1

Mutation: A heritable change in the genetic

material
– Mutations may be beneficial, neutral or harmful
_____________________________________________
– Mutagen: Agent that causes mutations
– Spontaneous mutations: Occur in the absence of a
mutagen.
• Repair of damaged DNA are ways developed by
organisms to minimize mutations.
 Mutations can also be described based on their effects
on the wild-type phenotype
 They are often characterized by their differential ability to survive.

 Mutations provide allelic variations [Positive or Negative]

Positive - are the foundation for evolutionary change needed for a species
to adapt to changes in the environment thus enhances the survival or
reproductive success of an organism .[Beneficial mutations ]

Negative - are much more likely to be harmful than beneficial to the

individual and often are the cause of diseases, decreasing the chances of
survival [Deleterious mutations]
The most extreme are lethal mutations.
 The environment can affect whether a given mutation is
deleterious or beneficial.
 Some mutations are conditional.

 They affect the phenotype only under a defined set of conditions

 An example is a temperature-sensitive mutation
Mutations Can Occur in Germ-line or in Somatic cells

 Germ-line mutations are those that occur

directly in a sperm or egg cell, or in one of
their precursor cells.

 Somatic mutations are those that occur

directly in a body cell, or in one of its
precursor cells
 Germ-line cell
mutation Gametes

The size of the patch

Somatic will depend on the
mutation
Embryo timing of the mutation
The earlier the
mutation, the larger
the patch

Patch of An individual who has

affected somatic regions that are
Mutation is area genotypically different
found Mature from each other is called
throughout individual
a genetic mosaic
the entire
Therefore, the body.
mutation can be
passed on to future Therefore, the mutation cannot be
generations passed on to future generations

Half of None of
the gametes the gametes
carry the carry the
mutation. mutation.

5
(a) Germ-line mutation (b) Somatic cell mutation
Somatic cell mutation
 Mutations
• Changes to DNA/gene are called mutations
– change the DNA
– changes the mRNA DNA 3’TACGCACATTTACGTACG5’
– may change protein
– may change trait mRNA 5’AUGCGUGUAAAUGCAUGC3’

protein aa aa aa aa aa aa aa

trait
The genetic code.
Mutations can be divided into three main types

1. Genome mutations
 Changes in chromosome number (ploidy)……... [give examples]

2. Chromosome mutations
 Changes in chromosome structure………………. [give examples]

Visible under microscopic examination

3. Gene mutations
 Relatively small change in DNA sequence that affects a single
gene.
 3. Gene Mutations
• Mutation: Permanent, heritable changes in the genome.
– Point mutation (base substitutions)
 Missense mutation
 Nonsense mutation (premature stop)
 Silent mutation

– Insertions/deletions
 Frameshift mutation
– Dramatic change in amino acids
– Run-ons, premature stops (nonsense mutations.)
The Creation of Mutation (mutagenesis)
1. Spontaneous mutation
= 1 in 109 (a billion) replicated base pairs or 1 in 106 ( a
million) replicated genes
2. Induced mutation: due to Chemical & Physical agents
(mutagens)
Mutagens increase mistakes to 10–5 (100 thousand) or 10–3 ( a
thousand) per replicated gene.
 Chemical mutagens
– Base pair changers - (nitrous acid)
– Base analogues - (e.g.. 5 bromouracil)
– Frameshift mutagens - (aflatoxin, benzpyrene)

 Radiation (physical agent)

– X rays, gamma rays break DNA, bases
– UV light causes knots in DNA strand
 Gene Mutations: Change in the DNA Sequence

 A point mutation is a change in a single base pair

 It can involve a base substitution
5’ AACGCTAGATC 3’ 5’ AACGCGAGATC 3’
3’ TTGCGATCTAG 5’ 3’ TTGCGCTCTAG 5’

 A transition is a change of a
C
pyrimidine (C, T) to another pyrimidine
or a purine (A, G) to another purine.
A G

 A transversion is a change of a T
pyrimidine to a purine or vice versa.
• Horizontal/vertical
are transitions,
 Transitions are more common than • Those to each side
transversions are transversions.
Gene Mutations Change the DNA Sequence cont

