Though derived
from Mendelian genetics, an understanding of the inheritance of polygenic
traits deviates from the simpler analysis of classical Mendelian examples. It
requires a unique set of concepts and definitions, the majority of which this
chapter has been dedicated to. The sheer number of genes that can possibly
participate in dictating the phenotypic variation of a complex trait makes it a
daunting task to find the genetic correlates of any trait under investigation. One
must factor in the added component of the possible permutation and combina-
tion of interactions that are possible between the genes regulating the expres-
sion of the trait. These can range from simple additive interactions to the more

(continued)
20 Quantitative Genetics 1073

Box 20.1 (continued)

elusive dominant and epistatic genetic interactions. Furthermore, interactions
between the genetic components and the environment of the organism further
complicate the matter of genetically dissecting a complex trait. One such trait
is aggression.
Aggression is an evolutionarily conserved, complex and universal
behaviour that influences access to food resources, escape from predators,
obtaining mates and establishing dominance hierarchies. It is considered to be
a quantitative trait and is influenced by both genetic and environmental factors.
Common genes and neural pathways have been implicated in aggressive
behaviour in Drosophila, mice and humans. The bioamine signalling pathway
plays a key role in regulating this behaviour. However, the genetic basis of the
phenotypic variation in aggression remains unknown. Using Drosophila as a
model system, Shorter et al. proceeded to find the genes responsible for
genotypic (or genetic) variation in aggression. They performed genome-wide
association (GWA) analysis in available inbred Drosophila lines after deter-
mining their aggression levels (Fig. 20.14). They also performed GWA analy-
sis on outbred lines. These outbred lines were derived from mating inbred
parental Drosophila lines which differed drastically in their levels of aggres-
sion (as ascertained from Fig. 20.14). One chosen parental line was a low
aggression line, while the other was a high aggression line. SNPs were used as
DNA markers to locate the position of possible QTLs. SNPs were scored for
co-segregation with the magnitude of aggression exhibited by the fly
harbouring that particular SNP. Genes significantly associated with aggression
post-analysis included those that are involved in motor activity, reproduction,
chemosensation and neurotransmitter reception. Interestingly, they calculated
H2 ¼ 0.69  0.07, but, h2 ¼ 0 for this trait. This suggests that though genetic
components of variance in aggression exist, they are predominantly
non-additive in nature. Furthermore, analysis of the data did not find much
overlap between the candidate genes identified in the inbred parental popula-
tion and the outbred offspring population of Drosophila. This lack of overlap
also hints at the near absence of additive components of genetic variance in
aggression. In fact, the construction of a genetic interaction network showed
that the genes identified in the original parental population and the offspring
population were epistatically linked to each other (gene-gene interactions). In
order to validate the biological relevance of the candidate genes, insertional
mutants (of the candidate genes) were generated and RNAi knockdown
experiments were conducted in Drosophila. In nearly all the cases, disruption
of the expression of the candidate gene resulted in a reduction in aggression
(Fig. 20.15). Additionally, it was shown that epistatic interactions between
different candidate genes can also regulate the degree of expressed aggression.
It is now believed that aggression has an underlying polygenic genetic archi-
tecture dominated by epistatic gene action. It is yet to be determined how these
genetic interactions establish the neural underpinnings of aggression.
1074 A. Chatterjee

20.8 Summary

• Quantitative genetics is the study of phenotypic traits which express a range of

values. Such traits are usually regulated by more than one gene and hence are also
called polygenic traits. They are considered to be multifactorial in nature as both
genetic and environmental factors can contribute to the range of values (variation)
observed in the trait. Such traits can exhibit meristic, threshold, or continuous
characteristics. They are distinct from oligogenic (or Mendelian) traits. Examples
of such traits include kernel colour in wheat, ear length in maize and skin colour
in humans.
• Quantitative or polygenic traits are studied at a population level. The collective
variation exhibited by the individuals of a population (or sample) is described
using various statistical terms such as mean, standard deviation and variance. The
relationship between two different phenotypic traits is computed using correlation
and regression analysis. ANOVA helps in the estimation of the degree by which
measures of two traits are different from each other.
• Variation in a phenotypic trait can be caused due to variation originating from the
genes as well as variation arising from environmental factors. Genetic variation
can be due to additive and dominance factors operating within a locus, or due to
epistatic interactions between loci. Environmental factors can be internal or
external in nature. They can also be special or general in nature. Other factors
contributing to phenotypic variation include gene-environment interaction, gene-
environment covariance and intangible variation.
• Heritability measures the contribution of genetic factors to phenotypic variation
(variance). It can be estimated as broad-sense heritability and narrow-sense
heritability. The former takes into consideration all genetic sources of phenotypic
variation, while the latter only considers the additive genetic component of
phenotypic variance. There are multiple ways of calculating heritability. These
include elimination of one variance component, parent-offspring regression and
comparing phenotypic variance in individuals with varying degree of relatedness.
• Anthropomorphic manipulation of the mean value of phenotypic traits is artificial
selection. Such selection is usually done for commercially important traits such as
milk yield, egg production and wool yield. Quantities such as genetic gain,
selection intensity and genetic advance help describe the selection process.
There are limits to artificial selection as excessive change in the magnitude of
one trait usually has an undesirable effect on the phenotype of another trait. This
is due to the phenomenon of phenotypic correlations.
• Localizing the multiple genetic elements responsible for regulating the pheno-
typic expression of a trait is known as QTL mapping. The consistent
co-segregation of molecular markers in the genome, such as RFLPs and SNPs,
with a specific magnitude of the quantitative trait of interest helps in deciphering
the possible location of genes controlling a polygenic trait.
20 Quantitative Genetics 1075

References
Arranz JJ, Coppieters W, Berzi P, Cambisano N, Grisart B, Karim L, Marcq F, Moreau L, Mezer C,
Riquet J, Simon P (1998) A QTL affecting milk yield and composition maps to bovine
chromosome 20: a confirmation. Anim Genet 29:107–115
Bidinost F, Roldan DL, Dodero AM, Cano EM, Taddeo HR, Mueller JP, Poli MA (2008) Wool
quantitative trait loci in merino sheep. Small Rumin Res 74:113–118
Grisart B, Coppieters W, Farnir F, Karim L, Ford C, Berzi P, Cambisano N, Mni M, Reid S,
Simon P, Spelman R (2002) Positional candidate cloning of a QTL in dairy cattle: identification
of a missense mutation in the bovine DGAT1 gene with major effect on milk yield and
composition. Genome Res 12:222–231
Gutiérrez-Gil B, El-Zarei MF, Alvarez L, Bayón Y, De La Fuente LF, San Primitivo F, Arranz JJ
(2009) Quantitative trait loci underlying milk production traits in sheep. Anim Genet 40:423–
434
Liu Z, Sun C, Yan Y, Li G, Wu G, Liu A, Yang N (2018) Genome-wide association analysis of
age-dependent egg weights in chickens. Front Genet 9
Roldan DL, Dodero AM, Bidinost F, Taddeo HR, Allain D, Poli MA, Elsen JM (2010) Merino
sheep: a further look at quantitative trait loci for wool production. Animal 4:1330–1340
Russell PJ (2014) iGenetics, a molecular approach, 3rd edn. Pearson New International Edition,
Harlow
Shorter J, Couch C, Huang W, Carbone MA, Peiffer J, Anholt RR, Mackay TF (2015) Genetic
architecture of natural variation in Drosophila melanogaster aggressive behavior. Proc Natl
Acad Sci 112:E3555–E3563
Sinha NK, Singh SK (1997) Genetic and phenotypic parameters of body weights, average daily
gains and first shearing wool yield in Muzaffarnagri sheep. Small Rumin Res 26:21–29
Snustad DP, Simmons MJ (2012) Principles of genetics, 6th edn. John Wiley and Sons, Inc.,
Hoboken, NJ
Vesely JA, Peters HF, Slen SB, Robison OW (1970) Heritabilities and genetic correlations in
growth and wool traits of Rambouillet and Romnelet sheep. J Anim Sci 30:174–181
Wolc A, Arango J, Settar P, Fulton JE, O’sullivan NP, Preisinger R, Habier D, Fernando R, Garrick
DJ, Hill WG, Dekkers JCM (2012) Genome-wide association analysis and genetic architecture
of egg weight and egg uniformity in layer chickens. Anim Genet 43:87–96
Yuan J, Sun C, Dou T, Yi G, Qu L, Qu L, Wang K, Yang N (2015) Identification of promising
mutants associated with egg production traits revealed by genome-wide association study. PLoS
One 10:e0140615
Zhang Q, Boichard D, Hoeschele I, Ernst C, Eggen A, Murkve B, Pfister-Genskow M, Witte LA,
Grignola FE, Uimari P, Thaller G (1998) Mapping quantitative trait loci for milk production and
health of dairy cattle in a large outbred pedigree. Genetics 149:1959–1973

Further Reading
Falconer DS, Mackay TFC (1996) Introduction to quantitative genetics, 4th edn. Addison Wesley
Longman Limited, Harlow
Griffiths AJ, Wessler SR, Lewontin RC, Gelbart WM, Suzuki DT, Miller JH (2011) An introduc-
tion to genetic analysis, 10th edn. W H Freeman and Company, New York
Klug WS, Cummings MR, Spencer CA, Palladino MA (2012) Concepts of genetics, 10th edn.
Pearson Education, Inc., San Francisco, CA
Lynch M, Walsh B (1998) Genetics and analysis of quantitative traits. Sinauer Associates, Inc.,
Sunderland, MA
1076 A. Chatterjee

Pierce BA (2010) Genetics: a conceptual approach. W H Freeman and Company, New York
Powar CB (2003) Genetics (volume 1), 1st edn. Himalaya Publishing House, Mumbai
Singh BD (2005) Genetics, 1st edn. Kalyani Publishers, New Delhi
Tamarin RH (2001) Principles of genetics, 7th edn. The McGraw-Hill Companies, New York
Population Genetics
21
Payal Gupta

21.1 Identification of Genetic Variation

With the exception of identical twins, all humans show variations in their looks,
features and habits. The basis of these fundamental differences lies in the genetic
make-up of all individuals. The gigantic genome and the multiple processes of
recombination, mutation, random assortment, linkage, etc. provide each individual
with his/her unique genetic make-up. Therefore, as humans we have the same basic
genomic structure, but every human has a different genetic constitution.
Thus genetic variation defines the differences in DNA sequences between
individuals of the same population or the gross sequence differences between two
populations. It can also imply genetic differences between members of the same
species or members of different species. Genetic variations are at the core of all the
natural diversity that we observe within a population or between populations.
The variations can arise from differences in sequences within coding or
non-coding segments of the genome. Since most of the genes that code for important
peptides are relatively conserved, variations in non-coding sections are usually more
informative.
When certain variations present themselves at specific sites along the chromo-
some and can be uniquely characterized using techniques such as polymerase chain
reaction (PCR) and gel electrophoresis, they can serve as molecular markers. Also,
the genetic distance between molecular markers that are linked can be estimated
from the outcomes of crosses.
A single molecular marker can exist in more than two “forms,” that is, the same
site can have more than two types of sequence variations. This property is termed
polymorphism, literally meaning existing in multiple forms. If a specific locus in the
genome of species always has a fixed nucleotide/s, it is said to be monomorphic.

P. Gupta (*)
University of Calcutta, Kolkata, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 1077
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_21
1078 P. Gupta

However, if the genomes of different members of that species show sequence

variation at that locus, then the locus is said to be polymorphic. A particular variation
for a locus should be present in at least 1% of the population to qualify as a
polymorphic marker. The study of distribution of polymorphic markers in a given
population provides vital information about the population. Further, polymorphic
markers serve as important connotations for specific genes and traits, allowing us to
perform genetic association studies to predict genetic identity of the linked gene.
In population genetics, polymorphic markers generally display as observable
variations in a trait within a particular population. Population geneticists have been
interested in variations in traits that can be observed with naked eyes, like differences
in colour, shapes and patterns. A classic example is the Hawaiian happy-face spider
that displays an umbrella of colour and pattern polymorphism (Fig. 21.1). The
abdominal colour and pattern of the Maui population range from plain yellow to
varying red, white and/or black patterns on a yellow background, and the colour
morphism is controlled by simple Mendelian alleles.
In this discussion two dimensions of polymorphism become important: pheno-
typic polymorphism and genetic polymorphism. Phenotypic polymorphism
describes two or more morphs/variation of a single trait that is visibly noticeable
in individuals of a population. Genetic polymorphism describes changes or
variations at genetic level in a population. Genetic polymorphism may not always
be evident as phenotypic variations.

21.1.1 Single Nucleotide Polymorphism (SNP)

Single nucleotide polymorphism (SNP, frequently pronounced as “snips”) denotes

genetic variation present at a single nucleotide position. When a single nucleotide
position is polymorphic among individuals, it is recognized as SNP. SNPs are fairly
common in all genomes. They are extremely useful in the mapping of disease-
causing alleles and genes that contribute to quantitative traits. In humans alone,
SNPs represent 90% of all the variation in sequences that are found in different
individuals. A classic example of how polymorphism at a single base can result in
stark phenotypic changes is the human β-globin gene (Hb). The example shown in
Fig. 21.2 shows that the Hb gene can exist as two morphs HbA and HbS both of
which differ from each other at single base position; however, the recessive HbS
allele can cause sickle cell disease.

21.1.2 Microsatellite

Molecular markers can also present themselves as short tandem repeats (STRs), also
known as microsatellites, simple sequence repeats (SSR) and simple sequence length
polymorphisms (SSLP). These are di-, tri-, tetra- or penta-nucleotide sequences
repeated multiple times in a stretch of a chromosome. Many such stretches of
short tandem repeats are found across the genome, and their length varies
21 Population Genetics 1079

Fig. 21.1 Colour polymorphism displayed by the Hawaiian happy-face spider Theridion grallator.
Maui spiders display an array of colour and pattern polymorphism with different colours and
patterns appearing on a yellow background on the abdomen
1080 P. Gupta

Fig. 21.2 The single nucleotide polymorphs of the human β-globin gene. The β-globin gene has
two alleles, HbA (top sequence) and HbS (middle sequence), which differ from each other at single
nucleotide position (highlighted base pair) and serve as an example of single nucleotide polymor-
phism. However, a 5-bp deletion (bottom sequence) can result in a different loss of function allele

considerably between individuals. For instance, a dinucleotide sequence—(CA)n,

where n is the number of times CA sequence is repeated and can be anywhere
between 5 and 50—is commonly found in humans. In fact, thousands of different
DNA segments contain the (CA)n microsatellite. During replication of a microsatel-
lite region, the replication machinery often slips and causes copy number variation of
the repeat region. For this reason, the microsatellite segment is regarded as hyper-
variable. This makes them a very informative molecular marker and one that rapidly
evolves. They are in fact more polymorphic than SNPs and are more helpful in
detailed genetic polymorphism studies. Microsatellites are inherited in Mendelian
patterns, are often co-dominant and are easily characterized. Small amount of
samples can be used to conduct PCR for microsatellites, and the information can
help in pedigree analysis for the study of population structure.
21 Population Genetics 1081

21.1.3 Haplotype

Today innumerable genetic markers are available at one’s disposal. However, it is

more informative to analyse multiple markers simultaneously to make mapping and
genetic analysis more robust. Therefore, the inheritance of multiple markers as a
single linked haplotype becomes essential. Haplotype is a set of genes present on a
chromosome that are transferred from parents to progeny en bloc or as a single block
unit. This inheritance of a group of genes as a single cluster occurs due to tight
genetic linkage. Similarly, a set of SNPs or other polymorphisms can also be
inherited as a block due to their close genetic proximity. The study of haplotype
structures can help in various ways. Haplotype mapping can help us to identify a
disease-carrying allele by virtue of its proximity to a particular morph of a haplotype
(Fig. 21.3). It can also help in the identification of genetic variants that are responsi-
ble for different traits in a population.
When markers and alleles show linkage frequencies considerably higher than one
would expect out of a random cross, the phenomenon is known as linkage disequi-
librium (LD). A study of STR and Alu deletion polymorphism at the CD4 locus at
42 populations worldwide first outlined the importance of LD in tracing population
history.

21.1.4 The International HapMap Project

The understanding of the usefulness of SNPs on a block gave rise to The Interna-
tional HapMap Project. Each haplotype block of SNPs may contain many SNPs, but
a combination of a few variants itself can give rise to a pattern unique to an

Fig. 21.3 Schematic representation of disease allele mapping using haplotype. Four sites (1–4,
and) are linked and occur as a haplotype along a particular chromosome. These sites have SNP
variants (A, B and C) that serve as their markers. If a founder disease-causing mutation arises near to
the second locus which harbours the SNP variant “C”, the disease allele becomes tightly linked to
the “C” variant at the second locus. Thus the presence of the “C” variant at this locus would indicate
a higher probability of disease allele
1082 P. Gupta

Fig. 21.4 Tag SNPs can help identify haplotype variants. (a) The same chromosomal segments
from four different people show SNP variations. The DNA sequence is identical for maximum
bases in these chromosomes, but three bases exhibit sequence variation. Each SNP has two possible
alleles; the first SNP in panel (a) has the alleles C and T. (b) A haplotype comprises a specific set of
alleles linked to proximal SNPs. The figure shows only the variable bases consisting of 20 SNPs
that stretch of 6000 bases of DNA and mark a particular haplotype. This includes the three SNPs
shown in panel (a). For this segment of DNA, the majority of population would have haplotypes
1–4. (c) The three highlighted SNPs serve as tag SNPs such that genotyping only these three tag
SNPs out of the 20 SNPs is enough to uniquely identify these four haplotypes

individual and a haplotype. These few SNPs that can successfully and specifically
identify a haplotype are called tag SNPs. The genomic map of these tag SNP
haplotype blocks is known as a HapMap. The HapMap is crucial as it narrows the
nearly ten million known SNPs to roughly 50,000 SNP tags that are required to
examine the entire genome for association. This makes genome scan for identifica-
tion of linked disease alleles both efficient and comprehensive as now less number of
SNPs needs to be mapped (Fig. 21.4).
To execute this task of mapping haplotypes, researchers from academic centres,
non-profit biomedical research groups and private companies in Canada, China,
Japan, Nigeria, the United Kingdom and the United States collaborated to undertake
the International HapMap Project. A meeting on October 27 to 29, 2002, marked the
beginning of the project, and it was projected to take about 3 years.
The International HapMap Project aims at determining the common features in
DNA sequence variation in the human genome. This involves characterization of
sequence variants, their prevalence and associations between them. For this purpose
DNA samples from populations with ancestral lineage from parts of Africa, Asia and
Europe were analysed. The project makes the expense of whole genome sequencing
dispensable by allowing the indirect association approach of marking the linkage of
a trait to a particular haplotype. This can be applied to any functional candidate gene
21 Population Genetics 1083

in the genome, to any DNA segment suggested by family-based linkage analysis, or

finally to the entire genome for scans for disease risk factors.
Apart from the study of genetic associations with diseases, the HapMap can also
be applied to the study of the genetic factors that contribute to variation in response
to environmental factors and susceptibility to infection and in the physiological
responses to drugs and vaccines in specific populations or individuals. These studies
are based on the understanding that contributing genetic variants occur with a higher
frequency in a group of people having a certain disease or exhibiting a particular
response to environmental factors, drugs or pathogens than in a group of people that
do not exhibit the disease or response phenotype. The read-outs from analysis of tag
SNPs can indicate chromosomal segments displaying different haplotype
distributions in the two groups of people, those with a disease or response and
those without. Each segment can be further analysed in greater detail to identify the
exact variants or genes in the genetic region contributing to the disease or response
phenotype. This can eventually lead to more effective interventions and also allow
the development of diagnostic markers that can predict which drugs or vaccines
would be most effective in individuals with particular genotypes for genes affecting
drug metabolism.

