Molecular Basis of Inheritance

Nucleic acids are polymers of nucleotides, and DNA and RNA are the
two types of nucleic acids in living systems. DNA serves as the genetic
material in most organisms, while RNA can also act as genetic material
in some viruses.
The structure of DNA, its replication, transcription (making RNA from
DNA), the genetic code, translation (protein synthesis), and the basic
principles of their regulation are essential topics to be studied and
discussed.

Deoxyribonucleic Acid (DNA):

• First identified as an acidic substance in nucleus; by Friedrich Miescher in 1869
• Lengths of DNA varies, e.g., bacteriophage ф×174 has 5386 nucleotides; bacteriophage
lambda has 48502 bp; E. coli has 4.6 × 106 bp; human DNA has 3.3 × 109 bp
• Nucleotide consists of three components - a nitrogenous base, a pentose sugar (ribose
for RNA and deoxyribose for DNA), and a phosphate group
• Types of nitrogenous bases: two types - purines (adenine and guanine) and pyrimidines
(cytosine, thymine)
• Nucleosides has a nitrogenous base linked to OH of 1' C of the pentose sugar through N-
glycosidic linkage
• When a phosphate group is linked to 5' C of a nucleoside through phosphoester linkage,
forms nucleotide, phosphate group gives negative charge to the DNA
• Polynucleotide chain - more than two nucleotides linked together; has a free phosphate
moiety at 5'-end and a free OH group at 3'-end of the sugar
• Double helix model of James Watson and Francis Crick - has two polynucleotide chains
with sugar-phosphate backbones and paired nitrogenous bases; chains have anti-
parallel polarity; hydrogen bonds between bases; Purines always pair with
pyrimidines; (adenine with thymine and cytosine with guanine)
• Two chains coiled in right-handed fashion, with a pitch of 3.4 nm; 10 bp in each turn
• Central dogma: proposed by Francis Crick; states that genetic information flows from
DNA → RNA → protein

Packaging of DNA Helix

• Length of DNA in mammalian cell, calculated by multiplying total number of bp with the
distance between consecutive bp
• In prokaryotes - DNA organized in 'nucleoid'; held in large loops by proteins
• In eukaryotes - DNA organized around positively charged histone proteins, form histone
octamer; DNA wrapped around histone octamer - nucleosome

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Molecular Basis of Inheritance

• Nucleosome: Contains 200 bp of DNA helix; repeating units in chromatin form a "beads-
on-string" structure
• Chromatin - Coiled and condensed at metaphase stage to form chromosomes
• Non-histone chromosomal (NHC) proteins: Additional proteins involved in the higher-
level packaging of chromatin.
• Euchromatin - Loosely packed, transcriptionally active; heterochromatin - densely
packed, transcriptionally inactive

Experiments
• In 1928, Frederick Griffith conducted experiments with Streptococcus pneumoniae and
concluded that the transforming principle from the heat-killed virulent; S-strain had
transformed the non-virulent R strain, allowing it to become virulent
• Avery, MacLeod and McCarty concluded that proteases or RNases did not affect
transformation, however DNase inhibits transformation, suggesting that the DNA is the
transforming principle
• Alfred Hershey and Martha Chase in 1952 did blending experiments using
bacteriophages and E. Coli and established that DNA is the genetic material

DNA versus RNA

Characteristic DNA RNA
N2 Bases contains thymine contains uracil
Chemical stability stable due to lack of 2'-OH less stable due to the presence of 2'-
group on deoxyribose sugar OH group on ribose sugar
Structure double helical, linear or circular usually single stranded, linear
Duplication yes Yes
Mutation rate mutates at a slower rate mutates at a faster rate
Protein synthesis not directly involved, needs directly codes for proteins
RNA
Major role storage of genetic information transmission of genetic information

Ribonucleic Acid world

• RNA - first genetic material; acted as both - genetic material and catalyst; unstable;
processes like metabolism, translation, and splicing, evolved around RNA

Replication
• Mechanism of DNA replication is semiconservative
• Separation of two DNA strands would act as templates for the synthesis of new
complementary strands
• Result of DNA replication - Two DNA molecules, each with one parental strand and one
newly synthesized strand

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Molecular Basis of Inheritance

Experimental Proof
• Experiment - Meselson and Stahl performed experiment on E. coli; used heavy isotope
of nitrogen, this experiment proves that the DNA replication obeys the semi-
conservative mode of replication in which 50% of the DNA conserve for every next
generation .