 Mutations may also involve the deletion or addition of

short sequences of DNA

5’ AACGCTAGATC 3’ 5’ AACGCTC 3’
3’ TTGCGATCTAG 5’ 3’ TTGCGAG 5’
Deletion of four base pairs

5’ AACGCTAGATC 3’ 5’ AACAGTCGCTAGATC 3’
3’ TTGCGATCTAG 5’ 3’ TTGTCAGCGATCTAG 5’

Addition/insertion of four base pairs

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 Gene Mutations Can Alter the Coding Sequence
 Mutations in the coding sequence of a structural
gene can have various effects on the polypeptide

1. Silent mutations - those nucleotide substitutions that do

not alter the amino acid sequence of the polypeptide
Due to the degeneracy of the genetic code

2. Missense mutations - those nucleotide substitutions in

which an amino acid change does occur.
 Example: Sickle-cell anemia (Glu to Val)

 If the substituted amino acid has no detectable effect on protein

function, the mutation is said to be neutral (Thr to Pro). This can
occur if the new amino acid has similar chemistry to the amino
acid it replaced
 Normal red blood cells Sickled red blood cells
10 μm 10 μm

Micrographs of red blood cells

NORMAL : NH2 – VALINE – HISTIDINE – LEUCINE – THREONINE – PROLINE – GLUTAMIC ACID – GLUTAMIC ACID...
SICKLE
CELL : NH2 – VALINE – HISTIDINE – LEUCINE – THREONINE – PROLINE – VALINE– GLUTAMIC ACID...
1 2 3 4 5 6

A comparison of the amino acid sequence between

normal b-globin and sickle-cell b-globin
 Gene Mutations Can Alter the Coding Sequence cont.

3. Nonsense mutations - are those nucleotide substitutions that change

a normal codon to a stop codon

5’ UUU UUU UGC UUU UUU3’

Phe Phe Cys Phe Phe

5’ UUU UUU UGA UUU UUU3’ (Stop = UAA, UGA, UAG)

Phe Phe STOP -----------------

NB: There are NO tRNAs in cells with anti-codons that

recognize STOP codons in mRNA
 Nonsense mutations insert a stop codon which results in
premature termination.

 Truncated polypeptide usually results in loss of function

for polypeptide
 Nonsense suppressor mutations
 Result from a mutation in the anti-codon loop of a specific tRNA gene

 It allows the tRNA to recognize a nonsense codon and base pair with it.
DNA

Gene encoding tRNATRP

• Point mutation occurs in the anticodon loop OF the tRNA

• This allows this tRNA to base pair with a stop codon and deliver the
amino acid that was expected?
Trp Trp

AUG AUC
---UAC---UAG ---UAG---UAG
Normal tRNA Mutant tRNA
 Gene Mutations Can Alter the Coding Sequence cont.

4. Frameshift mutations - involve the addition or deletion of a

number of nucleotides that is not divisible by three.

 This shifts the reading frame so that translation of the

mRNA results in a completely different amino acid
sequence downstream of the mutation

Transposons/insertion sequences- jumping genes

 Mutations
• Changes to DNA/gene are called mutations
– change the DNA
DNA 3’TACGCACATTTACGTACG5’
– changes the mRNA
– may change protein
– may change trait mRNA 5’AUGCGUGUAAAUGCAUGC3’

protein aa aa aa aa aa aa aa

trait
 Frameshift mutations
mRNA without
mutation

Protein

Deletion of U

mRNA, Frameshift
mutation

Protein

Wild type THE BIG CAT ATE THE RAT

Deletion THE BIG CTA TET HER AT
Insertion THE BIG CBA TAT ETH ERA T
 Mobile Genetic Elements (mostly transposons):
• DNA that can move to a new location - (illegitimate
recombination).
• Movement is usually accompanied by replication and is
usually a rare event.
• Included: Insertion sequences (IS's), Transposons (Tn's)

the element "jumps"

They are found in bacteria and eukaryotes.