21.2 The Gene Pool Concept

Since population genetics involves the study of genetic variation in a group of

individuals over time, it becomes mandatory to look at the genetic make-up of the
entire population rather than the genetic composition of an individual. From this idea
evolves the Gene Pool Concept. The gene pool essentially comprises all of the
alleles of every single gene present in a given population. It is precisely a collection
of all the genetic information that a population harbours, and this determines the set
of all genetic variants from which the genetic make-up of forthcoming generation
will be determined. The gene pool of a population determines the genetic
characteristics of the next generation except if there is a change in the composition
of the gene pool due to a founder mutation, migration or any of the forces of
evolution. With each passing generation, the dynamics of the gene pool will change
depending on allelic variations and evolutionary forces.
To completely comprehend the concept of gene pool, it becomes important to
discuss the meaning of “population” from the perspective of a geneticist. For a
sexually breeding species, a population signifies a group of interbreeding individuals
of the same species that inhabit the same region. A species may occupy a wide
geographic area wherein they are subdivided into smaller, discrete populations.
Therefore smaller local populations or demes can make up a larger population,
with the members of a deme having a higher probability of interbreeding.
The aim of a population geneticist is to understand the prevalence of different
alleles of a polymorphic gene in a given population, study the changes that these
alleles undergo from generation to generation and identify the underlying causes for
such changes. In order to make an objective study, the population geneticist works
1084 P. Gupta

with two fundamental concepts: allele frequency and genotype frequency. Allele
frequency of a particular allele is the ratio of the number of copies of that allele in the
population to the total number of all alleles for that gene in the said population. The
genotype frequency is denoted as the ratio of the number of individuals with a
particular genotype in a population to the total number of individuals in that said
population.
This can be understood with an example of a particular population. Let us
suppose that there is a population of 100 mice with the following genotype:

64 mice have black coat colour with the genotype BB.

32 mice have grey coat colour with the genotype Bb.
4 mice have white coat colour with the genotype bb.

Therefore, for homozygous individuals an allele frequency calculation would

account for two copies of that allele, while for heterozygous individuals, it will
account for one copy of that allele. Likewise, while calculating the frequency of the
“b” allele, the heterozygotes will be counted once, but the homozygous “bb”
individuals will be counted twice for that allele. Hence the frequency of allele “b”
would be as follows:

32 þ 2ð4Þ
b¼
2ð64Þ þ 32 þ 2ð4Þ
40
or, b ¼ ¼ 0:2, or 20%
200
This tells us that the frequency of the “b” allele is 20% or that the “b” allele
represents 20% of the total alleles of this gene in this particular population.
Let us now calculate the genotype frequency of “bb” in this population of mice.

4
bb ¼
64 þ 32 þ 4
4
or, bb ¼ ¼ 0:04, or 4%
100
This indicates that 4% of the total mice in this population have “bb” genotype and
therefore have white coat colour.

21.3 The Hardy-Weinberg Equilibrium

With the understanding that a population inherits the alleles for all genes from an
ancestral gene pool, it becomes essential to address how this inheritance works in
context to the frequency of these alleles and the genotypic proportions. It is also vital
to derive a mathematical correlation between these concepts of change of genotype
21 Population Genetics 1085

and allele frequency over generations. Godfrey Harold Hardy, a British mathemati-
cian, and Wilhelm Weinberg, a German Physician, independently proposed a math-
ematical expression that could help estimate how allele and genotype frequencies
change over generations. This came to be known as the Hardy-Weinberg
equilibrium.
The Hardy-Weinberg equilibrium postulates that given that there are no evolu-
tionary forces acting on a geographically isolated interbreeding population, the
genotype and allele frequencies remain constant over generations. Since there is
no predicted change in the allele and genotype frequencies, hence the term “equilib-
rium” applies.

21.3.1 Assumption of the Law

The Hardy-Weinberg equilibrium provides a mathematical proof of concept that

allele and genotype frequencies remain constant over generation for a population
which is not undergoing any evolutionary changes. However, this derivation is based
on an exclusive set of assumption, and the law of equilibrium applies only to a
population which fulfils the following assumptions:

1. No new mutations: The genes of interest should not undergo any new mutation.
2. No genetic drift: Random sampling should not affect the allele frequency, and
therefore only infinitely large populations are considered.
3. No migration: The population of interest is assumed to be geographically
isolated, such that there is no immigration or emigration.
4. No natural selection: No genotype (dominant or recessive) should be favoured by
the environment, and all genotypes should have equal chances of survival and
mating.
5. Random mating: There is no mate selection or mating preference, and the
members of the population mate randomly with regard to their genotype and
phenotype such that the gene of interest is evenly mixed and distributed in the
population.

21.3.2 Prediction of the Law

When all of the above assumptions hold, then the allele and genotype frequency of
the gene of interest would be constant over generations. If we follow the example of
the mice with black (BB), brown (Bb) or white (bb) coat colour, the Hardy-Weinberg
Law would predict that the allele frequency of “b” would remain to be 0.2 or 20%
and the frequency of the “bb” genotype would remain 0.4 or 40% in the next and
forthcoming generations of the population in question. Therefore, Hardy-Weinberg
Law provides a quantitative relationship between allele and genotype frequencies in
a population.
1086 P. Gupta

21.3.3 Derivation of the Law

To further understand the Hardy-Weinberg equilibrium, let us reconsider the hypo-

thetical example of mice population that possesses polymorphic gene for the coat
colour trait with alleles “B” and “b”. Let the allele frequency for “B” be denoted by
“p” and the allele frequency for “b” be denoted by “q”. According to the Hardy-
Weinberg Law,

pþq¼1

Therefore, if the frequency of “b” (q) is calculated to be 0.2 or 20%, then the
frequency of “B” ( p) would be 0.8 or 80% so that their frequencies would add up to
1 or 100%. Since Hardy-Weinberg equilibrium assumes random mating, therefore
every individual in this biallelic system would inherit two alleles, and these would be
randomly and independently assorted. Therefore, we can apply the product rule and
multiply individual probability sums, p + q together. This would be applied as
follows to the earlier equation:

ð p þ qÞ ð p þ qÞ ¼ 1

or, p2 þ 2pq þ q2 ¼ 1

In our example, p2 ¼ genotype frequency of “BB”; 2pq ¼ genotype frequency of

“Bb”; q2 ¼ genotype frequency of “bb”. So if we say that p ¼ 0.2 and q ¼ 0.8, then,
based on the Hardy-Weinberg equilibrium, we can calculate the allele frequencies as
follows:

BB ¼ p2 ¼ (0.8)2 ¼ 0.64
Bb ¼ 2pq ¼ (2) (0.8) (0.2) ¼ 0.32
bb ¼ q2 ¼ (0.2)2 ¼ 0.04

Therefore, the allele frequency for “B” is 0.8 or 80%, and the allele frequency for
“b” is 0.2 or 20%. The genotype frequency for “BB” is 0.64 or 64%, for “Bb” is 0.32
or 32% and for “bb” is 0.04 or 4%.
The Hardy-Weinberg equilibrium can also be explained with the help of a
Punnett’s square used to calculate offspring genotype frequencies from randomly
combining gametes. The frequency of an allele is equal to the frequency of gametes
carrying it in the population. In our example, the frequency of the allele “B” is 0.8,
and so the frequency of the gametes carrying allele “B” is also 0.8. We can now
apply the product rule to calculate the probability of the genotype “BB” as
0.8  0.8 ¼ 0.64 (Fig. 21.5). The frequency of “Bb” or heterozygote (given as
2pq) would be (2) (0.8) (0.2) or (0.16) + (0.16) or 0.32.
The Hardy-Weinberg equilibrium forms the basis of our understanding of how
the alleles of a gene mix and change in a population. It can help us predict changes in
the frequency of one allele or genotype depending on the changes that the other
21 Population Genetics 1087

Fig. 21.5 Punnett’s square

can be used to estimate allele
and genotype frequencies

alleles go through. For instance, when the frequency of the “b” allele is low, the
genotype “BB” will be predominant. Conversely when the frequency of allele “b” is
high, the genotype “bb” will have a higher probability of representation in a
population.

21.3.4 Extension of the Law with More than Two Alleles

Given the stringent assumptions, no population can practically follow the Hardy-
Weinberg equilibrium; however, the Law can be extended and modified to fit
practical examples like multiple allelic systems. The basic understanding is that
the Hardy-Weinberg equations predict the probability of finding a particular geno-
type combination if the frequency of alleles is known. Since the premise of indepen-
dent assortment still stands, therefore the probability of finding a particular genotype
is calculated by applying the product rule using individual allele frequencies. Let us
understand this better with the help of the example of the ABO blood grouping with
three alleles, IA, IB, IO with their frequencies represented as pA, pB, pO, respectively.
Let us suppose that the values for the frequencies are as follows:

pA ¼ 0.3
pB ¼ 0.1
pO ¼ 0.6

So we see that the sum of allele frequencies, i.e. pA + pB + pO would be equal to

1 [0.3 + 0.1 + 0.6 ¼ 1] according to the basic principle of Hardy-Weinberg
equilibrium. Now we can draw a Punnett’s square similar to the one used for a
1088 P. Gupta

Fig. 21.6 Punnett’s square for calculation of allele frequencies in a tri-allelic system of ABO blood
grouping

trihybrid cross for gamete combination. Again, it is implicit that the frequency of a
particular allele in a population is equivalent to the frequency of that gamete in that
population. As shown in Fig. 21.6, the frequency of genotypes can be calculated as
follows:

Frequency of the homozygote ¼ square of allele frequency;

Frequency of the heterozygote ¼ (2) (frequency of the first allele) (frequency of the
second allele).

Therefore, the frequency of the IA homozygote can be calculated as:

(pA)2 ¼ (0.3)2 ¼ 0.09

Also, the frequency of the IAIB heterozygote genotype can be calculated as:

(2) (pA) (pB) ¼ (2) (0.3) (0.1) ¼ 0.06

The above basis of product rule can be applied to any number of alleles for a gene
in a population.
21 Population Genetics 1089

21.3.5 Extension of the Law to X-Linked Alleles

In our discussions so far, we have focussed on autosomal genes. However, applying

the principles of Hardy-Weinberg equilibrium to alleles on X-chromosomes in
organisms, such as Drosophila and humans, in which females are homozygous
and males are hemizygous for the X-chromosome, requires a different approach.
The fundamental principles remain the same. Since females carry two
X-chromosomes, the frequency of a genotype would be calculated by simply
applying the logistics of the Hardy-Weinberg equations as follows:
Frequency of homozygous females ¼ (Frequency of allele)2.
Frequency of heterozygotes females ¼ (2) (frequency of first allele) (frequency of
second allele).
However, since males are hemizygous for the X-chromosome, the frequency of
the genotypes is equal to the individual allele frequency. Hence, X-linked traits are
more common in males compared to females. It is now important to appreciate that
in a randomly mating population, the males will receive the X-chromosomes from
the female parent, while the females will receive one X-chromosome from each male
and female parent. Hence, the X-linked allele frequency in females will be the
average of both parents. Therefore, the frequency pattern of the alleles between
two sexes will alternate in each generation.

21.3.6 Test for Hardy Weinberg Proportion

Since Hardy-Weinberg’s equilibrium applies only to populations that follow specific

assumptions and do not undergo any evolutionary changes, hence it might be
expected that very few examples follow this in practice. However, a chi-square
test can be performed to test if a particular population is following Hardy-
Weinberg’s equilibrium or not. If the equilibrium is not in place, then one can
analyse which of the five assumptions have been violated.
Let us test the Hardy-Weinberg law on a hypothetical population using the
chi-square (χ2) test. In our hypothetical population, a gene with alleles “R” and
“W” controls the colour of wings in a species of butterfly population. The allele
frequency of “R” is denoted by “p”, while the frequency of “W” is denoted by “q”.
Now let us assume that in the population of our interest, the number of “RR”
butterflies is 12, the number of “RW” butterflies is 53 and the number of “WW”
butterflies is 12. The allele frequency “p” can be calculated as follows:


2  number of “ RR” homozygotes þ number of “ RW” heterozygotes
p¼
2  total number of individuals
Therefore,
1090 P. Gupta

ð2  12Þ þ 53
p¼ ¼ 0:5
ð2  77Þ

And,

q ¼ 1  p ¼ 1  0:5 ¼ 0:5

Using the above values for “p” and “q”, we can calculate the expected genotype
frequencies as per Hardy-Weinberg law.

p2 ¼ (0.5)2 ¼ 0.25
2pq ¼ (2) (0.5) (0.5) ¼ 0.5
q2 ¼ (0.5)2 ¼ 0.25

To implement a chi-square test, we need to calculate the actual numbers for the
individual genotype populations and compare them with the numbers one would
expect if the Hardy-Weinberg law were true for the population. The same has been
shown in Table 21.1. We can then compute the chi-square (χ2) value by calculating
the value of “d” or the deviation for each genotype by subtracting the expected
values (e) from the observed values (o). Then d2 is estimated followed by estimation
of d2/e values for each genotype. Since two degrees of freedoms are lost, the
probability ( p-value) for degree of freedom 1 and chi-square (χ2) value, from
Tables 21.1, 10.98 is estimated from the chi-square (χ2) table. The chi-square (χ2)
value 10.98 for 1 degree of freedom indicates a p-value of 0.01 which implies that
there is less than 1% probability that the difference between observed and expected
values is due to chance alone. Therefore, we can conclude that our hypothetical
population does not follow the Hardy-Weinberg law.

21.4 Mating System

As we have discussed, one of the basic assumptions of the Hardy-Weinberg equilib-

rium is random mating, which implies that individuals will choose their mating
partners, regardless of their genotype and phenotype. This ensures that there is even
mixing and shuffling of alleles in a population. However, in an actual scenario, this is
often not the case. There are many different types of mating systems that exist in
real-life populations. Mating system essentially describes the norms that define the
sexual behaviour and mate selection patterns of a group under the given
circumstances. One system that we have already discussed is random mating. This
system entails that there will be no bias in the mate choice and that any given
individual will choose a mate from a population by complete chance and in a random
fashion. The opposite of this would be the concept of non-random mating. In this
type of mating system, there are various factors that govern the mate choice of an
individual in a population. Due to this phenomenon of mate preference, even
 21
Population Genetics

Table 21.1 The chi-square (χ2) value table for testing of the Hardy-Weinberg equilibrium
d χ2
Genotype Genotype frequency Expected number (e) Observed number (o) ¼ (o-e) d2 d2/e ¼∑(d2/e)
RR p2 ¼ 0.25 0.25  77 ¼ 19.3 12 7.3 53.29 2.76 10.98
RW 2pq ¼ 0.5 0.5  77 ¼ 38.5 53 14.5 210.25 5.46
WW q2 ¼ 0.25 0.25  77 ¼ 19.3 12 7.3 53.29 2.76
1091
1092 P. Gupta

shuffling of alleles does not take place. The non-random mating system can be
divided into the following categories based on the criterion for mate selection:

• Mating according to index values.

• Mating according to relationship.
• Mating between breeds—crossbreeding.

In the following section, we will discuss different types of non-random mating at

length and also understand their subcategories.

21.4.1 According to Index Value

In this type of non-random mating system, the mate is chosen based on the
phenotypes. This is also known as assortative mating. Certain phenotypes are
desirable in a prospective mate, while others are undesirable. This often happens
in natural populations, but it is a tool which is extensively exploited by breeders for
the purpose of creation and maintenance of a practically desirable population. Each
desirable trait has certain index value, and the breeding mate is chosen based on the
sum of the index values. Assortative mating ensures that certain phenotypes are
preferred over others; thus, even mixing of alleles is not possible in this type of
mating system. Based on the type of selection, assortative mating can be divided into
two groups: positive assortative mating and negative assortative mating.

21.4.1.1 Positive Assortative Mating

Positive assortative mating means the mating of “like-to-like.” In this system of
mating, individuals with similar phenotypes are more likely to mate. This is often
observed in human populations, where a tall man would prefer a tall woman as a
mate and a short man and short woman would be more likely mating partners. This
would result in more genetic and phenotypic variability than that in the case of
random mating. Further, positive assortative mating would result in higher propor-
tion of homozygotes.

21.4.1.2 Negative Assortative Mating

Negative assortative mating is the system where individuals with dissimilar
phenotypes have higher probabilities of mating. This mating system would produce
offspring that would represent the mean of the population. For example, the contin-
uous mating of a tall and short individual for over generations would finally give rise
to a population with a height which represents the mean of the tall and the short
heights. Therefore, genetic and phenotypic variability is likely to be reduced.
Negative assortative mating (or disassortative mating) is a system wherein the
males with highest index value are likely to mate the lowest ranking females and
vice versa. This would result in a higher proportion of heterozygotes.
21 Population Genetics 1093

21.4.2 According to Relationship

In this type of non-random mating, mates are chosen based on their genotype. In
other words, genetically related individuals are chosen as mating partners. This type
of non-random mating is also known as inbreeding or consanguinity. This is a
common practice in some human societal structures and often takes place in nature
when the population strength is low. Since inbreeding involves the mixing of similar
types of alleles, it favours homozygosity. Highly inbred populations would have an
exceptionally high proportion of homozygous individuals.

21.4.2.1 Line Breeding

In this type of inbreeding, the mating partners come from the same line of descent.
Line breeding generally involves the mating of genetically related individuals apart
from the individual parents or brothers and sisters. Typical line bred mates are
grandchild/grandparent, great-granddaughter/great-grandson, uncle/niece, aunt/
nephew and cousin. Line breeding is a less intense form of inbreeding. It is widely
practiced by a number of animal breeders who want to concentrate a certain desirable
trait and therefore an allele within the population without undertaking conventional
inbreeding route.

21.4.2.2 Deliberate Inbreeding

As discussed before, populations with small number of individuals, certain societal
structures and animal breeders favour inbreeding. In the latter two cases, the choice
of an inbred mating partner is deliberate and is often made so that a certain trait or
allele remains “fixed” in the prospective generation. However, in the process of
trying to “fix” one or many traits, other allelic positions also start to become
homozygous. So many unfavourable recessive traits that were previously not
expressed owing to heterozygosity become homozygous and prevalent in a popula-
tion. This results in the reduction of mean fitness of the population and is termed as
inbreeding depression. In small populations, because there is a lack of choice in
terms of mating partners, inbreeding is an unavoidable choice. In such populations,
recessive diseases start becoming rampant and often lead to the extinction of these
populations. Conservation biologists try to overcome this problem by introducing
members from other populations into the inbreeding population.