Mechanism
• DNA replication - Requires a set of enzymes, DNA-dependent DNA polymerase,
Primase, DNA helicase, DNA ligase; occurs at replication fork; begins at specific regions
called origins of replication (ori)
• DNA polymerases - Highly efficient and accurate; work in 5' to 3' direction, continuous
and discontinuous replication occur on leading and lagging strand respectively
• Deoxyribonucleoside triphosphates act as substrate & provide energy
• Discontinuously synthesized Okazaki fragments are joined by DNA ligase

Transcription
• Process of copying genetic information from DNA into RNA
• Adenine forms base pair with uracil and cytosine forms base pair with guanine
• Only a fragment of one of the DNA strands (template strand with 3’→5’ polarity) is
transcribed while the other; the coding strand (5’→3’ polarity) is displaced
• Simultaneous transcription of both strands hinders protein translation

Transcription unit at template strand consists of Promoter (located towards 5’ end, provides
binding site for RNA polymerase; determines which strand of DNA serves as the template
strand), Structural gene (gene to be copied), Terminator (located towards 3'-end, marks the of
the transcription process)
• Structural genes may be monocistronic (eukaryotes) or polycistronic (prokaryotes)
• In eukaryotes structural genes are referred as split genes as they have both Exons
(coding sequences that appear in mature RNA or proteins) and Introns (intervening
sequences, do not appear in mature RNA or proteins)
• Regulatory sequences - do not code for any RNA or protein; have role in gene regulation

Process of Transcription
• Three typical types of RNAs are present in the cell’s mRNA (messenger RNA), tRNA
(transfer RNA), and rRNA (ribosomal RNA)
• In bacteria single DNA-dependent RNA polymerase catalyses transcription of all three
types of RNA while in eukaryotes three RNA polymerase are found; RNA Pol I for
(rRNAs), RNA Pol II (for mRNA and hnRNA), and RNA Pol III (tRNA, 5srRNA, and snRNAs)
• The enzyme RNA polymerase can initiate transcription at promoter region with the help
of σ factor, elongates RNA chain and terminates transcription with the help of rho
factor
• Splicing - the process of removing introns and joining exons in a defined order to create
mature mRNA in eukaryotes

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Molecular Basis of Inheritance

• hnRNA (heterogeneous nuclear RNA) undergoes capping (methyl guanocine

triphosphate cap at 5’ end) and tailing (at 3’ end) before becoming functional mRNA
• Fully processed mRNA is transported out of the nucleus for translation

Genetic Code
• George Gamow proposed that triplet codon code for 20 different amino acids
• Har Gobind Khorana's artificially synthesised RNA
• Marshall Nirenberg's cell-free tanslation system helped decipher to the triplet code
• Genetic code - a triplet code; 61 codons coding for amino acids; 3 are stop codons
• Some amino acids are coded by more than one codon, making the code degenerate
• Codon - read in mRNA in a contiguous fashion without punctuations
• Universal, with exceptions in mitochondrial codons and some protozoans
• Codon AUG codes for Methionine (Met); acts as the initiator codon
• Stop codons - UAA, UAG, and UGA; signal the end of translation
• Insertion or deletion of one or two bases changes the reading frame cause frameshift
mutations which can lead to significant changes in the amino acid sequence and protein
function
• Insertion or deletion of three or its multiple bases does not change the reading frame
• tRNA reads genetic code via anticodon loop with complementary bases
• tRNA has an amino acid acceptor end where amino acids binds
• tRNA are specific for a particular amino acid, no tRNAs for stop codons
• tRNA secondary structure - clover-leaf like; actual (3D) structure looks like an inverted L