Well studied in corn (Ac and DS elements), Drosophila (copia and P elements),
and in people (All sequences- 500,000 copies in our DNA!)
 Point vs Frameshift Mutations
1. Which type of mutation is more serious?

1. Frameshift mutation affects every amino acid after

the mutation

1. Point mutation affects only the amino acid at the

mutation
Gene Mutations outside of coding sequences can still affect phenotype

 Mutations in the core promoter can change levels of gene expression.

Mutations in the promoter, splicing junction or ribosome binding site are
also mutagenic.
 Up mutations increase expression.
 Down mutations decrease expression

• Possible consequences of Gene Mutations

Outside of the Coding Sequence:

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 consequences of mutations on
splicing A 1. Exposure of
B
C
a premature
stop codon
1 2 3 4 5 2. A disruption
of a reading
frame

no mutation mutation A mutation B mutation C

normal mRNA truncated exon 3 longer exon
mRNA skipped 4
1 2 3 4 5 1 2 1 2 4 5 1 2 3 4 5

Normal Truncated Pprotein of different size (smaller or

protein protein longer)
active inactive inactive or aberrant function
Gene Mutations and Their Effects on Genotype
and Phenotype

 In a natural population, the wild-type is the relatively

prevalent genotype. Genes with multiple alleles
may have two or more wild-types.

 A forward mutation changes the wild-type

genotype into some new variation.

 A reverse mutation changes a mutant allele

back to the wild-type - It is also termed a reversion
 OCCURRENCE AND CAUSES OF MUTATION
Mutations can occur spontaneously or be induced
 Spontaneous mutations
[Spontaneous Mutations are Random Events]
 Result from abnormalities in cellular/biological processes
– E.g., Errors in DNA replication

 Underlying cause originates within the cell

 Induced mutations
 Caused by environmental agents
 Agents that are known to alter DNA structure are termed
mutagens
These can be chemical or physical agents
 Mutation Rates and Frequencies
 Mutation rate - the likelihood that a gene will be altered
by a new mutation.
 It is commonly expressed as the number of new mutations

in a given gene per cell generation

 It is in the range of 10
-5 to 10-9 per generation

 The mutation rate for a given gene is not constant

 It can be increased by the presence of mutagens

 Mutation rates vary substantially between species

and even within different strains of the same species
 Mutation Rates and Frequencies cont.
 Within the same individual, some genes mutate at
a much higher rate than other genes

 Some genes are larger than others

 This provides a greater chance for mutation

 Some genes have locations within the chromosome that

make them more susceptible to mutation.
 These are termed hot spots i.e., Specific bases or regions that
are more likely to be the site of a mutation within a gene

 NB: Hot spots can be also found within a single gene

 Causes of Spontaneous Mutations
 Spontaneous mutations can arise by three types of
chemical changes

 1. Depurination The most common

 2. Deamination

 3. Tautomeric shift

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 Depurination - involves the removal of a purine
(guanine or adenine) from the DNA.

 The covalent bond between deoxyribose and a

purine base is somewhat unstable:

 It occasionally undergoes a spontaneous reaction with

water that releases the base from the sugar.
 This is termed an apurinic site.

 Fortunately, apurinic sites can be repaired.

 However, if the repair system fails, a mutation may result during
subsequent rounds of DNA replication.

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5′ 3′ 5′ 3′
C G C G
A T A T
T A Depurination T A Apurinic site
C G C
G C G C
3′ 5′ 3′ 5′

(a) Depurination

5′ 3′
C G Three out of four (A, T and G)
A T are the incorrect nucleotide
T A
5′ 3′ C G
C G G C There’s a 75% chance
A T 3′ 5′ of a mutation
DNA replication
T A
C 5 3′
G C C G
3′ 5′ A T
T A X could be
X A, T, G, or C
G C
3′ 5′
(b) Replication over an apurinic site

Spontaneous depurination 31
 Mutations Due to Trinucleotide Repeats

 Several human genetic diseases are caused by an

unusual form of mutation called trinucleotide repeat
expansion (TNRE)
 The term refers to the phenomenon that a sequence of 3
nucleotides can increase from one generation to the next
(Anticipation)
 These diseases include
 Huntington disease (HD)
 Fragile X syndrome (FRAXA)