21.4.2.3 Inbreeding Avoidance

To minimize the effects of inbreeding depression, conservation biologists and
breeders tend to minimize the inbreeding coefficient by the process of outcrossing.
Outcrossing is a term given to matings within a breed that are as unrelated as
possible. The purpose is to avoid inbreeding but also to maximize heterozygosity
of gene loci to capitalize on non-additive genetic effects.
1094 P. Gupta

21.4.3 Crossbreeding

Another type of non-random mating is crossbreeding which implies the mating of

individuals from different breeds, varieties or populations within a species. The
underlying idea is that each of the selected breeds has been line bred for several
generations. By doing so, favourable genes have become “fixed” in that breed, and
these are fundamentally different from those that have become fixed in partner breed.
Thus, by mixing breeds, the favourable alleles of each breed are combined in the
heterozygous offspring. Heterozygosity reaches its maximum in the offspring gen-
eration. A key feature of this type of mating is heterosis, H, which is defined as the
superiority of crossbred offspring compared to the average of the two parental
breeds. Heterosis is also known as hybrid vigour.

21.4.3.1 Single Cross

This system of crossbreeding involves two pure bred strains which are interbred.
This is also known as rotational crossbreeding system. For example, suppose there
are two breeds of cows “A” and breed “B” each having a set of desirable traits. In a
single crossbreed system, cows of breed “A” will be mated with bulls of breed “B”
and vice versa. The first generation offspring will be a true hybrid and possess
maximum hybrid vigour. In subsequent crosses, only the crossbred females will be
mated with pure bred males and never the reverse. However, the alleles of the pure
bred will be concentrated, and an allelic equilibrium will be achieved by the seventh
generation of mating.

21.4.3.2 Terminal Sire System

As we have seen that in single cross breed system, the heterosis is eventually lost,
therefore to circumvent this problem, a terminal crossbreed system is often used. In
this system, there are two rotational crossbreds. The female offspring resulting from
the mating of the crossbreeding is then mated to the male of a third cross breed. The
resulting offspring has highest heterosis and is not used for further breeding purpose.
Therefore, this type of breeding is known as terminal sire system of breeding.

21.4.3.3 Composite Breed

When animals from two or more breeds with similar genetic composition are mated
to each other and selection for further breeding mates is applied within this group,
the crossbreed is known as composite breeding system. The crossbred animals
become a composite breed. Composite breeds show more phenotypic variance
compared to either purebreds or F offspring. This happens because of the random
segregation of alleles. The more the number of breeds used to establish the compos-
ite breed, the more is the heterosis. A composite breed is created to have the “good”
qualities of each breed that has gone into it.
21 Population Genetics 1095

21.5 Measurement of Genetic Variation

Now that we understand and appreciate how the frequencies of different alleles
change in a population, we must move on to addressing a fundamental question of
population genetics. Most geneticists are concerned with understanding how much
genetic variation actually exists in a population. There are a number of reasons why
this question is so central to the understanding of changes in population genetics
dynamics. First, the amount of genetic variation determines the potential of a
population to adapt to evolutionary changes. This adaptability plays a significant
role in the survival or extinction of a particular population. Second, the variations
give us an idea of the types of evolutionary forces acting on the population as some
forces increase variations while others work to decrease it. The genetic variation can
be measured at the protein or DNA levels as discussed below.

21.5.1 At Protein Level

Genetic codes translate into proteins which are finally responsible for functional
execution. These are also perceived as phenotypic variations. In order to understand
variation and polymorphism at protein levels, a population-wide analysis of
differences in forms of proteins is conducted for a particular locus. This can be
achieved by electrophoretically resolving the proteins for a particular gene. This will
separate all different variants of the protein based on their charge and mass. Signifi-
cant changes in amino acid will result in protein bands in different positions.
However, the silent or same sense mutations at the DNA level will not translate
into different proteins, and therefore these variations will not be noted. Understand-
ing protein variation at population is basic and informative. However, it is not as
robust as an analysis of DNA sequence variation.

21.5.2 At DNA Level

We have already discussed at some length about the various polymorphisms

observed at DNA sequence levels. These polymorphisms at most loci are very
informative as they can account for synonymous mutations (those that do not
translate into a structurally or functionally different protein) and non-synonymous
mutations (that which translate into functionally and/or structurally different pro-
tein). Polymerase chain reaction (PCR) is a uniquely useful tool in this direction.
For example, restriction fragment length polymorphism (RFLP) is a process where
changes in length of PCR products that can indicate sites of polymorphisms.
Variations or polymorphism in this case results in the addition or deletion of a
restriction enzyme cut site. Further, DNA fingerprinting studies are performed to
analyse differences in DNA banding patterns. This will lead to identification of
various alleles and variations in the genetic sequence. Another technique which is
now being increasingly used to locate polymorphic loci is DNA sequencing. With
1096 P. Gupta

the advent of next-generation sequencing techniques, obtaining whole genome

sequence data is becoming feasible, and significant changes in the defining genetic
pool of a population can be assessed.

21.6 Modulation of Genetic Variation

Population genetics involves both experimental and theoretical aspects. On the

experimental side, the actual genetic variations are recorded, and an estimate of
the mean heterozygosity in the population is made. The theoretical facet of popula-
tion genetics deals with understanding how the current trends in genetic variation
will change with respect to time and circumstances. It also involves making
predictions about what the genetic composition should be and what changes are
expected in response to evolutionary forces. Therefore, it is important to appreciate
how different forces of evolution concert to modulate genetic variation in a
population.

21.6.1 Mutation

One of the most important factors that can modulate genetic variation is mutation.
Mutation is defined by a sudden, random and irreversible change in the genetic
make-up of an individual. If the mutation is in the germ line, it will also become
heritable. It is perhaps the strongest tool of evolution for the creation of new alleles
and for the generation of population-wide genetic variation. Mutation creates new
genetic variations; however, the fate of these variations is decided by the environ-
ment and the forces of evolution acting upon it. Whether a mutation is neutral or
detrimental or beneficial to the organism is decided by the environment it is in and
the functioning of natural selection. Therefore, mutation provides the raw material
for the forces of evolution to work on. Let us take the example insecticide resistance
in a pest population. If an insecticide “X” is widely used for the eradication of the
insect “Y”, the population will grossly suffer. However, if a mutation is able to
confer resistance to the insecticide “X”, then the insects with the mutation will have a
survival advantage, and the frequency of this allele responsible for the resistant
phenotype will start to rise. Therefore, mutation creates variation, and the process of
natural selection decides which mutations would be retained or propagated and
which ones would perish.

21.6.2 Genetic Drift

Any change in the relative frequencies of different genotypes in a small population

occurring due to chance factors is termed as genetic drift. When such change is
random in nature, it is known as random genetic drift. There are multiple reasons
behind such observations. We must appreciate that a basic assumption of the Hardy-
21 Population Genetics 1097

Weinberg equilibrium is that the size of the population is infinitely large. An

infinitely large population ensures that there is very little effect of chance factors,
like a catastrophic event, or sampling errors on the observed frequencies of different
genotypes. However when the population size is small, the expected and observed
probabilities of genotypic distribution show a considerable change. For example, let
us suppose that on an island there are ten individuals, five with black hair and five
with blond hair colours. Also, let us assume that hair colour is controlled by a single
gene inherited in Mendelian proportions. Now as an earthquake hits the island, and
50% of the population dies, there is a chance that all five individuals with black hair
perish as the colour of hair cannot confer any survival advantage in this case. Thus
the genetic variant resulting in black hair colour will be lost. Now, suppose that the
population size were 1000, with 500 people having black and 500 having brown hair
colour. In the event of an earthquake, it becomes very improbable that of the 50% of
the population dying out, all 500 will have black hair colour.
This example also illustrates an important aspect of the factor that gives rise to
genetic drifts. It is known as the bottleneck effect. A population bottleneck is an
event which randomly wipes out a major segment of the population and considerably
decreases its size. The event could be a natural disaster or a man-made disaster (like
hunting). As discussed, in the event of a catastrophe, if the population size is small,
the probability that the genotypic frequencies would reduce in proportion to the
reduction in the population is extremely low. It may in fact so happen that certain
variants become extinct and others become more concentrated in the surviving
population.
The flip-side of this is the founder’s effect which defined the loss in genetic
variation that occurs due to the population arising from a small ancestral subset of a
larger population. In other words, if few members of a large population migrated and
established their own line of descent, the descendants would carry only the alleles
that the few members had and the variants from the original population would be
lost. A very compelling practical example of this phenomenon is the high prevalence
of rare disease alleles in Ashkenazi Jews. The current estimates suggest that every
one in three Eastern European descent Ashkenazi Jews is a carrier for certain rare
genetic disorders, like Gaucher disease. A strong theory that explains this phenome-
non is that Ashkenazi genetic diseases arise because of the common ancestry many
Jews share. While people from any ethnic group can develop genetic diseases,
Ashkenazi Jews are at higher risk for certain diseases because of specific gene
mutations.

21.6.3 Balance Between Mutation and Drift

We have now discussed two important regulators of genetic variation: one being
mutation and the other being genetic drift. However, these factors affect the variation
in a population in reciprocal manner. While mutation is responsible for the creation
of new alleles and thus in the increase in variation, genetic drift is responsible for
sampling errors and chance factors resulting in decrease in genetic variation. It
1098 P. Gupta

becomes interesting to understand how these forces together would influence genetic
variation dynamics in a population. This is often understood by the mutation-drift
balance concept.
Given that the rate of mutation and the effective population size are relatively
stable, the amount of genetic variation will tend towards an equilibrium known as
mutation-drift balance in which the rate at which variation is lost through drift is
equal to the rate at which new variation is created by mutation.

21.6.4 Migration

An important assumption of the Hardy-Weinberg equilibrium is that there is not

migration between populations of the same species. This ensures that the gene pool
and relative frequency of alleles are stabilized in the given population. However, in
practicality, migration is an important factor influencing genetic variation in a
population. Migration implies the movement of organisms. For the purpose of this
discussion, we will consider the movement of the organisms of the same species
between two different populations. An immigration or addition of a new member
from a different population would imply the addition of new genetic variants into the
population of interest. Therefore, we concern ourselves with the movement of genes
or alleles from donor to recipient population, and this can happen when a new allele
migrates from one population to a recipient population. This is also known as gene
flow.
Gene flow introduces a new allele into the recipient population. Given that
mutation is a rare event, a mutation that arises in one population would remain
limited to that population only if the process of gene flow does not happen.
Therefore, gene flow helps in the spread of a new allele and the creation of genetic
variation across receiving populations. Further, the gene frequencies of the migrating
population and recipient populations are generally different. Through exchange of
genes, gene flow helps in homogenizing the frequencies of alleles across
populations. This ensures that two separate populations do not amass gross genetic
differences between them.

21.6.5 Natural Selection

So far, we have focussed on factors that can affect changes in the genetic variations
within a population, viz. mutation, drift and migration. However, these factors can
create or change variations, but they alone cannot affect the adaptability of the
change thus created. Adaptation, which implies the tendency of an organism to
adjust to its habitat and environment, is controlled by the process of natural
selection. Natural selection is a process by which organisms that can adapt better
to their environment survive and reproduce. Natural selection is the driver of
evolution. Migration, mutation and drift affect and modulate the pattern of adapta-
tion, but adaptation arises from natural selection.
21 Population Genetics 1099

Let us go back to the example of the insecticide-resistant pests. Mutation gives

rise to the allele for resistance. However, if there is no insecticide in the environment,
the resistant insects will not gain any survival advantage, and given the low number
of initial mutants, the frequency of such allele will be extremely low. However, if
there is rampant use of the insecticide, the frequency of the resistant allele will rise
considerably as the mutants will gain a survival advantage. This process which
decides the acceptability and propagation of a variation is natural selection.

21.6.6 Balance Between Mutation and Selection

Now that we understand how variations are created and selected, let us look at how
natural selection and mutation function to balance the frequency of alleles. This is
often referred to as the mutation-selection balance. There is a pertinent mutation
pressure on a population. Mutations arising naturally are often neutral or detrimental.
Beneficial mutations are rare in nature. So, the process of mutation is continually
adding detriment genes into the pool. On the other hand, the process of natural
selection works to weed out unfavourable alleles by hindering their survival or
reproducibility. Therefore, by the process of natural selection, the frequency of a
detrimental allele will continue to decrease until it becomes rare. When the allele
becomes extremely rare in a population, no significant change in its frequency is
brought about by natural selection. So, when these opposing forces of mutation and
selection work on a population, detrimental alleles are added by mutation and
reduced by natural selection. Eventually, the population will achieve a mutation-
selection balance, where the addition of a detrimental allele by mutation will be
counter-balanced by the removal of alleles by selection. Consequently, a state of
equilibrium will be established, and no effective change in allele frequency will be
observed unless there is a new factor that is introduced into this dynamics.

21.7 Conservation Genetics

All the discussions so far have been involving genetic variability in a population and
how the allele frequencies respond to evolutionary forces. One of the main areas
where the application of such understanding is focussed is conservation genetics or
the conservation of a gene pool of a species. This branch of population genetics aims
at working out how the changes in allele frequencies and gene pool of a population
can affect the viability of this population. For this purpose population viability
techniques are employed, and researchers work out ways to prevent a population
from going extinct.
The environment has suffered a great change. Part of it is natural, but human
activities account for the majority of the detrimental changes to the environment. As
such many species have lost their habitat and have become extinct. This process of
extinction takes time and generally happens with gradual decrease in the size of the
population in a particular habitat. The aim of a conservation geneticist is to
1100 P. Gupta

determine the minimum number of members in a population required for the species
to maintain its gene pool and a genotypic frequency. A population or a particular part
of genetic variation can be maintained either in situ or ex situ.

21.7.1 Ex Situ Conservation: Captive Breeding

Ex situ conservation essentially means the conservation of the genetic diversity

outside of its natural habitat and ecosystem. The genetic diversity is preserved in the
form of a population of species, their germ line cells or somatic cell lines. These are
preserved, maintained and kept available.
One method of doing so is captive breeding, where small populations of a species
are captured and bred outside of their natural habitat and maintained in controlled
environments. Some examples are wildlife reserves, zoos, botanic gardens, etc. This
is done to prevent the species from extinction due to gross changes in their ecological
environment. However, the fruitful preservation of a species in such environments
requires a deep understanding of genetic, ecological, behavioural and ethical issues.
One must consider the effect of genetic drift on a small population maintained
outside of its habitat. The impact of founder’s effect and bottleneck effect is higher
in small population especially outside of its natural habitat.

21.7.2 Ex Situ Conservation: Gene Bank

Another way of ex situ conservation is the creation of a gene bank which acts as a
repository of genetic material. This could be done in the form of cutting and freezing
parts of plants, storing seeds in a seed bank and freezing and maintaining germ line
of somatic cells of organisms.
Gene banks help in the preservation and rehabilitation of genetic diversity. For
example, frozen plant material could be revived and propagated artificially. Further,
frozen mammalian sperms can be used for artificial insemination, surrogacy and
revival of the mammalian species.

21.7.3 In Situ Conservation

In situ conservation is the process of preserving populations of organisms as they

occur in nature. The population is maintained in a natural state and in its natural
habitat. This helps not only in the preservation of the organism itself but also in the
preservation of the evolutionary processes that help maintain the adaptability of the
population to its environment. For example, large ecosystems may be left intact as
protected reserve areas with minimal intrusion or alteration by humans.
In situ conservation adds to the ex situ method of conservation. It is extremely
advantageous as it maintains not only a population but an ecosystem as a whole. As
such, it safeguards many genetic variations that we might not be aware of. It also
21 Population Genetics 1101

offers a window through which a biologist can study the forces of evolution in
action. However, maintaining in situ conservatories requires the combined efforts of
the local and government level support.

Box 21.1: Scientific Concept: Analysis of Genetic Variation and Potential

Applications in Genome-Scale Metabolic Modelling – Joao G. R. Cardoso
et al.
An understanding of genetic variability in microorganisms is very crucial in
the current scheme of things. The advent of next-generation sequencing (NGS)
has reduced the cost of sequencing and made re-sequencing possible.
Re-sequencing implied the sequencing of closely related strains in search of
genetic variations. Re-sequencing has implication in biotechnology, under-
standing of pathology of microorganism-based diseases, evolution in environ-
ment and effect of human activity on microorganism diversity. It also helps in
the making of in silico genome-scale metabolic models (GSMs) which
consists of gene-protein reaction (GPR) association maps that help in deter-
mining how biochemical reactions relate to the genome and proteome of a cell.
In a very comprehensive review, Cardoso et al. talked about the various
tools that are used for analysis of genetic variants. They also discussed
techniques that could detect both synonymous and non-synonymous single
nucleotide variants (SNVs) in coding regions or variants in promoter or other
regulatory regions.
They focus on different in silico tools that can be used to determine how a
particular variation in the genetic make-up of an organism can affect its
phenotype. It is important to appreciate that the effect of a variation is not
binary in nature for practical purposes, i.e. a single variation can lead to
multiple types of effects. A variation can have no effect on the phenotype; it
could lead to a loss in function of the coded protein; or it could improve the
protein’s activity. The software uses a different criterion to analyse the effect
of a genotype on a particular phenotype. Figure 21.7 depicts the different
softwares available and the criterion they use for analysing the effects of a
variation.
The article also discusses deep mutational scanning, a technique that uses
traditional methods like error-prone PCR to construct an exhaustive library of
all possible variants. The variants are sequence using NGS methods and then
assigned scores based on fitness measure (e.g. growth rate, ligand binding or
product fluorescence) for each amino acid substitution discovered in the
selected pool.
This article describes, in extensive details, the emergence of in silico,
in vivo and in vitro technologies that help in the prediction of genetic
variations and their possible phenotypic outcomes.
1102 P. Gupta

Fig. 21.7 Different criterion used in software to analyse the effect of a genotype

21.8 Chapter Summary

In this particular chapter, we have tried to explore the various facets of population
genetics and genetic variation.

• Genetic variation is the key to the diversity present in nature and is often evident
in the form of polymorphism.
• Polymorphism is alternating forms of a genetic sequence present in at least 1% of
a population. If the variation is at a single nucleotide level, it is known as single
nucleotide polymorphism (SNP).
• Microsatellites are short sequences that display copy number polymorphism and
can be indicative of genetic variability.
• Haplotype is a segment of DNA that is inherited en bloc and thus displays very
tight linkage. A map of haplotypes can indicate which polymorphisms are tightly
associated with traits, and very few polymorphs can thus be informative of a
considerable segment of the genome.
• All the alleles and polymorphs present in a population are known as its gene pool.
Allele frequency and genotypic frequency are crucial parameters to understand
the dynamics of inheritance of this gene pool in the population.
• The Hardy-Weinberg equilibrium explains the dynamics of genetic inheritance
through a mathematical expression. Given that no evolutionary forces are acting
on a population, the allele frequency for a particular gene inherited in a Mendelian
pattern would remain constant over generations.
• Genetic drift, mutation, migration and natural selection act on a population and
have distinctive effects on the genetic variability and genotypic frequency of the
population.
21 Population Genetics 1103

• The random mating system forms a basic assumption of the Hardy-Weinberg

equilibrium. However, non-random mate selection is prevalent in nature.
• Assortative mating is a form of non-random mating where the mates are selected
on the basis of presence or absence of a certain panel of phenotypes.
• Inbreeding is the mating of individuals with common ancestry. It works to
decrease heterozygosity in a population.
• Outcrossing is the opposite of inbreeding and requires mating of individuals from
different ancestral lineages.
• Given that the gross changes in the environment are leading to the loss of genetic
variation and extinction of certain populations, genetic conservation becomes
necessary.
• Gene banks and captive breeding are forms of ex situ conservation where
individuals are conserved outside of their natural habitat.
• In situ conservation is the conservation of a population in its natural habitat.