Translation
• Process of polymerizing amino acids to form a polypeptide; determined by sequence of
bases in mRNA
• Amino acids activated in the presence of ATP and linked to their respective tRNA, known
as charging of tRNA or aminoacylation
• Ribosome - Responsible for protein synthesis; consists of structural RNAs and proteins,
two subunits: small subunit encounters mRNA and initiates translation; two sites in
large subunit act as a catalyst; 23S rRNA in bacteria acts as a ribozyme
• Translational unit - sequence of RNA flanked by start codon (AUG) and stop codon,
codes for a polypeptide
• Untranslated regions (UTRs) - found at both the 5'-end and 3'-end; not translated
• Initiation - Ribosome binds to mRNA at start codon (AUG), recognized by initiator tRNA;
elongation - amino acids linked to tRNA bind to appropriate codon in mRNA by forming
complementary base pairs with tRNA anticodon; termination - release factor binds to
stop codon

Gene expression
• In eukaryote gene expression can be regulated at transcriptional, processing, transport,
and translational levels

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Molecular Basis of Inheritance

• Transcriptional level regulation involves controlling the formation of the primary

transcript; processing level regulation includes the control of splicing of primary
transcripts; transport level regulation involves the movement of mRNA from nucleus to
cytoplasm; translational level regulation affects the process of protein synthesis
• In prokaryotes gene expression primarily occurs at the transcriptional initiation stage
• RNA polymerase activity is regulated by activators or repressors
• Each operon has a specific operator and repressor

Lac operon exhibits negative regulation

• Has one regulatory gene (i gene) and three structural genes (z, y, and a)
• i gene codes for repressor protein; z gene codes for beta-galactosidase (hydrolyses
lactose into galactose and glucose); y gene codes for permease, (increases the cell's
permeability to β-galactosids), a gene encodes transacetylase
• Lactose - substrate for β-galactosidase; serves as an inducer; regulates lac operon
• Repressor protein binds to operator region; inhibits transcription
• Inducer (like lactose) inactivates repressor, allowing transcription to proceed
• Glucose and galactose cannot act as inducers for the lac operon

Human Genome Project (HGP) - 1990

• Goals - identifying genes, DNA sequences of 3 billion base pairs, database creation, data
analysis tools, and addressing ethical, legal, and social issues
• Two main approaches: Expressed Sequence Tags (ESTs) & whole-genome sequencing
(Sequence Annotation)
• Sequencing required isolation of total DNA, fragmentation, cloning into suitable host
organisms with the help of BAC and YAC; automated DNA sequencers
• Genetic and physical maps use restriction endonuclease sites and microsatellites to
understand genome structure

Salient features of human genome

• Human genome contains 3,164.7 million bp
• Largest human gene is dystrophin has 2.4 million bases
• Nearly all (99.9%) nucleotide bases are identical in all individuals
• Contains very high percentage of repetitive sequences which have no coding functions
• Chromosome 1 has most genes (2,968); Y chromosome has fewest genes (231)
• Numerous locations where single base DNA differences called SNPs (single nucleotide
polymorphism) occur

DNA fingerprinting
• Developed by Alec Jeffreys, identifies unique differences in DNA sequences
• Satellite DNA (repetitive DNA sequences) used for DNA fingerprinting
• Satellite DNA do not code for any protein and classified into; micro-satellites Short
Tendem repeats, mini-satellites (VNTR) etc.

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Molecular Basis of Inheritance

• Polymorphism arises due to mutations; basis for genetic mapping and DNA
fingerprinting
• Variable Number of Tandem Repeats (VNTR) - satellite DNA used in early DNA
fingerprinting; VNTR probes yield characteristic patterns unique to individuals
• Technique involves DNA isolation, restriction enzyme digestion, electrophoresis,
blotting, hybridization, and autoradiography
• Polymerase chain reaction (PCR) has increased sensitivity
• Used in forensics, genetic diversity studies, and population analysis

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