References
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Evolutionary Genetics
22
Ankita Dua and Aeshna Nigam

22.1 Concept of Evolution

Nothing in biology makes sense except in light of evolution—Theodosius Dobzhansky

Evolution may be defined as the changes in gene pool which will lead to
progressive adaptation of the population to the environment. The concept of natural
selection proposed by Charles Darwin and Alfred Russel Wallace combined with
Mendelian inheritance gives an insight into the mechanism of evolution. Evolution is
basically a two-step process which can only occur when there are heritable variations
in the gene pool.
In words of Darwin, evolution is ‘descent with modification’ that species change
over time giving rise to new species sharing a common ancestor. Each species has its
own set of heritable differences from the common ancestor, which accumulate over
periods of time gradually. In the ‘tree of life,’ repeated branching events produce a
multilevel tree that links all organisms. Evolution, with respect to extent of change
and geological time scales, can be classified as:

• Microevolution is defined as a systematic change in allele frequencies and

chromosomal segments in local populations.
• Macroevolution involves changes of greater magnitude. Changes of macro extent
occur in the development of characteristics that distinguish groups such as genera,
families, orders, classes and phyla. These take place over a long span of geologi-
cal time scale.

A. Dua · A. Nigam (*)

Shivaji College, University of Delhi, New Delhi, India
e-mail: aeshnanigam@shivaji.du.ac.in

# The Author(s), under exclusive license to Springer Nature Singapore Pte 1105
Ltd. 2022
D. Kar, S. Sarkar (eds.), Genetics Fundamentals Notes,
https://doi.org/10.1007/978-981-16-7041-1_22
1106 A. Dua and A. Nigam

Various evidences have supported the theory of evolution. Homologous

structures are evidence of presence of a common ancestor, whereas analogous
structures evolve independently without common descent and are similar due to
inhabitance of similar environment as a result of convergent evolution. Species
relatedness can be evaluated by similarities and differences at the molecular level,
assessing the DNA and protein structure. Study of biogeography shows distribution
of organisms all over the globe and gives clues about relationships between them.
One of the most valuable evidences comes from fossil records that unfold the story
of timelines of existence of species over various periods (Futuyma 1998; Hall and
Hallgrimsson 2008; Ridley 2004).

22.1.1 Theories of Evolution: Lamarckism

Jean Baptiste de Lamarck is a French naturalist of the nineteenth century, known for
his speculations on evolution, published in his book The Philosophie Zoologique in
the year of 1809. ‘Inheritance of Acquired characters’ is his significant proposition
albeit unaccepted widely.
Lamarck’s strong doctrine is, species undergo modifications concerning the
environment, contradicting its fixity. He claims domestication of plants and animals
modifies their structure unrecognizable to the wild variety. For instance, domestic
ducks and geese lost their ability to fly compared to the wild birds of their race due to
prolonged captivity. If the captivity is extended, even more, there might not be a
change only in their ability but also in their morphology, claims Lamarck. He further
endorsed, Ranunculus hederaceus of terrestrial habitat grown in a damp soil has
been found to have a smaller stem and devoid of small segmental leaves which are
dissimilar to the same species, Ranunculus aquatilis of aquatic habitat. According to
Lamarck, the impact of the environment on living species causes an imperceptible
alteration in structure and organization. Unconcealed modifications in animals can
be observed with substantial changes in the environment leading to novel
requirements. The emergence of new habits due to these long-lasting changes in
the habitat will lead to the development of a new pertinent organ which further
develops stronger and larger with perpetual use. These modifications in animals
curtail inefficient organs to disuse. The disappearance of inefficient organs is
coupled with prolonged disuse. Lamarck also observed these permanent
modifications become inherited giving rise to distinct species. In a nutshell, external
stimulus causes a heritable beneficial genomic mutation in species for adaptation
(Fig. 22.1). Based on his extensive studies and observation of species, he discerned
two laws of nature:
First Law: ‘In every animal which has not passed the limit of its development,
more frequent and continuous use of any organ gradually strengthens, develops and
enlarges that organ, and gives it a power of the proportional length of time it has been
so used; while the permanent disuse of any organ imperceptibly weakens and
deteriorates it, and progressively diminishes its functional capacity until it finally
disappears.’
22 Evolutionary Genetics 1107

Fig. 22.1 Lamarck’s theory of evolution. (Adapted from Koonin and Wolf 2009)

Lamarck has demonstrated the authenticity of the laws with various examples and
believed these laws are certainly true and permanent. He gave definitive examples of
use and disuse of organs rising due to contemperory habits. Certain changes in the
habitat of animals have induced swallowing of the feed without primitive mastica-
tion, eventually leading to the absence of teeth in vertebrates (e.g. whale, anteater).
Further, Lamarck articulates, disuse of eyes had constricted the organ in moles.
Living beneath the soil, where sunlight is arduous to percolate, mole has tiny eyes
based on its utility. In addition to that, Spalax which lives in the similar habitat of
mole rats is blind with the vestiges of the organ, as a result of lack of utility. Snakes
are the exceptional class of reptiles which do not have four limbs like crocodiles,
frogs and turtles. Lamarck reasoned this unique feature of snakes with two facts:
(1) Their peculiar adoption to crawl with the elongated body helped them to hide in
the grass and move in confined places with ease (2). Their long legs have put to
perpetual disuse, which eventually disappeared. It is unlikely for a snake to have
short legs which makes them incompetent, and they cannot have more than four legs
under the reptile criteria.
Lamarck illustrated his opinion on the development of a new organ or stronger
and prominent development due to recurrent use of an existing organ supporting
altered environment with exemplifications. Perpetual use of skin between the three-
digit feet to capture aquatic life forms of prey has given rise to the palmate or webbed
foot essential for swimming in ducks and geese. Lamarck explained, in a few cases,
birds developed long stretchy legs with feathers above the thighs when they are
reluctant to swim and depend on prey in the shore. In forests, prey-predator stress is
inevitable and especially ruminants like deer need to protect themselves from
predators as well as hunters. Lamarck claims, the ‘inner feeling’ of the animals to
safeguard them from dangers has allowed the secretion of a blend of horny-bony
substance which gave rise to antlers and horns. He claims giraffe has developed long
necks compared to its ancestors. The gradual transformation of African forest
grasslands to arid areas coerced the animals to depend on trees for nourishment.
This obligation has resulted in elongated necks and limbs in giraffe. Lamarck’s other
doctrine is, the adaptations in species that help in survival are inherited and are called
use-inheritance. This gives rise to the second law of Lamarckism.
Second Law: ‘All the acquisitions or losses wrought by nature on individuals,
through the influence of the environment in which their race has long been placed,
1108 A. Dua and A. Nigam

and hence through the influence of predominant use or permanent disuse of any
organ; all these are preserved by reproduction to the new individuals which arise,
provided that the acquired modifications are common to both sexes or at least the
individuals which produce the young.’
This theory of use-inheritance is considered improbable due to lack of evidence
by Lamarck. This theory was condemned by many people, and experiments were
conducted to prove or disprove it. August Weismann, a German Evolutionary
biologist, was the first person to propose the germplasm theory in
animals (Weismann 1893). He proposed any metamorphosis in the somatoplasm
does not affect the germplasm. Weismann argued Lamarck’s proposition of ‘Inheri-
tance of acquired characters,’ claiming germ cells give rise to somatic cells, therefore
for a variation to occur, preliminary change must occur in the germplasm to be
inherited. During one of his lectures delivered in 1888 on ‘A supposed transmission
of mutilations,’ he presented results of his experimental investigation on mutilation
inheritance in mice (Weismann 1891). In the first generation, he amputated tails of
12 mice comprising 7 females and 5 males. The offspring from the first generation
were found with perfectly grown tail, and even subtle presence of the acquired
inheritance was not found. Surprisingly, the fifth generation with 901 offspring
developed from the mutilated parents did not show the trivial presence of rudimen-
tary tail defects or tail-less condition. Weismann set forth a plausible assumption that
the expression of mutilations in the progeny might take place after many generations.
Unfortunately, use-inheritance can be widely accepted if there was at least one proof
to support this theory.
Furthermore, McDougall in 1938 (McDougall 1938) conducted experiments on
learning as an acquired inheritance. He designed a T-shaped tank, with two exits:
one exit with the electric shock was illuminated, whereas the free exit was kept dim.
Rats which chose the lighted pathway received an electric shock for 3 s, and animals
which chose the dim exit were rewarded. He trained the rats six times daily to
accustom to the experiments and halted the training only when the rats learnt to
discriminate the exits and chose the dim exit successively. He bred these rats for the
second generation. McDougall found mistakes reduced gradually from generation to
generation and claimed learning is an acquired trait. Drew (1939) criticized
McDougall experiments for biased learning in animals, and inheritance of avoidance
behaviour interlinked with various factors is impossible. When repeated, contrast
results were obtained by Crew and Agar (Agar et al. 1954; Crew 1936). Further,
technical errors found in McDougall’s experiment led to severe criticism.

22.1.2 Theories of Evolution: Darwinism and Neo-Darwinism

Charles Darwin and Alfred Russel Wallace independently developed theories of

evolution based on natural selection which were communicated to a meeting of
Linnaean Society of London on July 1, 1858. Both went on voyages of discovery
before stating their ideas: Darwin went around the world on H.M.S. Beagle
(Fig. 22.2), and Wallace travelled to Brazil, Malaysia and Indonesia.
22 Evolutionary Genetics 1109

Fig. 22.2 The voyage of HMS Beagle. The path traced by HMS Beagle in 1831 in its 5-year
journey that led to Darwin’s postulates of natural selection and origin of species. (Adapted from
Campbell et al. 2008)

Wallace is considered to have begun the study of biogeography, and both of them
were posthumously awarded the ‘Gold Medal’ by Linnaean Society of London for
the 50th anniversary of their publication. Darwin’s voyage spanned a period of
5 years from 1826 to 1830 that enabled him to observe the wide range of species and
geological forms around the globe. His breakthrough discovery was the exotic
collection of flora and fauna evolving on the Galapagos Islands off the coast of
Ecuador. These are located in the Pacific Ocean, approximately 960 km west of the
South American coast, straddling the equator at the 90th meridian west. The
archipelago was made of 13 major islands, 6 smaller islands, over 40 islets and
many smaller unnamed islets and rocks, for a total of approximately 8000 km2 of
land spread over 45 000 km2 of water.
He noted that different islands with similar habitats were not always occupied by
identical species. He proposed that:

• Excess reproduction and limited resources lead to competition, and as a result of

natural selection, only the organisms best adapted to the habitat could survive and
pass their characters to the next generation.
• Changing environments and hereditary variations and natural selection together
result in modification of existing characters or origin of new characters that
become established throughout a species.

Darwin’s work was documented in his book The Origin of Species in 1859 which
is said to have revolutionized the foundation of evolutionary biology.

The most curious fact is the perfect gradation in the size of the beaks in the different species
of Geospiza, from one as large as that of a hawfinch to that of a chaffinch, and . . . even to
that of a warbler. . . . Seeing this gradation and diversity of structure in one small, intimately
related group of birds, one might really fancy that from an original paucity of birds in this
1110 A. Dua and A. Nigam

Fig. 22.3 Phylogenetic analysis of Darwin’s finches. Combined analysis of the cytb and cr
sequences of Darwin’s finches done by neighbour-joining tree construction method. Shape of the
beak is illustrated by the drawings made on the right side (Sato et al. 1999)

archipelago, one species had been taken and modified for different ends.—Darwin
(1839) (Abzhanov 2010)

He studied the concept of adaptive radiation (diversification of a founder popula-

tion into a collection of species differentially adapted to diverse habitats) under
natural selection of 14 closely related finch species (belonging to the Avian order
Passeriformes). These were diverse in their beak shapes and sizes. Later on, he asked
John Gould at the museum of Zoological Society, London, to catalogue these
species. Gould realized that these diverse species actually reflected differences in
diets that were closely related to each other and distantly to a South American
mainland species. Developmental basis of this variation was analysed by a compar-
ative analysis of expression patterns of the growth factor Bmp4 of the species of
genus Geospiza (Abzhanov et al. 2004; Parent et al. 2008). The expression of Bmp4
in upper beak mesenchyme correlated with the deep and broad beak morphology.
When this protein was misexpressed in chick embryo, it caused morphological
transformations similar to beak morphology of the large ground finch (Abzhanov
et al. 2004). In another analysis, two mitochondrial DNA segment sequences (cytb,
cytochrome b, and cr, control region) have been used for evolutionary history of the
group. The results reveal that Darwin’s finches are a monophyletic group with the
closest species to the founder being Warbler’s finch, followed by vegetarian finch
and two sister groups of ground and tree finches. The Cocos finch found on the
Cocos Island of the Pacific Ocean is related to the tree finches (Fig. 22.3). The
mtDNA and microsatellite data found was consistent with the theory that the finches
originated from a single common ancestor from a possible founder population that
22 Evolutionary Genetics 1111

reached the islands from South/Central America (Bluestone 2009; Bowman

1961; Parent et al. 2008).
Post Darwin, a number of workers such as J. Huxley, T. Dobzhansky,
J.B.S. Haldane, S. Wright, T.H. Morgan, G.G. Simpson, G.L. Stebbins, E. Mayr
and C. Darlington came about the theory of ‘Modern Synthesis’ or Neo-Darwinism.

Neo-Darwinism is an attempt to reconcile Mendelian genetics, which says that organisms do

not change with time, with Darwinism, which claims they do.—Lynn Margulis

The term ‘Neo-Darwinism’ was coined by physiologist George Romanes in

1883. It is the modern concept of evolution where a gene pool of a population is a
unit of selection versus the individual. There is a synthesis between Darwin’s theory
of natural selection and gene mutations playing a central role in production of
variation in a population. By this time, Mendel’s work had also been rediscovered,
and it was significant in adding to the theory. A connection was established between
genes as a unit of evolution and natural selection as the mechanism. Evolution is a
gradual process which is a result of variations accumulated at the genetic level in
populations over a period of time (via mutations). The result of these variations is the
phenotypic changes in the population and in allele frequencies as a result of natural
selection (Denis 2011).
The pioneer workers who strengthened the ‘Concept of Modern Synthesis’ were
as follows:

• Theodosius Dobzhansky worked on evolution of fruit fly populations (Drosoph-

ila melanogaster). His major publication was Genetics and the Origin of Species
in 1937.
• E.B. Ford worked on ‘Ecological Genetics,’ and his book titled the same was
published in 1964.
• H.B.D. Kettlewell was the pioneer worker on industrial melanism in peppered
moth Biston betularia.
• Julian Huxley wrote the book ‘Evolution: The Modern Synthesis’ in 1942 that
brought to light the concepts introduced by Fisher, Haldane and Wright.

22.2 Genetic Variation in Population

Variation is an interplay of heredity and environment and is the most impressive

characteristic of any sexually breeding population. The capacity to undergo variation
is due to a complex set of heritable traits. Variations are the reason present-day
organisms have evolved from a few primordial forms of life and diversified into
complex life systems; they are seen at phenotypic, chromosomal and molecular levels.
Phenotypic variation is seen in different varieties of species in animals as well as
in different flowers in the flower kingdom. These visible morphological or pheno-
typic differences are called polymorphism, meaning ‘many forms.’ For example,
land snails have different coloured bands on their shells, mammals have different
coloured coats, and insects have patterned wings. The human species have also been
understood to be polymorphic; the most interesting example being their blood
1112 A. Dua and A. Nigam

groups where the antigen coded is present on the surface of the blood cell. The
antigens are polymorphic. In the Duffy blood typing system, there are two antigens
present on the surface of cells. Alleles coding these antigens called the ‘Duffy
alleles’ encoded by a gene on chromosome 1 are often polymorphic. Various
human ethnic groups have varied status of Duffy polymorphism (Anstee 2010).
Variation in chromosomes is often an indication of polymorphism at the phenotypic
level. Researchers found abundant comparative data on comparing polytene
chromosomes from various species of Drosophila (Zykova et al. 2018). These
chromosomes develop from diploid nuclei chromosomes by successive duplication
of each chromatid without the segregation. The formed elements associate length-
wise and form a cable-like structure. In Drosophila melanogaster, they are >100
times longer than regular metaphase chromosomes. Here, level of variation in
chromosomes can be studied at an unparalleled level. In every polytene chromo-
some, banding patterns are significant as there is alternation between compacted and
decompacted regions of chromosomes known as bands and interbands. Dobzhansky
and his team members identified various patterns of banding in Drosophila species.
Variations are also seen at the nucleotide and at the protein level. Genetic variation in
natural population was studied by R.C. Lewontin, J.L. Hubby and H. Harris by
application of gel electrophoresis to study amino acid differences in proteins of
various species. Amino acids are building blocks of proteins, and their differences in
shape, molecular weight and charge can be studied while migrating in gels. This
technique was applied to various other creatures as well, and different forms of
proteins could be studied as the mobility of a protein was specific through the gel.
The ultimate data on genetic variation is obtained on DNA sequencing. All
sequences—exons or introns—can be sequenced and analysed. At present high
end sequencing technologies have been successful in decoding even the billion
base pair human genome.
Variations have been classified by various workers into a number of categories:

22.2.1 Continuous Vs. Discontinuous Variations (Table 22.1)

Evolutionary biologists also classify variations as follows.

Table 22.1 Comparing continuous and discontinuous mutations

Continuous variations Discontinuous variations
• Numerous, small-scale variations occurring • Sudden, drastic changes in traits of
between individuals of a population with individuals of a population
respect to characters such as height, weight, • Usually referred to as mutations which are
skin colour, texture, etc. responsible for variations that lead to
• These are fluctuations on either side of the formation of a new species
mean value of a character.
22 Evolutionary Genetics 1113

22.2.2 Environmental Variations

These are acquired changes and may not be inherited at the gene level. Environmen-
tal influences act on nutrition, competition, disease and biotic and abiotic factors.
Phenotypic plasticity is defined as the ability of a genotype to produce more than one
phenotype when exposed to different environments. For example, in the semiaquatic
plant Ranunculus, the leaves that are submerged in water have a dissected leaf
lamina, whereas those above water have a single lamina (Cook and Johnson 1968).

22.2.3 Mutational Variations

According to T.H. Morgan, the ultimate source of heritable variation is mutation,

and he stressed on the fact that the latter are changes in single genes with effects
ranging from minute to severely pronounced. The term ‘gene’ was coined by Danish
botanist Johannsen in 1909, and Wilhelm Waagen gave the term ‘mutation.’ How-
ever, it was Hugo de Vries and William Bateson who described mutations as major
changes in genes at the molecular level leading to deviations from the parental types.
Mutations occur in genes bringing about variation, and natural selection favours the
advantageous variants and weeds out the disadvantageous ones (Marshall 2002).
Mutations can be point mutations or chromosomal aberrations:

• Gene/point mutations (discovered by T.H. Morgan during his work on white-

eyed trait in a laboratory population of Drosophila) are the elemental source of
variations as they bring about changes in a pure line gene of a parent and lead to
origin of a new gene that is inheritable. Genes are generally stable and mutate at a
slow rate. These changes could be in composition—transition and transversion—
or change in number by addition, deletion and frame shift. Mutation rate is
defined as ‘probability that an allele copy changes to another allelic form in one
generation.’ As mutation is a spontaneous process, the rate of change in gene
frequency from mutation is very low. Mutation of a single gene usually has
minimal effect on the population; it is amplified by interaction with other
genes, for example, cases of multiple allele interactions, epistasis, pleiotropy
and polygenic effects. Generally, mutations that are less severe occur at a higher
frequency, and a mutant gene may not display its effects under normal environ-
mental conditions but may be deleterious in a different environment.

Table 22.2 Change in number of chromosomes along with amount of DNA

Aneuploidy Loss or gain of individual chromosome of a normal diploid set of an organism. It
can be nullisomy (2n
2), monosomic (2n
1), trisomic (2n + 1), tetrasomic
(2n + 2) and polysomic
Haploidy Loss of an entire set of chromosome
Polyploidy Gain of one or more sets of chromosomes. It can be triploidy (3n), tetraploidy (4n)
and polyploidy
n refers to the haploid set of chromosomes
1114 A. Dua and A. Nigam

• Aberrations on the other hand refer to loss or gain of genes and change in
placement or position within the chromosome or between different chromosomes.
Structural changes involve deficiency (loss), duplication (repetition of a DNA
segment) and polyteny (multiple copies of entire DNA strands) which bring about
changes in the amount of total DNA (Table 22.2). Changes in location of genes
(no change in DNA amount) are done by inversion (reversal of gene order within
same chromosome)—paracentric/pericentric (depending on presence/absence of
centromere in the inverted segment) and translocation. Loss or gain of genes with
change in amount of DNA is done by change in chromosome number.

22.2.4 Recombination

Variations introduced as a result of permutations and combinations of existing genes

are termed as recombination, and these are the raw materials for natural selection and
evolutionary changes. Such events may add up with point mutations, and their
combined effect makes a great impact. Recombination maybe a result of:

• Crossing over of two homozygous gametes to give rise to a heterozygous

offspring.
• Random assortment of genetic material.

22.3 The Neutral Theory of Molecular Evolution

22.3.1 Neutral Theory

The neutral theory of evolution was given by Motoo Kimura in 1968 and it states:

This neutral theory claims that the overwhelming majority of evolutionary changes
at the molecular level are not caused by selection acting on advantageous
mutants, but by random fixation of selectively neutral or very nearly neutral
mutants through the cumulative effect of sampling drift (due to finite population
number) under continued input of new mutations (Kimura 1991).

As sequencing technologies developed, more data was available to test this

hypothesis. Kimura predicted that in protein molecules the substitutions of amino
acids that occur more often are conservative. These replacements do not affect the
function of the protein as the changed amino acid shares similar biochemical
properties to the original one. Introns and pseudogenes also evolve at a high rate
similar to these base substitutions.
There were several similarities of the neutral theory with Darwin’s postulates:
22 Evolutionary Genetics 1115

• Natural selection is the driving evolutionary force and results in adaptation of

organisms to their environment.
• Mutations in the functionally important regions are deleterious, and selection
rapidly removes them from the population.

Kimura’s theory challenged Darwin’s work as:

• It was proposed that intraspecific nucleotide differences are a product of neutral

mutations rather than genetic drift.
• Most intraspecific polymorphisms are also neutral.

Alterations or substitutions in protein-coding DNA sequences can be classified as

synonymous (do not affect encoded amino acids) and non-synonymous (affect
encoded amino acids). Synonymous substitutions are mostly neutral as they do not
alter the protein sequence.
The neutral theory focuses on three main ingredients: mutation, genetic drift, and
purifying selection:

• Mutations are the driving forces of evolution in proteins as well as DNA. Every
generation, approximately 108–109 events of mutation occur. As discussed,
mutations can be beneficial or detrimental to the fitness of an organism or may
be selectively neutral.
• If the mutations are advantageous, they end up getting fixed in the population.
The negative mutations are eliminated from the population by the action of
purifying selection.
• Selectively neutral mutations have no effect on fitness, and their fate is dependent
on random genetic drift. Most are lost from the population shortly after they
appear.

Evolution rate according to the neutral theory depends on the neutral mutation
rate which is constant in different lineages over time. Highest rates of evolution are
found in molecules in which any mutational change may have least effect on the
function. On the contrary, the lowest rate is found in the molecules where selection
pressure is the highest (Duret 2008).

22.3.2 Rate of Neutral Substitution and Molecular Clock

Mutations in DNA can be of three types: deleterious (which may affect the fitness of
the individual negatively), may increase efficiency of organism and can be neutral.
When a mutation has no effect on survival and reproduction of an individual, it is
termed as a neutral mutation.
Investigations about the molecular clock started with study of the proteins—
haemoglobin, cytochrome c and fibrinopeptides in the early 1960s. E. Zuckerkandl,
L.B. Pauling, E. Margoliash, R.F. Doolittle and B. Blomback concluded that
1116 A. Dua and A. Nigam

Fig. 22.4 Amino acid changes in cytochrome c, haemoglobin and fibrinopeptides. All three
proteins display different rates of changes per unit time, but the rate is constant for each. (Adapted
from Yi, S.: Neutrality and Molecular Clocks. Nature Education Knowledge. 4(2), 3 (2013))

differences in the protein sequences of different species were in accordance to their

timeline of divergence (Fig. 22.4). Regular ticks of clocks were considered synony-
mous to accumulation of amino acid/nucleotide substitutions over time in a lineage
and hence the name ‘molecular evolutionary clock’ coined by Zuckerkandl and
Pauling in 1965. Evolutionary rate or rate of molecular clock is the number of
substitutions per a defined unit of time.
The concept of molecular clock measures the absolute time of evolutionary
change based on the observation that various genomic regions evolve at steady
rates. It talks about orthologous and paralogous genes. Both are homologous, that is,
arising from a common ancestor, where orthologous genes diverged post a specia-
tion event and code for proteins with similar function in different species and
paralogous genes diverge within the same species and encode for proteins with
similar not identical function. Nucleotide substitutions in orthologous genes are
22 Evolutionary Genetics 1117

proportional to the speciation event where the species diverged from the common
ancestor and in paralogous genes to the time of duplication.
Initially it was hypothesized that the evolutionary force driving these
substitutions was natural selection. However, Kimura said that the changes at the
molecular level were neutral, that is, no consequence over fitness, and these occurred
completely by random chances. Hence, it could not be predicted whether a specific
neutral mutation will be or not fixed in a population.
Rate at which neutral substitutions occur in a population depends on the
mutation rate and can be predicted as:

Total number of haploid individuals in a population ¼ N.

Rate of neutral mutations ¼ u/individual/generation.
Total number of mutations in one generation ¼ Nu.

As all the mutations are neutral, their success rate is dependent on simply chance
probability. Hence all mutations have an equal chance of getting fixed (equivalent to
substitution).

Probability that each mutation is fixed ¼ 1=N:

Rate of substitution ¼ Number of new mutations in each generation ðNuÞ

 probability that each mutation gets fixed ð1=NÞ¼ u

Therefore, for neutral mutations, rate of substitution ¼ rate of mutation.

It can be predicted that according to the neutral theory, if mutation rates are
constant over time, substitutions will occur constantly as well and clock-like regular
rates of substitutions will occur constantly over time.

22.4 Natural Selection

Natural selection is defined as a directional, non-random and guiding force that leads
to evolution of organisms to a better state of adaptiveness. Natural selection is the
differential reproduction of genotypes; it is measured by the relative reproductive
successes (fitness) of genotypes.
Salient features of natural selection:

• Key force in driving evolution.

• Shifting of allele frequencies in a large population.
• Fitness is an attribute of a genotype due to its genetic contribution to the future
generation. High fitness is a measure of high rate of reproductive success and
hence increased genetic contribution to the gene pool. Natural selection clearly
favours individuals with higher fitness (Orr 2009).
• Intensity of natural selection acting on genotypes in a population is defined as
selection coefficient.
1118 A. Dua and A. Nigam

• If a mutation is selected for or against in a population, it is termed as ‘positive or

negative’ selection.

Modes of selection in nature vary at different levels of biological organization

leading to varied evolutionary outcomes:

A. At the population level:

• Directional selection.
• Stabilizing selection.
• Disruptive selection.
B. At the local population level or within species:
• Kin selection.
• Group selection.
C. At the level of individuals:
• Sexual selection.

Fig. 22.5 Modes of selection. A hypothetical deer mouse population (with heritable variation in
fur colour from light to dark) as an example of three types of natural selection. White arrows
indicate patterns of evolution, selective pressures against certain phenotypes: A: Original population
graph where frequency of individuals is plotted against the fur colour phenotypes. B: Directional
selection favours the extreme phenotypes, in this case the dark fur individuals. The darker mice are
saved from their predators as they take refuge under dark rocks in the environment. C: Disruptive
selection favours variants at both ends of the spectrum. The mice inhabit patches of light and dark
coloured rocks, and the mice of an intermediate colour are at a disadvantage. D: Stabilizing
selection favours the intermediate/average phenotypes. If the environment consists of intermediate
colour rocks, the light and dark mice will be eliminated. (Adapted from Campbell et al. 2008)
22 Evolutionary Genetics 1119

22.4.1 Directional, Stabilizing and Disruptive Selection

Effect of the genotype at various loci combined with environmental effects define the
phenotype of an organism (Fig. 22.5).

22.4.1.1 Directional/Progressive Selection (Under Changing

Environmental Conditions)
Under pressure of changing environmental conditions (biotic and abiotic factors), a
species population is faced with the challenge of adjusting and keeping pace with
change. Hence the genetic variables that were ideal in the older environment cannot
continue to do so in an altered one. With respect to the change in the environment, a
new adaptive peak is reached, an extreme being favoured and others eliminated.
Also known as progressive selection, a unidirectional change in the genetic compo-
sition of the gene pool occurs where the peripheral variants with weak selective
advantages originally may find themselves to be better adapted. Phenotypes at one
end of the spectrum are selected causing the allele frequency to continuously shift in
one direction.
The classical example of directional selection is the phenomenon of industrial
melanism in England in the nineteenth century. The role of natural selection in
evolution of melanic forms in peppered moths (Biston betularia) was studied in
detail by H.B.D. Kettlewell and E.B. Ford. Peppered moths exist in two forms—
melanic (black in colour; dominant allele C) and non-melanic (mottled grey; homo-
zygous for recessive allele c). Prior to the industrial revolution, the melanic forms
were a rare sight, and the non-melanic light coloured moths were abundant in the
natural habitat. Predators could easily spot and prey upon the dark melanic forms
that rested on the light coloured lichen-covered trees. The non-melanic moths easily
blended with the environment and were not easily visible. With the start of the
industrial revolution, these numbers underwent a dramatic change as did the envi-
ronment. The sooty smoke ejected from the factories (heavy industrial areas around
Manchester and Birmingham) resulted in attributing a black cover to the trees and
prevented growth of lichens. This resulted in protection of the melanic forms and
more visibility of the light coloured moths to the predators. Industrial melanism
owes its name to the increase in proportion of the melanic forms as selection
favoured the moths for the specific character—protective coloration. Kettlewell
and Ford who studied this phenomenon in detail reasoned that a single dominant
gene for the melanic colour was present in the population and its frequency increased
from a mere 1% to 90% with the onset of the revolution in less than 50 generations of
the insect. Therefore, changing environment led to change in the genetic constitution
of the population as in the changed environment the alleles favoured by selection
were different from the ones found in the earlier environment (Cook and
Saccheri 2013).
The action of natural selection on a character such as body size which is
continuously distributed is interesting to study. Based on the assumption that
small-sized individuals have higher fitness (greater reproductive success) compared
to larger individuals, natural selection is then directional and favours these, and if the
1120 A. Dua and A. Nigam

character is inherited, a decrease in body size is seen over the generations. The same
assumption can be vice versa, i.e., if large-sized individuals had higher fitness.
For example, the pink salmon (Oncorhynchus gorbuscha) in the Pacific North-
west known for performing extensive migrations has been decreasing in body size.
In 1945, fisherman chose the salmon based on not their number but by their pound
weight. And for such a screening, they increased the use of gill netting that selects for
larger fish. This further led to the increased survival of the smaller fish, and as a result
of such a selection, the average weight of salmon decreased by about one-third in the
next 25 years.

22.4.1.2 Stabilizing/Normalizing/Centripetal Selection (Under Constant

Environmental Conditions)
This selection takes place in a uniform environment, with the sole aim of increasing
the adaptive peak by eliminating peripheral variants on both sides of the mean
population. The mean population is well adapted to the given environment that
remains stable, and the variants arise by mutation, gene flow, recombination and
chromosomal segregation. The ill-adapted variants are weeded out, and the well-
adapted are preserved and increase in number over time. Hence, this selection aims
to keep the population genetically constant for hundreds or thousands of generations.
In such conditions, individuals with extreme measurements of any trait are at a
disadvantage in comparison to the ones with average measurements. It is unspectac-
ular, ubiquitous and most common mode of selection in nature.
At the genetic level, variability is preserved by stabilizing selection. The interme-
diate phenotype having the average characteristics is usually heterozygous compared
to the extremes that are homozygous and usually disadvantageous.
The most spectacular study of stabilizing selection was published by American
scientist H.C. Bumpus at Rhode Island. Post a severe snow storm, he studied
136 immobilized sparrows in his lab of which 72 were revived but 64 died. He
identified the sex and measured several morphological traits of the sparrows. His
conclusion was that ‘the birds which perished had either very long/ short wings as
compared to those with average structural measurements.’ This indicated that
individuals on the extremities of the populations do not survive abnormal or
catastrophic events.
Another example of stabilizing selection is the data regarding birth and survival
rate of human babies. Karn and Penrose (1952) showed that mortality for infants is
greater for those who are either underweight or overweight. Newborns born at
average weight (7.5–8.5 lb) show minimum mortality. The underweight babies are
born premature (not ready for independent existence), and the overweight babies
often suffer physical damage during delivery.

22.4.1.3 Disruptive/Diversifying/Centrifugal Selection (Under

Heterogeneous Environmental Conditions)
In disruptive selection, both the extreme phenotypes in a population are favoured,
and the intermediates are selected against. Here, genetic uniformity becomes a
drawback when a population encounters a range of ecological discontinuities. The
22 Evolutionary Genetics 1121

population as a result splits into several groups with different sets of genotypes, each
capable of successfully exploiting a different environment. This leads to adaptive
polymorphism with respect to ecological opportunities. Diversifying selection hence
facilitates a polymorphic population to adapt to different niches of a heterogeneous
environment (consisting of different microhabitats). Individuals of the intermediate
category have lower fitness compared to the extremes (homozygous for various
alleles) and fail to survive. Hence, disruptive selection promotes genetic diversity as
a previous homogenous population gets split into different adaptive forms as a result
of being subjected to divergent selection pressures.

22.4.2 Kin and Group Selection

Kin selection and group (multilevel) selection are two evolutionary phenomena
which form the framework explaining the social behaviour in animals.

22.4.2.1 Kin Selection

Charles Darwin while explaining his theory of natural selection proposed that
individuals have a tendency towards increasing the chances of their own survival
and reproduction. However, cases have been seen in nature where the close genetic
relatives aid each other selectively. This social phenomenon known as altruistic
behaviour was a Darwinian puzzle. Why should the individuals save their close
relatives at the risk of their own fitness? It was several years later that this question
was answered by J.B.S. Haldane, one of the founders of population genetics. This
phenomenon was termed as ‘indirect selection’ by Jerry Brown. This is in contrast to
direct selection where the individual increases its own fitness by self-reproduction.
Another term ‘kin selection’ was introduced by John Maynard Smith which includes
the parental aid given to the offspring (which are descendent kin) as well as altruistic
behaviour for close relative (non-descendent kin). Kin selection has been mainly
used to explain why the phenomenon of social and altruistic behaviour has evolved
in animals.
The total contribution of an individual including direct and indirect fitness is
termed as inclusive fitness. Direct and indirect fitness can be expressed in the same
genetic unit. For example, an individual A has one offspring and adopts three
nephews. The total genetic contribution of A to the next generation would be:

Inclusive fitness ¼ Direct fitness þ Indirect fitness

¼ 1  0:5 þ 3  0:25
¼ 0:5 þ 0:75 ¼ 1:25 genetic units

The mathematical concept of inclusive fitness by W.D. Hamilton introduced in

1954 can be used to calculate the adaptive value of altruistic behaviour. The altruistic
behaviour would evolve only when the indirect fitness gained through the altruistic
1122 A. Dua and A. Nigam

allele is greater than the direct fitness gained by self-reproduction. This is stated as
Hamilton’s rule and can be represented as:

rb B > rc C

where

‘r’ is the coefficient of relatedness.

‘B’ is the number of genetic relatives that have survived due to the altruistic act of the
individual.
‘C’ is the number of offspring produced by the individual.

Using the equation for the previous example:

r b B ¼ 0:25  3 ¼ 0:75
r c C ¼ 0:5  1 ¼ 0:5

Through calculations we conclude rbB > rcC; thus, any allele associated with this
altruistic act would increase in frequency.
Studies have been carried out to show the universality of kin selection.
Łukasiewicz et al. (2017) worked on bulb mites (Rhizoglyphus robini) to show
how the effect of relatedness promotes female productivity and cooperation in sex.
They carried out experiment in two evolutionary groups: one of relatives and the
other of non-relatives in the laboratory during the reproductive phase of the cycle.
The result was in sync with the kin selection theory, where the evolution in the group
of relatives resulted in increased reproductive output by the females (Kin
slection, http://nectunt.bifi.es/to-learn-more-overview/kin-selection/; Kramer and
Meunier 2016).

22.4.2.2 Group Selection

Group selection or multilevel selection, as against the Darwinian concept of selec-
tion, refers to the idea that selection does not take place at the individual level but can
act on multiple levels of biological organizational level including a whole group of
organisms. Group selection looks into the direction and strength selection acting on
multiple hierarchical levels. There are several arguments which emerge and discard
the theory of group selection.

22.4.3 Sexual Selection

Sexual selection is a subcategory of natural selection which provides advantage to

certain individuals over the others to be able to successfully mate. It accounts for the
evolutionary phenomenon of the existence of expensive secondary traits which make
them much more vulnerable to the predators. Sexual selection is also the evolution-
ary phenomenon which has also resulted in sexual dimorphism. A common example
22 Evolutionary Genetics 1123

Fig. 22.6 Sexual selection in peacocks. (a) The relation between the tail length of the peacock and
fitness; (b) the correlation in the exaggeration of character and fitness results in bell-shaped curve.
The peak is the optimal value. The modern peacocks lie on the right of the optimal value (Adapted
from Ridley 2004)

of this is the peacock and peahen. Peacock has elaborate tail feathers and is brightly
coloured which is costly for its survival. Firstly, there is immense physiological
investment in development of these tail feathers. The elaborate courtship display
through these colourful plumages consumes time and energy. Lastly these feathers
make it easy for the predators to spot them. In contrast the peahen is drab coloured
with a short tail exhibiting sexual dimorphism between male and female. Why have
these secondary sexual traits evolved in male? Darwin argued that even though these
traits are expensive and may shorten the life, but it ensures that the individual is able to
contribute its genes to the next generation, thus increasing their fitness. The peacock
with the bigger, colourful and elaborate tail feathers would be able to perform an
elaborate courtship display, thus ensuring successful reproduction. A peahen would
choose such a peacock as the mate because the elaborate and colourful plumage and
courtship display would indicate healthy genes, ensuring good genes are passed onto
her offspring. The male ornamentation is basically targets of female choice.
There are two types of sexual selection: intersexual (female’s choice) and
intrasexual (male-male competition). Intersexual selection is when the female has
a choice of the mate, and she chooses the mate on the basis of the sexual traits or the
exhibition of male dominance (Byers and Waits 2006). The gain obtained could
either be direct such as resources and safety, or in other case the gain could be
indirect, i.e. creation of offspring with superior quality of genes. The above-stated
example also exhibits mate choice of peahen of peacock with highest number of
ocelli in the tail feathers, thus exhibiting superior genes. Fisher attempted to explain
this evolution of costly characters with his ‘runaway theory.’ Earlier before the
evolution of female choice, the peacock might not have been prevalent with long
tails. Randomly a mutant female chose peacock with long tail which also had higher
fitness associated with it. The peahen will now produce peacock with averagely long
1124 A. Dua and A. Nigam

tail and higher fitness. Slowly, the population will be replaced by peacocks with long
tail and peahen who choose the peacock with this attribute. The evolution of long tail
of peacock and peahen (with mother’s genes of choice and father’s genes for long
tail) with such preferential choice would reinforce each other resulting in the
evolution of long tails (Fig. 22.6).
A study was carried out on the pronghorns of the National Bison Range in
northwestern Montana. The sample population was ear tagged and genotyped to
ten microsatellite loci. In the study which spanned for 4 years, it was seen that 59%
of the fawns were fathered by a small group of males who were physically attractive.
Male attractiveness was associated with the offspring survival to weaning with the
help of general linear latent and mixed models program, a statistical model. Fawn
deaths are basically caused by vulnerability to coyote predation. 50 days post their
birth, the fawns are able to gain speed in their sprint and thus escape predation. Thus
there would be a differential growth rate of the fawns depending on the rate of
weaning. The study showed that the fawns of preferred attractive males had faster
growth (hind foot length was measured) and thus greater chances of survival. Thus
female investment in mate sampling results in higher fitness by producing offspring
with superior genes.
Intrasexual selection mainly includes competition among male to be able to mate
with the female. The male-male competition is usually observed in polygynous
mating system. This competition often results in intense contests to prove the
superiority of the individual. Sometimes the competition is extremely intense
resulting in fights. This has resulted in evolution of large body size or modes of
fighting (such as horns and antlers). The winner of these intense competitions
usually gets access to the males as they display superior and good quality genes.

22.4.4 Concept of Fitness, Selection Coefficient, Genetic Load

and Genetic Death

22.4.4.1 Fitness
Fitness in the simplest term may be defined as the ability of an organism, rarely a
population or species, to survive and reproduce in its adapted environment. If the
organism reproduces successfully, it consequently contributes its genes to the next
generation.
Thus, in order to estimate fitness, it is thus important to understand the various
components of fitness:

• Viability which defines the survival of the newly formed zygote up to the
reproductive age.
• Fecundity which defines the number of offspring produced in the next generation.

Together these components decide the extent of contribution of a genotype or

allele to the next generation. Each allele does not enjoy the same fitness through time
but changes with the changing environment. The terms dominant and recessive
allele, only define the expression of the phenotype at a particular locus. The
22 Evolutionary Genetics 1125

changing frequency of allele however is influenced by the caring for young one. The
fancy display of feather by male might seem to endanger the survival of the adult, as
they become vulnerable to the predator. They are important in attracting the opposite
sex and ensuring the survival of the young ones (Alcock 2005). This ensures the
increased fitness of the individual and contribution of its genes in the next
generation.
Fitness mathematically involves two terms: absolute and relative fitness.
Absolute fitness is the total fitness of a genotype which includes viability,
successful reproduction, no. of viable offspring produced, etc.; it is represented as
W and can be greater than or equal to 1.
The geneticists, however, more often use the term ‘fitness’ for relative fitness of
an organism. It is represented by w and may be defined as the survival/reproductive
rate of a genotype in comparison to the maximum survival/reproductive rate of other
genotypes in the environment. It is also known as survival or adaptive value.
For example, wAA ¼ 1 represents the relative fitness of the genotype AA,
wAa ¼ 0.8 represents the relative fitness of genotype Aa and waa ¼ 0.7 represents
the relative fitness of genotype aa. This means all individuals of genotype AA, 80%
of genotype Aa and 70% of the genotype aa would survive in the given environment.
Of the three genotypes, AA is considered to be most fit.

22.4.4.2 Selection Coefficient

The measure of fitness and effect of selection is measured in terms of selection
coefficient. It is the measure of the extent of natural selection against the contribution
of a genotype to the next generation. It is symbolized as ‘s’ and lies between 0 and
1. Let us look at a hypothetical case where the population comprises the genotype
AA, Aa and aa to understand this concept. These genotypes have different chances
of survival as seen in Table 22.3.
The selection acts against the recessive allele ‘a’, and thus there will be a smaller
number of surviving adults of genotype ‘aa’. If s ¼ 0.1 then the chances of survival
of genotype ‘aa’ are 90% as against 100% survival of genotypes AA and Aa. This is
a mathematical model; however, in a real environment, the chances of survival, from
birth to reproductive age, of an organism of even the best genotype are only 50%.

22.4.4.3 Genetic Load and Genetic Death

The concept of genetic load was introduced by Haldane where he observed the loss
of population fitness due to selection-mutation balance. Therefore, in order to
understand genetic load, we need to once again refer to our concept of fitness. As
we have already discussed, a population comprises genotypes of varying fitness. The
genotype with highest fitness is assigned a relative fitness value of 1. The rest of the
genotype have relative fitness values less than 1. In spite of the fact that the selection

Table 22.3 Chances of Genotype Chances of survival

survival of different
AA, Aa 1
genotypes
aa 1-s
1126 A. Dua and A. Nigam

Table 22.4 Relative fit- Genotype Survival to reproduce Relative fitness

ness of different genotypes
AA 40% 0.8
Aa 50% 1
aa 30% 0.6

is continually acting, these alleles with suboptimal fitness remain in the gene pool
either because the allelic variant resulting in reduction in fitness is being replenished
in the gene pool due to mutation (known as mutational load) or because they remain
in combination with advantageous alleles (known as segregational load). We can
also measure the average fitness of a population. It is defined as mean fitness (w) and
equals to the frequency multiplied by fitness of genotype. The genetic load basically
measures the relative chance that an average individual will die before the reproduc-
tive age and thus have no contribution to the next generation because of the
deleterious allele in it. It can also be defined as the sum of deleterious genes in the
genome. It is symbolized as ‘L’ and lies between 0 and 1.

L ¼ 12w

If all the individuals of a population have fitness 1, then there is no load on the
population.
Let us look at an example to understand genetic load: The frequency of the two
alleles A and a is 0.5.
Calculating from Table 22.4:

Mean fitness ¼ 0:25ð0:8Þ þ 0:5ð1Þ þ 0:25ð0:6Þ ¼ 0:85

The load of the population L ¼ 1
0:85 ¼ 0:15

That means 15% of the offspring will die before the reproductive age, i.e. undergo
genetic death.
The failure of individuals to produce offspring and contribute to the next genera-
tion is called genetic death. High genetic load can put the population in the danger
of extinction. The marine life has been observed to have maximum genetic load in
comparison to freshwater or terrestrial species. The bivalves among the marine
species show highest genetic load due to small population size and high
mutation rate.

22.5 Population Genetics

A population is a group of organisms of the same species living in a defined

geographical area and can reproductively interbreed. Population genetics is an
important aspect of evolution and speaks about gene and allele frequencies. It is
22 Evolutionary Genetics 1127

the study of genetic make-up of the population, i.e. gene pool and change in the gene
pool over time.
Evolution and variation in gene pool are population phenomena so they are best
understood as changes in allele frequencies. There are four characteristics which
account for most of the changes in allele frequencies:

1. Mutation: produces genetic variation in gene pool and contributes to the first step
of evolution.
2. Natural selection: adaptive, directional changes in allele frequencies.
3. Genetic drift: random, non-adaptive and non-directional changes in allele
frequencies.
4. Migration: presence of gene flow.

The rates of mutation are very low in nature, so the primary contribution of
mutation is only in production of genetic variation. It is migration, genetic drift or
natural selection which acts on these genetic variations to produce change in the
allele frequencies.
It might seem that a population which is well adapted to a geographic area would
actually harbour high levels of homozygosity. But to the surprise of many evolu-
tionary biologists, there is considerable genetic variation in the gene pool. In fact, for
a population to succeed, it should have genetic variability.

22.5.1 Hardy-Weinberg Model

While studying single gene locus in a population, we observe the changes in allele
and genotype frequencies. The determination of the change in allele and genotype
frequencies of the population, from one generation to the other, forms the major
study of the population geneticists. The relation between changing allele frequencies
leading to change in genotype frequencies has been explained by Hardy-Weinberg
law. G.H. Hardy and Wilhelm Weinberg in 1908 independently formulated a simple
equation which can be used to trace the allele and genotype frequencies of the
population in an ideal scenario. The Hardy-Weinberg law states that in an ideal,
infinitely large population with random mating, on which no evolutionary force is
acting, the allele frequency does not change and the genotype frequencies stabilize
after one generation.
Assumptions made in Hardy-Weinberg law:

1. Infinitely large population.

2. Random mating.
3. Absence of evolutionary forces like:
(a) Natural selection—thus all individuals have equal rates of survival and
reproduction.
(b) Migration—absence of gene flow.
(c) Mutation—as a result no new alleles are created.
1128 A. Dua and A. Nigam

Fig. 22.7 Punnett square sperm f(A)=p f(a)=q

showing permutation and
combinations of genotype egg
produced by random matings.
Each allele has equal f(A)=p p x p= p2 p x q=
probability of pairing with
other alleles producing pq
genotypes p2, 2pq, q2
f(a)=q q x p= qp q x q= q2

A population which meets the above criteria are said to be in Hardy-Weinberg

equilibrium. The Hardy-Weinberg law is a mathematical model and uses the Men-
delian laws of segregation.
If two alleles A and a of a single locus are considered, then the genotype
frequencies AA, Aa and aa if in Hardy-Weinberg equilibrium would be p2, q2 and
2pq, where the frequency of A ¼ p and frequency of a ¼ q.
The above genotype can only be obtained when there is random mating in the
population resulting in the random combination of gametes. This can be understood
by the study of Punnett square often used by the twentieth-century geneticists to
predict probability of genotypes.
From the Punnett square (Fig. 22.7), it is clear that each allele has equal proba-
bility with pairing with the other producing the genotypes p2, 2pq and q2 if there is
random mating.
The Hardy-Weinberg equilibrium equation is:

p2 þ 2pq þ q2 ¼ 1

Since we are taking into consideration a single locus with two alleles (A and a),
these alleles should account for the 100% frequency of the gene in the gene pool.
Or in other words, p + q ¼ 1
To demonstrate how Hardy-Weinberg law of equilibrium can be used, let us
consider a population with T ¼ 0.7 and t ¼ 0.3.

f ðT Þ þ f ðt Þ ¼ p þ q ¼ 0:7 þ 0:3 ¼ 1

If the population undergoes random mating, then we attain three genotypes TT, Tt
and tt in the proportion as discussed above: p2, 2pq and q2:

f ðTT Þ ¼ p2 ¼ 0:7  0:7 ¼ 0:49

22 Evolutionary Genetics 1129

f ðtt Þ ¼ q2 ¼ 0:3  0:3 ¼ 0:09

f ðTt Þ ¼ 2pq ¼ 2  0:3  0:7 ¼ 0:42

0.49 + 0.42 + 0.09 ¼ 1 showing that we have accounted for all zygotes formed.
Assuming they follow Hardy-Weinberg law, these offspring have equal chances
of survival, and they become adults. They also have equal probability to reproduce
and mate randomly. Forty-nine percent of the gametes would be contributed by the
genotype TT, 42% by Tt and only 9% by tt.
The gamete T would be contributed by both the genotypes TT and Tt. Thus

f ðT Þ ¼ 0:49 þ ½ 0:42 ¼ 0:7

The gamete t would be contributed by both the genotypes TT and Tt. Thus

f ðt Þ ¼ 0:09 þ ½ 0:42 ¼ 0:3

The initial gene pool that we started with had the frequency of alleles T and t in
the proportion 0.7 and 0.3. After a generation of random mating, the alleles remain in
the same proportion. Thus, we can say in absence of evolutionary force, this
population has remained in Hardy-Weinberg equilibrium and has not exhibited
any change in allele frequencies.
Thus, we can draw two inferences from this model:

1. Allele frequencies remain the same from one generation to another.

2. Genotype frequencies stabilize after one generation of random selection.

However, the real population is rarely in Hardy-Weinberg equilibrium. Then why

is this model of great importance to the population geneticists? The Hardy-Weinberg
model helps the population geneticists to understand what the evolutionary forces
are acting on the real-world population and changing the allele
frequencies (Jankowska et al. 2011; Klugs et al. 2012).

22.5.1.1 Application of Hardy-Weinberg Law on Human Population

Hardy-Weinberg law can be applied on human population to test if the population is
undergoing evolution or not. It is used to calculate the percentage of population
carrying autosomal recessive alleles causing inherited diseases and for biomedical
research. One such study was done on population of the USA to study the occurrence
of phenylketonuria (PKU), a metabolic disease due to homozygosity of recessive
allele. It results in mental retardation, stunted growth and other symptoms. The
frequency of occurrence of this disease is 1 in every 10,000 babies born in the USA.
Considering that no new mutations for PKU are being added into the gene pool, we
can use this data to calculate the percentage of carriers in the population.
Using the Hardy-Weinberg equation: p2 + 2pq + q2 ¼ 1
q2 ¼ 1/10000 ¼ 0.0001
1130 A. Dua and A. Nigam

q ¼ √0.0001 ¼ 0.01
p+q¼1
p ¼ 1 2 q ¼ 1 2 0.01 ¼ 0.099
The number of carrier (heterozygotes) ¼ 2pq ¼ 2  0.099  0.01 ¼ 0.198.
Approximately 2% of the population are the carriers of the recessive allele. This
estimate is not exact, but we can get an idea of an approximate idea of the carriers in
the population.
Another such genetic disease is haemophilia, a genetic disease which impairs the
body’s ability to make blood clot. The occurrence of haemophilia A and B in the
population is 1/12000. Using Hardy-Weinberg’s equation, we can estimate the
carriers in population.
q2 ¼ 1/12000 ¼ 0.00008333
q ¼ √0.00008333 ¼ 0.0091
p+q¼1
p ¼ 1 2 q ¼ 1 2 0.0091 ¼ 0.9909
The number of carrier (heterozygotes) ¼ 2pq ¼ 2  0.9909  0.0091 ¼ 0.01803.
Thus the frequency of occurrence of carriers in the population is 0.01803 which is
very low (Klugs et al. 2012; Pierce 2012).

22.5.2 Genetic Drift

When the size of the population is small, then chance alone can result in the change
in allele frequency. The smaller the size, the greater can be the degree of fluctuation
of allele frequencies. Any random, non-adaptive, non-directional change in allele
frequency occurring in a small population is called genetic drift. Once it begins, the
phenomenon of genetic drift will continue in subsequent generations till an allele is
either completely lost or fixed in a population. The concept of genetic drift was
introduced by one of the founding fathers of population genetics, Sewall Wright in
1931, and is also known as Sewall Wright effect (Wade 2008).
This concept can be understood by random sampling in genetics. Let us consider
a single locus gene with two alleles A and a, in a population of ten individuals. The
allele frequency of A and a is 0.5 each in a gene pool of genotype AA, Aa and
aa. Each of these genotypes has equal probability of survival and reproduction, i.e. in
absence of natural selection, the fitness of all the genotypes is 1. In accordance with
the Hardy-Weinberg principle, the allele frequency should remain the same in the
next generation. However random sampling may change the scenario, and by chance
the allele ‘A’ might get a better environment for reproduction resulting in increase in
the frequency of allele ‘A’. The allele A has no adaptive value for the environment,
and this increase in frequency is just a matter of chance. Such random change in
allele frequency is significant only in a small population and results in subsequent
changes in genotype frequencies.
The genetic drift can act through two phenomena:

(a) Founder’s effect.

(b) Bottleneck effect.
22 Evolutionary Genetics 1131

22.5.2.1 Founder’s Effect

The establishment of new population formed by a few founding members from the
parental population which are not representative of the original gene pool is known
as founder’s effect. This concept was introduced by Ernst Mayr in 1942.
Several evidences of founder’s effect have been seen in humans such as preva-
lence of some recessive diseases in selective populations. These usually occur in
small or isolated population as observed in Finns. There are 30 recessive diseases
which are common in Finland than rest of the world. Diseases like cystic fibrosis and
phenylketonuria are common in Caucasian population. Studies have shown that
these diseases have arisen from one remote mutation in the immigrants of the
founder’s population, thus exhibiting the founder’s effect (Peltonen 2001).

22.5.2.2 Bottleneck Effect

Bottleneck effect occurs usually because of a disaster or natural calamity which
drastically reduces the population. The surviving population does not represent the
genetic make-up of the parental population.
An example illustrating bottleneck effect is the loss of genetic variation in
Northern elephant seals. Due to unchecked hunting, their number reduced to as
low as 20 at the end of the nineteenth century. Conservation acts resulted in rebound
of their population but with low genetic variation showing the mark of bottleneck
effect.
Similar loss of genetic diversity has also been observed in cheetah (Acinonyx
jubatus). Cheetahs are inbred with extremely low genetic diversity. Investigations
have been carried out by Stephen J. O’Brien and his colleagues where they examined
the genomes of seven cheetahs, four from Namibia and three from Tanzania.
Genome sequencing confirmed the fact that there is very low genetic diversity in
the gene pool of this animal. Low genetic diversity poses a threat to the extinction of
the species.
The initial loss of diversity happened around 10,000 years ago, when they just
managed to survive the ice age. Studies have been carried out on hypervariable
minisatellite loci and mitochondrial DNA to date this bottleneck event to the late
Pleistocene epoch. Modern times have increased the problems of habitat encroach-
ment, hunting and poaching making them vulnerable to extinction. Conservation
biologists are working hard to repopulate them, but their gene pool exhibits low
genetic diversity due to these events (Raymond and O’Brien 1993; Emerling 2016).

22.5.3 Role of Mutation and Migration in Changing Allele Frequency

22.5.3.1 Mutation
Mutation is any change in DNA sequence of the gene within the chromosome which
occurs due to error in DNA replication. Mutation in fact is the only phenomenon
which can produce new alleles. All other evolutionary forces only reshuffle the gene
pool to produce variable genotypes. Mutations are very important as they produce
variation in the gene pool on which evolution acts. In other words, mutations are the
1132 A. Dua and A. Nigam

raw material for evolution or are the engine of evolution. Mutations are random
events without any adaptation value. They can either be selected and become
abundant or completely lost from the gene pool.
Mutation, in itself, is a weak evolutionary force to change allele frequency but a
strong force to create genetic variation. However, it is difficult to measure mutation
in a diploid organism as most mutations are recessive in nature.

22.5.3.2 Migration
Migration is the movement of a subpopulation from one place to another. The
migrating population carries its own ancestral genes and interbreeds with the native
subpopulation resulting in sudden influx of alleles. This transfer of genes results in
gene flow. Thus if the two populations have different set of genes and in absence of
any selection phenomenon, migration alone can result in change in genotype fre-
quency. In order to understand it, let us look at an example where a hypothetical
species has two alleles A and a. The species also has two populations, one residing in
the mainland and the other in the island. The frequency of A on mainland is
represented by pm and frequency of A on island by pi. If migration takes place
from the mainland to island, then the frequency of A in the next generation on island
pi1 is represented by:

pi1¼ ð1
mÞ pi þ mpm

where m represents the migrants from mainland to island. Putting in the value of
pm ¼ 0.7 and pi ¼ 0.3 and that at a time 10% of the migrants have moved from
mainland to island,

pi1 ¼ ð1
0:1Þ 0:3 þ 0:1  0:7

¼ 0:9  0:3 þ 0:07
¼ 0:27 þ 0:07
¼ 0:34

These calculations show change in allele frequency due to migration from 0.3 to
0.34 in one generation itself.
There is considerable influence of human migration in distribution of ABO blood
group (Mourant et al. 1976). Karl Landsteiner, an Austrian physician, has been
credited with the discovery of ABO blood group. The ABO blood group is con-
trolled by a single gene with three alleles, present on chromosome 9. The native
Americans can be traced to a founder population of 10–20 individuals who had
migrated to the American mainland. This has been illustrated through study of
mtDNA and Y chromosome. The Americans thus have a high percentage of O
blood group. The Di blood group polymorphism tracks the migration of humans
from East Asia to America. Another such example is the prevalence of Di antigen in
South East Poland which provides the measure of extent of invasion of Mongolians
in recent times in Europe.
22 Evolutionary Genetics 1133

22.5.4 Impact of Positive Selection

Selection can be of two types: positive and negative or purifying selection. Purifying
selection prohibits the spread of deleterious mutations in the gene pool.
Positive selection is also known as Darwinian selection. It is the phenomenon of
natural selection by which advantageous mutation becomes fixed in a population, or
in other words it promotes the spread of advantageous mutation in a gene pool.
Positive selection thus promotes the emergence of new phenotype.
Charles Darwin in his explanation of selection had stated that those organisms
which have the best attributes are the ones that survive in an environment. He was
mainly concerned with phenotypic evolution. As we are looking at evolution in
terms of genetics, let us redefine this outlook. The organisms that harbour mutations
which increase their fitness in the environment, are the ones which survive and
reproduce (Forsdyke 2007).
Whether the mutations that occur in the nucleic acid sequence result in positive or
negative selection depends on which part of the gene product or protein they are
affecting. If a mutation occurs in the active site of the enzyme such that it lowers the
catalysing rate of the enzyme, it might result in lowering the fitness of the organism.
In other case, mutation in the antigen might enhance the ability of the pathogen to
invade the host, thus increasing its fitness and resulting in positive selection.
Extensive studies have been done on positive selection. One such study reviews
large number of genes of the human population which have undergone positive
selection (Wu and Zhang 2008). Darwinian selection has intensively acted in the
modern human population resulting in high genetic diversity which has resulted in
differences in appearances, metabolism of drugs and resistance to diseases. One such
set of genes are those that are involved in the development of the brain. The brain
size has increased in primates specially in the Homo sapiens and the species closely
related to them. Some genes involved in development of brain have undergone rapid
positive selection. Microcephalin is the key regulating gene of the brain of human
and is still evolving. FOXP2 is another gene present both in human and birds. It
regulates the singing ability of the birds and speech expression in man. The copy of
FOXP2 in human has high evolutionary rate under the influence of positive
selection.
Another such example was a study carried out by Zhang et al. (2002), on the
evolution of a duplicated pancreatic ribonuclease gene (RNASE1) of leaf-eating
colobines. Like ruminants these old world monkeys extract nutrients by breaking
down the symbiotic bacteria with a set of enzymes including RNASE1. Phylogenetic
analyses of the RNASE1 gene of the non-colobine monkeys with the Asian colobine
douc langur (Pygathrix nemaeus) show the substantial difference in the sequence. A
closer examination shows that one copy of the gene RNASE1 has remained
conserved but the other copy of the gene RNASE1B has accumulated many
non-synonymous substitutions post recent duplication. These rapid substitutions
have accumulated due to positive selection pressure for adaptation of enhanced
ribonuclease activity at low pH of the colobine intestine (Zhang 2010).
1134 A. Dua and A. Nigam

22.6 Speciation

22.6.1 Definition of Species

Several workers have tried to define the concept of ‘species,’ and the most important
fact to be noted is that these individuals belonging to a species co-exist in a particular
span of time.

22.6.1.1 Typological Species Concept

This concept suggests that ‘variety in nature can be reduced to a few distinct types.’
Varied individuals often belong to a single type of species that is defined by a certain
set of norms. The concept was proposed centuries ago by Plato and fails to recognize
a species as a unit of evolution. Hence it was not accepted by modern evolutionists.

22.6.1.2 Biological Species Concept

The ‘Biological Species Concept’ proposed by Harvard evolutionist Ernst Mayr
(1963) states ‘Species are groups of interbreeding natural populations that are
reproductively isolated from other such groups.’ Species is an ecological unit
sharing resources of the environment and interacting as a unit with other such
groups. This concept focuses on species, in genetic terms as a gene pool, a unit
within which the gene frequencies can change. The movement of genes through a
species via mechanisms such as immigration, emigration and interbreeding is termed
as gene flow. Natural selection favours inheritance of genes that are beneficial to the
organisms as well as that help them in better co-existence with the environment. A
gene that does not favour these aspects is selected against. An important limitation of
the biological species concept is that it does not apply to the organisms that
reproduce asexually.

22.6.1.3 Recognition Species Concept

The concept states ‘species as a collection of individuals with shared specific mate
recognition system (SMRS).’ It was given by H. Paterson in 1993. The idea behind
placing individuals together as a species is interbreeding and production of a viable,
fertile offspring. And this concept highlights the method by which potential conspe-
cific mates are recognized for breeding. The sensory systems may be visual, acous-
tic, chemical, etc. An example citing the same is a population of crickets within a
single habitat in the USA. Their courtship begins with the males singing for
attracting the females. The songs are species specific and hence confine
interbreeding to a single species.

22.6.1.4 Morphological Species Concept

Species are characterized by body shape and other structural features and can be
applied to sexual as well as asexual organisms.
22 Evolutionary Genetics 1135

22.6.1.5 Ecological Species Concept

The ecological niche, that is, the microenvironment of a species, defines a species
population. The concept highlights the interaction of the members with the living
and non-living parts of their environment.
In a population that is sexually reproducing, speciation leads to the division of a
parental gene pool into two or more distinct gene pools. The concept of genetic
changes accumulating to form a new species is a macroevolutionary event. The
underlying cause of speciation is the adaptation of populations to discontinuous
ecological niches, leading to reproductive isolation that allows them to evolve
independently.

22.6.2 Modes of Speciation

Speciation is classified as allopatric, sympatric, peripatric and parapatric (Fitzpatrick

et al. 2009).

• If a geographical barrier is responsible for divergence of a population from its

ancestor, it is termed as allopatric speciation (Fig. 22.8). The physical barrier
prevents mixing of the population, interbreeding and hence gene flow. The
divided populations hence formed start developing independently of each other,
accumulating genetic differences between them as a result of mutations, genetic
drift and natural selection. The presence of related flora and fauna in Galapagos
Islands off the west coast of South America is a key evidence of allopatric
speciation. These have been discussed in detail by Charles Darwin in his book
Origin of Species. Over 14 species of finches are distributed all over these islands
that have accumulated differences from their ancestors with respect to their food
preferences and beaks. The antelope squirrel Ammospermophilus harrisii inhabits
the southern rim of the Grand Canyon and the northern rim by the white-tailed
Ammospermophilus leucurus. Small rodents are unable to cross such barriers
unlike birds and hence have diverged into two different species.
• Sympatric speciation occurs in geographically overlapping areas (Fig. 22.9).
Divergence occurs due to reduced gene flow by polyploidy, sexual selection and
habitat differentiation. An example is presence of cichlid species in East African
Great Lakes that are known to be monophyletic (Schliewen et al. 1994). Analysis
of their mitochondrial DNA revealed that these flocks have undergone sympatric
diversification. Sympatric speciation is also said to be underway in pest of apples,
North American maggot fly Rhagoletis pomonella. Its original habitat was the
hawthorn tree, but now it colonizes apple trees. Apples mature sooner compared
to hawthorn fruits; natural selection favoured the apple-feeding flies. The apple-
feeding flies show temporal isolation from the hawthorn feeders providing a
prezygotic isolating barrier to gene flow as well.
• Ernst Mayr described the founder effect speciation which was later termed as
peripatric speciation by him. His idea elaborated that in isolated populations
with restricted distributions, the peripherally located species to the parent species
1136 A. Dua and A. Nigam

Fig. 22.8 Sequence of events

in allopatric speciation. A: The
original parent population. B:
The population is split by a
geographical barrier. C:
Owing to the distance and
isolating mechanisms that act,
the populations become
genetically different by means
of drift and selection. D: As a
result of the events, the
populations have become
independent species, have
adapted to their unique niche
and cannot interbreed
anymore. Speciation is
complete when no gene flow
occurs even if they occupy the
same geographical area

tend to diverge genetically and form new genera. He reasoned that the allele
frequencies at various loci differed from the parent population due to genetic drift.
A common example is of the paradise kingfishers Tanysiptera in New Guinea.
The T. galatea is present throughout lowlands of New Guinea, whereas several
distinct forms (T. riedelii, T. carolinae) are distributed on the islands along its
coast.
• If gene flow is weak between populations residing in adjacent regions with varied
selection pressures, it leads to parapatric speciation. The hybrids so formed may
be weak, with lower fitness, inviable and sterile. Steady genetic divergence would
lead to complete reproductive isolation. An example is of the three-spined
sticklebacks (Gasterosteus aculeatus) in lakes (each with outlet streams) of
Vancouver Island in western Canada. Though there was an absence of any
physical barriers between streams and lake populations, the subpopulations
have evolved with various different morphological features. Genomic analysis
has shown that genetic differences between the populations were pronounced in
the central chromosomal regions (Roesti et al. 2012).
22 Evolutionary Genetics 1137

Fig. 22.9 Sequence of events

in sympatric speciation. A:
The original parent
population. B: The
subpopulations start getting
formed in the absence of any
geographical barrier and begin
their genetic differentiation.
C: The subspecies hence
formed become completely
reproductively isolated and
cannot interbreed, thus
forming independent species

22.6.3 Isolating Mechanisms

The barriers that lead to these events may be physical, geographical, physiological,
temporal or ethological and are collectively termed as reproductive isolating
mechanisms. These gene flow barriers evolve as a result of divergence between
populations that accumulate genetic differences over time. Accumulation of these
isolating mechanisms results in the process of speciation. The strength of isolation is
assessed if the species merge back into a single lineage on coming back into contact
with each other. Speciation is complete if no intermixing takes place and no hybrids
are formed. However, if isolating mechanisms are weak and overcome by gene flow,
they end up merging the species into a single lineage.
These are classified into two main categories with respect to their occurrence pre-
or post-mating (Table 22.5):

(a) Prezygotic isolating mechanisms: These act before fertilization and hence no
zygote formation takes place. As a result of these mechanisms, no mating can
take place.
(b) Post-zygotic isolating mechanisms: These act post fertilization, the population
members are willing to mate and the hybrid hence formed isn’t fit to be either
1138 A. Dua and A. Nigam

Table 22.5 Isolating mechanisms operating in nature

S. no. Mechanism Example
Pre-mating/prezygotic mechanisms: no mating, no zygote formation (interspecific pairs do
not form)
1. Geographical: Differences in preferred Two species of fern primula, P. vulgaris
habitat/biotope hinder mating in animals and P. elatior, have different soil,
and pollen exchange in plants temperature and moisture
preferences (Keller et al. 2016)
2. Seasonal/temporal: Members of different Species of toads: Bufo americanus and
populations differ in breeding time/time Bufo fowleri have different breeding times
of attaining sexual maturity or flowering (early and late in spring, respectively)
3. Ethological/behavioural/psychological: Behaviour, courtship signals are species
Isolation occurs due to incompatible specific and enable mate recognition. For
behaviour of the males and females of example, in birds and arthropods, songs,
different species courtship dances, display and tactics are
responsible for conspecific mating.
Marine blue footed booby birds are
recognized by their blue feet, a sexually
selected trait. They mate post a courtship
display that calls for the male to high step
that enables the female to focus on his
bright feet
4. Mechanical: Reproductive organs The ‘Lock & key’ theory has been
(genitalia in animals, stamen and pistil in proposed for genitalia of animals to
plants) of the species are physically correspond and mate with each other. For
incompatible entomophilous flowers, floral parts
become highly specialized to attract a
single kind of pollinator and prevent
interspecific pollination. Shells of two
species of snail Bradybaena spiral in
different directions (clockwise and
counter-clockwise). As a result, their
genital openings are not aligned, and
mating is unsuccessful
Post-mating prezygotic mechanisms: Mating takes place, but the gametes do not get
fertilized
1. Gamete viability: The sperms do not In some crosses of drosophila, an
survive in the female reproductive tract; insemination reaction can result in
pollen fails to germinate swelling in the vagina of the female and
hence unsuccessful fertilization (Turissini
et al. 2018)
2. Gamete recognition: Sperms and eggs of Sea urchins of the genus Echinometra
the two species are incompatible with have external fertilization, where the
each other; pollen and egg nuclei are also sperms and eggs are laid into surrounding
incompatible/fail to attract each other water and fuse to form zygotes. Hetero-
specific gametes do not fuse with each
other as the proteins on their surfaces
cannot bind to each other
Post-zygotic mechanisms: mating takes place, and hybrids hence formed are sterile/non-
viable
1. Hybrid inviability: Interspecific hybrids Salamander subspecies Ensatina
are weak, are non-viable with lower occasionally hybridize, and the resultant
hybrids either do not complete their
(continued)
22 Evolutionary Genetics 1139

Table 22.5 (continued)

S. no. Mechanism Example
fitness and hence get eliminated at any development or, if they do, are frail in
stage before attaining sexual maturity stature
2. Hybrid sterility: Hybrids are sterile due Genetic causes of such an event are at
to abnormal developments of their gonads three levels:
or failure of gametes to develop (a) Genic: Incompatible gene
combinations create a disharmony and
failure to form gametes (common in
animal hybrids)
(b) Chromosomal: Different gene
arrangements and chromosomal
aberrations (inversions, translocations,
etc.) lead to sterility (common in plant
hybrids)
(c) Cytoplasmic: Unfavourable
interactions between nuclear and
cytoplasmic factors
3. F2 breakdown: Inviability or sterility of F2 hybrid even if F1 is vigorous

viable or reproduce further. Hybrids are sterile, or even if they manage to

reproduce, the progeny does not have high levels of fitness. Post-zygotic
mechanisms are uneconomical as the hybrids hence produced have no genetic
contributions to the gene pool and hence a waste of sexual energy. Mostly the
parent populations have diverged so much that hybrids are inviable or sterile.

Reproductive isolation is not a product of a single gene mutation between two

isolated populations; rather, it arises by gradual accumulation of differences in gene
combinations, frequencies, arrangements and interactions between them.

22.6.4 Genetics of Speciation and Reproductive Isolation

Differences in various chromosomal segments and genes lead to strengthening of

isolating mechanisms and finally result in speciation (Noor and Feder 2006). The
hybrids formed due to a hetero-specific mating are inviable, weak and sterile as both
paternal and maternal sets of chromosomes form disharmonious association. This
may lead to a loss in function of several indispensable housekeeping genes due to
epistasis or occurrence of homologous alleles on different chromosomes. Prezygotic
mechanisms hence are more economical and do not allow investment of biological
energy which may lead to formation of a wasteful end product (Wolf et al. 2010).
In prezygotic isolation, several differences in gene combinations and chromo-
somal segments have been established firmly by natural selection over a long span of
time. When two such populations come in proximity, then do not seek potential
mates within each other. Post-zygotic mechanisms reveal that the two populations
under scrutiny haven’t yet accumulated enough differences yet. Strongest genetic
1140 A. Dua and A. Nigam

dissimilarities exist in the mechanism resulting in inviable hybrids compared to

others. Hence, natural selection is yet to intensify its divergent action on them.
Hence, it can be concluded that species do not arise at a single step, but through an
accumulation of various genic and chromosomal differences. At the genetic level,
four problems are known to cause hybrid inviability/sterility/breakdown: chromo-
somal rearrangements, incompatibility at the gene level, ploidy and interaction
between endosymbionts and the nuclear genome. When a gene at a specific locus
from one parent does not interact well with a gene at another locus from the other
parent, such genic incompatibilities affect the hetero-specific hybrid viability.
Also proposed in the 1930s was the Dobzhansky-Muller theory for post-zygotic
isolation. According to the theory, an ancestral population splits into different
populations in which gene flow is absent. Each population is well adapted to its
local environment as a result of genetic changes that have accumulated over time. If
the populations encounter each other later and mate, the genetic changes at the
various loci will now allow the hybrids to be successful, and hence they will be
sterile. The main aspect to be impressed upon is that post-zygotic isolation is due to
interaction of multiple loci. An interesting pattern in post-zygotic isolation was
inferred by J.B.S. Haldane, called Haldane’s rule, that ‘the heterogametic hybrid
of a population will have lower fitness compared to the homogametic one.’

22.7 Evolution of Man

Apart from fossil evidences, the origin and evolution of man have been widely
studied with the help of DNA sequences to trace the history of modern man. The
relationship among man and its nearest relatives ‘The great apes’ has long been
studied. There are major morphological differences including bipedalism, presence
of an apposable thumb and body proportions distinguishing chimpanzees, gorillas
and man.
All early hominid fossils have been procured from Africa, but the first fossils
found outside were of Homo erectus (China and Indonesia) (Fig. 22.10). It is
postulated that H. erectus gave rise to archaic European, Asian and African
populations. The best known of such hominids are the Neanderthals (Homo
neanderthalensis) that lived in Europe and Western Asia about 3,00,000 years
ago. These fossils were obtained from Feldhofer cave in Germany and Mezmaiskaya
cave in Caucasus Mountains east of Black Sea. Mitochondrial DNA analysis from
Neanderthal fossils with 2000 present-day human samples suggests that they did not
contribute to the mitochondrial DNA of Homo sapiens. They may have competed
with ancestors of modern humans and lost out in the competition and became extinct.
Another approach was the whole genome sequencing of the Neanderthal species.
Their genome is made up of 3.2 billion base pairs and is 99.7% identical to the
modern human genome. Comparisons of the Neanderthal genome, five present-day
humans and chimpanzee genome revealed that there are amino acid coding
differences in 78 genes. Interestingly it was revealed that 1–4% of Neanderthal
sequences were found in humans from Europe and Asia but not Africa. Phylogenetic
22 Evolutionary Genetics 1141

Fig. 22.10 Evolutionary history of the modern man. Homo sapiens evolved from a common
ancestor in parallel with chimpanzees as traced by fossil evidences procured from over the globe.
Uncertainties in the line are indicated by question marks. (Adapted from Snustad & Simmons; 6th
Edition)

analysis done to compare and analyse the divergence of the species revealed that
humans and Neanderthals last shared an ancestor 706,000 years ago (Fig. 22.11).
The mitochondrial genome of 12 Neanderthal specimens has been completely
sequenced, and these are quite different from the known human mtDNA. It is
unlikely that their mtDNA made any significant contributions to modern human
mtDNA. Hence, modern man and Neanderthal man are considered as clear
biological species (Hartl and Jones 2009).
The initial publication of the chimpanzee (Pan troglodytes) genome and compar-
ison with the human genome (The Chimpanzee Sequencing and Analysis Consor-
tium, 2005) has shed light on the formation of the human species and the complex
speciation between the two. In addition to this was the sequencing of the genome of
rhesus macaque (Macaca mulatta) by the Rhesus Macaque Genome Sequencing and
Analysis Consortium in 2007. It was possible to compare the three primate genomes
and construct an ancestral primate genome. Comparative genomics could also
1142 A. Dua and A. Nigam

Fig. 22.11 Divergence between the human and the Neanderthal species. The separation events led
to major evolutionary events in both populations. Data obtained on sequencing comparisons
between the genomes of modern humans and the Neanderthal DNA. (Adapted from Concepts of
Genetics by Klug – 10th Edition)

determine the regions of the ancestor that could have contributed to human evolu-
tion. Chimpanzees and humans have 98% nucleotide level similarity (Portin 2007).
The main difference between the haploid chromosomal sets of the two in a karyotype
is a big metacentric chromosome 2 in man vs acrocentric in chimpanzee. The
sequencing of the Y chromosome of both the species revealed that there is an
accelerated rate of evolution of the chromosome vs the entire genome. It is a huge
challenge till date to explain the reasons of emergence of man post their separation
less than 6.3 million years ago.
As of today, genetic data such as variations at the level of blood groups,
restriction fragment length polymorphisms, lengths of repeat DNA sequences and
DNA composition have been used to investigate relatedness amongst various
populations, races and ethnic groups. Most analysis of human evolution has been
done using mitochondrial DNA as it evolves faster than the nuclear DNA and is
passed on only through the maternal parent. Hence, in evolutionary terms
researchers can detect changes at a genic level over short period of time and trace
it back to a common female ancestor.
22 Evolutionary Genetics 1143

22.8 Concept of Molecular Phylogeny

The concept of classification and systematic organization of biological hierarchy is

an age-old concept introduced by the naturalist Linnaeus in the eighteenth century.
He classified mainly to arrange the organisms in a hierarchical pattern, but unknow-
ingly he also paved path for understanding phylogeny of organisms. Phylogeny is
the study of evolutionary relationships. Earlier the direct phylogenetic studies were
mainly based on the fossil evidence. Other studies included comparison of morpho-
logical traits. The main problem with these methods was that the fossil data was
often incomplete and comparison of morphological traits was often biased. More-
over, study on microorganisms could not use any of these mentioned methods. The
introduction of molecular phylogeny broadened the horizons where phylogenetic
relationships could also be derived among evolutionary distant organisms.
The molecular phylogeny uses the sequences of the biomolecules to look into the
evolution of organisms. It is basically selection acting on the mutation in the
biomolecules. The accumulated mutations over millions of years can act as molecu-
lar fossils. Though sequencing was first introduced only in the 1970s, but Nuttall had
use immunological assays in the early 1900s to understand the evolutionary position
of man in relation to other primates. Even though Nuttall had displayed the success-
ful use of biomolecules in understanding phylogeny as early as 1904 yet it gained
momentum only in the 1950s because of several technical challenges. There has now
been gradual shift towards molecular phylogeny, which involves the use of DNA,
RNA and protein as molecular markers to study modern phylogenetics. The major
reason of this shift is to be able to obtain large datasets for the studies. The evolution
of sequencing has also ensured easy availability of molecular data. The molecular
data can be easily converted into mathematical and statistical data which makes it
easier to analyse. Unlike the morphological traits, the molecular data (A, T/U, G, C
for DNA/RNA and amino acids for proteins) are unambiguous (Brown 2002).

22.8.1 Phylogenetic Tree

The molecular data has been largely used to construct phylogenetic trees. A phylo-
genetic tree (Fig. 22.12) is a visual display of the evolutionary relationships among
organisms. Even though the phylogenetic tree is being constructed now to display
phylogenetic events, but tree-like illustrations (Fig. 22.13) were also observed in
Darwin’s book Origin of Species, where he used it to show that accumulation of slow
modification can lead to speciation event.

22.8.1.1 Objectives of Molecular Phylogeny

1. To infer a tree displaying true phylogenetic relationships among organisms.

2. To study and recover the order of evolutionary events and represent it as
phylogenetic tree, which is a graphical representation of phylogenetic
relationship.
1144 A. Dua and A. Nigam

Fig. 22.12 A typical rooted phylogenetic tree. Diagram showing various parts of the tree—
terminal nodes or taxa (operational taxonomic unit, OTU) are the extant species, internal nodes
are the recent common ancestors, branching shows the event of divergence and root is the common
ancestor. Often the details of common ancestor are not available, so to root a tree an outgroup
species is used. Outgroup is the species which is distantly related to the group of organisms. The
pattern of branching of the tree is known as tree topology. Ninety-nine percent of the species on
earth have become extinct. A tree like this gives a visual display inferring what would have been the
phylogenetic relationship of the extant species with the extinct species

Fig. 22.13 Darwin’s illustration. Darwin too in his book Origin of species had made a tree-like
illustration which expressed evolution. (Adapted from Karen Dowell 2008)

3. To be able to estimate the evolutionary date of divergence of organisms from their

common ancestor.

Since it is thought that all organisms have arisen from the Last Universal Common
Ancestor (LUCA), objectively there should be a single tree of life. However, it is
22 Evolutionary Genetics 1145

Fig. 22.14 16S rRNA tree of life. This is a rooted tree of life made by analyses of 16 s rRNA gene.
It has three major branches—bacteria, archaea and eukaryotes. This is a phylogram (scale 0.1
changes per site) (explained in the next section) showing hypothetically how life originated 3.8 bya
from primordial soup and diverged into various life forms. (Adapted from Pevsner 2009)

close to impossible to construct a true tree of life; rather, we construct ‘inferred trees’
which are based on the mutation in biomolecules or available data, showing
hypothesized phylogenetic relationships.
The most popular tree of life has been constructed based on the phylogenetic
analyses of 16 s rRNA gene (molecular marker). This tree has three branches
showing the major divergence—bacteria, archaea and eukarya (Fig. 22.14).
A tree may be rooted showing the common or may be unrooted (Fig. 22.15)
without the common ancestor. Often the data for the ancestors is not available that is
1146 A. Dua and A. Nigam

Fig. 22.15 A comparison between unrooted and rooted tree. The unrooted tree does not have any
common ancestor. On the other hand, the rooted tree always shows the divergence from the
common ancestor

when the unrooted tree is constructed which just shows the phylogenetic relationship
among the organisms. A phylogenetic tree is not always constructed to observe the
phylogeny of various species; it can also be constructed to chart the evolutionary
path of the individual gene. Such a study is known as gene phylogeny. The
evolutionary path of the gene might not overlap with the speciation events. A
phylogenetic tree of species results from the evolution of genome (total genetic
make-up of the organism).

22.8.2 Types of Tree Representation

The topology of the tree can be defined in two ways: cladogram and phylogram. A
cladogram (Fig. 22.16) is a basic representative tree. It is a relative tree based on the
order of phylogenetic events. The branch is unscaled and of the same length.
Phylogram (Fig. 22.16) on the other hand has scaled branches. The branch represents
the amount of evolution that has taken place since the time of divergence from the
ancestor.

22.8.2.1 Clade
Clade is a group which includes the ancestor and the descendants. The tree also
exhibits different type of branching pattern or the types of clade formed. There are
three types of clade—monophyletic, paraphyletic and polyphyletic. Monophyletic
clade includes the recent common and all its descendants. The paraphyletic clade
excludes a few of the descendants, and polyphyletic clade includes distantly related
species (OTU) (Fig. 22.17).
22 Evolutionary Genetics 1147

Fig. 22.16 Types of tree representation. Cladogram which has unscaled branches and phylogram
which has scaled branches showing amount of evolution that has taken place. (Adapted from Jin
Xiong 2006)

22.8.3 Procedure for Tree Construction

The phylogenetic tree construction involves the following steps (Fig. 22.18):

1. Choice of the molecular marker and collecting data.

2. Alignment of the data.
3. Choice of evolutionary model.
4. Constructing the phylogenetic tree.
5. Testing the reliability of the tree.

Let us look into the details of each of these steps:

1. Choice of the molecular marker and assembling data: Molecular marker is the
biomolecule whose sequence would be taken into consideration to study the
evolution. It may be nucleotide or protein sequence. The correct choice of
molecular marker is an important step as it helps in construction of a true tree.
If we are working with closely related organisms, then the rapidly evolving
nucleotides should be the choice. For studying the evolution of slightly divergent
organisms, the relatively conserved rRNA gene should be used. For more diver-
gent organisms, protein sequences are used as they are relatively more conserved
1148 A. Dua and A. Nigam

Fig. 22.17 The three types of clades. Monophyletic (green), polyphyletic (blue) and paraphyletic
(pink). (Adapted from Karen Dowell 2008)

due to degeneracy of genetic code. DNA sequences are also biased than protein
due to preferential usage of codon in some organisms. The protein has 20 amino
acids as against only 4 bases of nucleotides and thus can be used for sensitive
alignment. Globins are popularly used as molecular marker and were one of the
first proteins to be sequenced. They are also used as molecular clocks (concept
explained in earlier section).
Molecular marker can be used to spot positive and negative selection. For this it is
important to distinguish between synonymous (results in no change in amino acid
sequences) and non-synonymous substitution (results in change in amino acid). If
non-synonymous substitution is higher, then it means a part of protein is
undergoing evolution to bring about change in function of protein.
Once the molecular marker is chosen, the next step is to assemble the data of
the organisms. For this there are several databases available from which the data
can be extracted. For DNA the databases are DNA Data Bank of Japan (DDBJ),
GenBank, etc. and for protein are SWISSPROT, etc. There are online tools like
BLAST which can carry out the search and extract the data from databases.
2. Alignment of the data: Once the data is collected, the next step is to align these
sequences according to homology in these sequences. The homology describes
the phylogenetic relationships. There are two types of homologs—orthologs and
paralogs. Orthologs are genes which have same ancestor but have diverged due to
speciation event. Paralogs are duplicated genes of the same ancestor. Sequence
22 Evolutionary Genetics 1149

Fig. 22.18 Brief overview of

the steps involved in Choose the molecular
phylogenetic tree
construction. The flowchart marker
enlists the various steps
sequentially described in the
text to construct a
phylogenetic tree
Retrieve data from the
database

Align the data according

to the homology

Construct phylogenetic
tree

Test the reliability of the

phylogenetic tree

alignment helps in the identification of homologous region and thus defines the
evolutionary path. The multiple sequence alignment can be done by several
tools—CLUSTAL, MSA, T-Coffee, etc.
3. Choice of evolutionary/substitution model: Substitution models are statistical
methods to analyse the amount of evolution taking place. Several models are
available for scoring the nucleotide substitution. One of the simplest models is
Jukes-Cantor model which assumes that each nucleotide is replaced with equal
probability. The other slightly more complex model is Kimura 2 parameter which
differentiates between transition (mutation from purine to purine or pyrimidine to
pyrimidine) and transversion (mutation from purine to pyrimidine and vice
versa). In accordance with this model, transition occurs much more frequently
than transversion, which is logical. For amino acid substitution, there are models
like PAM. However these models assume that all positions in sequence have
equal mutation rates. But this is not the case. For example, the wobble position of
the codon mutates at a faster rate than others.
4. Construction of phylogenetic tree: There are two basic methods for tree construc-
tion: character based and distance based. The character-based method considers
the molecular sequence as character, and after alignment each of these characters
shares homology. It is also assumed that each of these characters evolves
1150 A. Dua and A. Nigam

independently and thus is considered separate evolutionary units. The character-

based methods are maximum parsimony and maximum likelihood. On the other
hand, the distance-based method calculates the dissimilarity between the
sequences during sequence alignment and converts it into a matrix based on
which phylogenetic tree of the organisms can be constructed. The branch lengths
are additive, i.e. the evolutionary distance between two organisms can be
obtained by adding the length of all the branches connecting them. The com-
monly used distance-based methods are unweighted pair group method using
arithmetic average (UPGMA) and neighbour-joining method.
5. Testing the reliability of the tree: The last step of the phylogenetic tree construc-
tion involves statistically analysing the reliability of the inferred tree. This can be
done through a statistical analysis model, bootstrapping. It tests for the sampling
errors of the phylogenetic tree. This is done by repeated sampling of the datasets
by introducing slight changes in them. If there is error in alignment, it will result
in construction of biased tree. Random fluctuation in dataset will result in
formation of altered tree. However if the alignment is correct, this random
fluctuation in datasets will produce the same tree showing statistical confidence
in the tree. Bootstrapping is of two types—parametric and non-parametric
bootstrapping. When the changes introduced in the datasets are random, it is
known as non-parametric bootstrapping. However when the new datasets are
generated on the basis of particular sequential changes, it is known as parametric
bootstrapping. All phylogenetic trees constructed after bootstrapping are
summarized into a single consensus tree with each node of the branching pattern
displaying the bootstrap value. This value evaluates the confidence level of the
statistical analyses (Fig. 22.19) (Xiong 2006).

Box 22.1: Scientific Concept

Eweleit, L., Reinhold, K., Sauer, J.: Speciation Progress: A Case Study on the
Bushcricket Poecilimon veluchianus. PLoS One 10(10): e0139494. https://
doi.org/10.1371/journal.pone.0139494 (2015).
A speciation study was carried out on subspecies of the flightless
bushcricket Poecilimon veluchianus, endemic to Central Greece. These are
P. v. veluchianus and P. v. minor and are differentiated on body size, timing of
male signalling and sperm transfer rate. The two subspecies are parapatrically
distributed in a V-shaped zone in Central Greece. The Iti mountain could be a
geographical barrier to gene flow. Also, as speciation is a long process that
could span over centuries, it can be considered that fragmentations occurring
previously could be barriers to gene flow. Laboratory experiments done earlier
suggest that females do not differentiate between songs of the two subspecies
and there is presence of partial post-zygotic isolation that diminishes fertility

(continued)
22 Evolutionary Genetics 1151

Fig. 22.19 A representative dendrogram. It is a representation of tree where branches have a scale
showing evolutionary time showing evolution of globin family of genes (bootstrap values are
shown at branching points)

Box 22.1 (continued)

of F1 by reduction in amount of sperm transfer. Also, F1 female hybrids were
mostly fertile compared to males with lower sperm count. Hence, there is
evidence for presence of 'premating barriers vs post-mating barriers' and it
indicates that speciation is an ongoing process.
Genetic differences between the subspecies were evaluated in this study
using the sequences of mitochondrial control region (CR marker) and internal
transcribed spacers (ITS marker 1 and 2). As mentioned earlier, due to absence
of premating isolating mechanisms, hybridization is possible in the area of
contact zone that would encourage gene flow in comparison to the distantly
located sites. The occurrence of shared haplotypes was investigated in the
contact zone vs the distant sites to predict the site of hybridization. Single site
substitution differences were found in between various haplotypes. Also to

(continued)
1152 A. Dua and A. Nigam

Box 22.1 (continued)

determine if geographic isolation has shaped the population structure, a
distance-based redundancy analysis (dbDA) was done.
The results demonstrated that the aligned sequences of the CR dataset
contained 794 bp and the ITS dataset consisted of 706 bp and both showed
high levels of genetic variation especially due to low number of variable sites.
A characteristic of Poecilimon species is a high number of exclusive
haplotypes occurring at one of the different sampling sites in the mitochondrial
CR marker. In contrast there was lesser diversity in the nuclear ITS marker that
displayed a lower genetic variation.
The genetic analysis done based on the ITS marker revealed one main
barrier to gene flow, hence indicating incomplete reproductive isolation. The
contact zone has been proposed to extend from north-east of Central Greece to
the south-west. The CR marker in contrast does not clearly support the
speciation with formation of two subspecies, restricted gene flow and a clear
contact zone.
No influence of sex was observed on the genetic pattern for P. veluchianus
on investigation with dbRDA. But on testing for isolation by distance (IBD)
for both sexes, 19% variability was found in females versus 10% in males.
Hence, it can be concluded that IBD has a stronger impact on females. Males
produce sounds prior to mating, waiting for the females, and this result
indicates that females need to walk around to locate the correct partner.
Individuals of both subspecies are tougher to distinguish in the field as they
are phenotypically similar with the exception of body size. This feature
depends on the size of the mother, and hence the hybrids’ body size depends
on the identity of the mothers’ subspecies. Laboratory experiments have
revealed that hybrids with a P. v. veluchianus mother grow bigger than pure
P. v. minor individuals and hybrids with a P. v. minor mother stay smaller than
pure P. v. veluchianus individuals.
Speciation is in progress for these subspecies, as there is a lack of a strong
prezygotic isolation barrier between these two parapatrically distributed sub-
species. It could also be predicted that the species experienced a bottleneck and
are now in a phase of range expansion. IBD and sexual selection have shown
to have a great influence on the population structure, and it has been
hypothesized that P. veluchianus may be a case of widely distributed ring
species. To investigate this further, closely related species of Poecilimon are
necessary. Both subspecies are also distributed in various altitudes with P. v.
veluchianus occurring above 380 m and P. v. minor occurring also below this
altitude, and the lack of a strong prezygotic barrier probably supports a
secondary contact zone. It is suggested that a secondary contact after an
allopatric phase is likely for the two subspecies. The missing premating barrier
suggests a rather weak selection against hybrids and might also indicate
speciation in progress. Further study using microsatellite data as well as
AFLP could shed light on this ongoing speciation process.
22 Evolutionary Genetics 1153

22.9 Summary

• Evolutionary genetics is the modern field of study which integrates genetics with
the Darwinian view of evolution. It attempts to account for any change in nature
in terms of allele, genes and genotypes and how the variations at population level
can bring permanent variations in the species leading from microevolutionary to
macroevolutionary changes.
• Mutation, natural selection, genetic drift and migration are the microevolutionary
changes. Mutation is the most important variation which acts as the raw material
for the evolution of the gene pool. Most of the mutations are neutral, but some are
positive which might improve the fitness of the organism in its environment
resulting in adaptive evolution. The other variations may occur at the chromo-
somal level through recombination and aberrations.
• The Hardy-Weinberg law was introduced to understand how these variations
affect the allele and genotype frequencies in a population. However, Hardy-
Weinberg law functions under ideal conditions in an infinite population in
absence of evolutionary forces. But in a real, finite population, the evolutionary
forces like natural selection, genetic drift and migration act to affect the variation
in gene pool every generation.
• Natural selection is the force which results in the adaptation of the fittest in the
environment. It is directional in nature and acts on a large population. Selection
can occur at individual level, population level or sexual level.
• On the contrary genetic drift is non-adaptive and results in random fixation of
allele in a small population. Migration or gene flow also affects the variation in a
gene pool. Magnification of these variations over a long period of time will lead to
speciation.
• Earlier evolution was studied through fossil record. However fossil records were
often incomplete leaving a number of question marks. With the onset of new
technology and development of molecular biology techniques, the field of molec-
ular phylogeny gained momentum. Using biomolecules and genes as markers,
phylogenetic tree can be constructed which gives a bird-eye’s view of the
phylogenetic relationship among organisms.
• Human evolution studies have also been carried out using the mitochondrial DNA
which has helped chart out the divergence of Homo neanderthalensis and Homo
sapiens.

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