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 Certain regions of the chromosome contain
trinucleotide sequences repeated in tandem
 In normal individuals, these sequences are transmitted from
parent to offspring without mutation
 However, in persons with TNRE disorders, the length of a
trinucleotide repeat has increased above a certain critical size
 Disease symptoms occur
 In some diseases, it also becomes prone to expansion
 This phenomenon is shown here with the trinucleotide repeat CAG

CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG

n = 11
CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG

n = 18
• 1) While they are inherited, there is a connection to the
number of repeats and both inheritance and severity.
With Huntington,
– 10-35 CAG’s is normal;
– 36-120 results in an affected person.
– The # of repeats is connected to age at onset - low means later,
more means earlier.
– They are autosomal dominant.
– The problem is with people at the borders; you can be normal,
with 34 repeats, and produce offspring with 38, who have
Huntington.
2) The repeats can be in coding or non-coding regions;
Huntington is in a coding region; the repeated
glutamines are thought to result in cellular
defects in nerve cells.
Fragile X - are not in a coding region
 In some cases, the expansion is within the
coding sequence of the gene
 Typically the trinucleotide expansion is CAG (glutamine)
 Therefore, the encoded protein will contain long tracks of
glutamine
 This causes the proteins to aggregate with each other
 This aggregation is correlated with the progression of the disease

 In other cases, the expansions are located in

non-coding regions of genes
 Some of these expansions are hypothesized to cause
abnormal changes in RNA structure.
 Some produce methylated CpG islands which may
silence the gene.
 There are two particularly unusual features that
some TNRE disorders have in common:
 The severity of the disease tends to worsen in future
generations. This phenomenon is called anticipation

 Anticipation usually depends on whether the disease is

inherited from the father or mother
 In Huntington disease, the TNRE is more likely to occur if inherited
from the father.
 In myotonic muscular dystrophy, the TNRE is more likely to occur if
inherited from the mother.
 This suggests that TNRE can occur more frequently during
oogenesis or spermatogenesis, depending on the gene involved.
The “DNA” cause of TNRE is not fully understood

 TNREs contain at least one C and one G

 This allows formation of a hairpin
 During DNA replication, a hairpin can lead to an increase
or decrease in the length of the DNA
 Polymerase can slip off DNA
 Hairpin forms and pulls strand back
 DNA polymerase hops back on begins synthesis from new location

 These changes can occur during gamete formation

 Offspring will have very different numbers of repeats
 Can also increase repeats in somatic cells
 This can increase severity of the disease with age
 Mechanisms of trinucleotide repeat expansion or deletion

One DNA template strand prior to DNA replication One DNA template strand prior to DNA replication
TNRE TNRE

DNA replication begins

and goes just past the TNRE.
Hairpin forms in template strand
DNA prior to DNA replication.
polymerase

DNA polymerase slips off

the template strand and a
hairpin forms. DNA replication occurs and
DNA polymerase slips over
the hairpin.

DNA polymerase resumes

DNA replication.

DNA repair occurs.

DNA repair occurs.

TNRE is longer. TNRE is shorter.

OR TNRE is the same length. OR TNRE is the same length.

(a) Mechanism of trinucleotide repeat expansion (b) Mechanism of trinucleotide repeat deletion
 Mutagenic agents are usually classified as
chemical or physical mutagens
Examples of Mutagens
Chemical mutagens

Physical mutagens

Concerned about mutagens:

 1. Mutagens are often involved in the development of human cancers
 2. Mutagens can cause gene mutations that may have harmful effects in future
generations
Chemical mutagens: Come into three main types

1. Base modifiers

2. Intercalating agents

3. Base analogues

Mutagens Alter DNA Structure in Different Ways

1. Base modifiers
 Base modifiers covalently modify the structure of a
nucleotide.

 For example, nitrous acid, replaces amino groups with

keto groups (–NH2 to =O).
 This can change
o Cytosine to Uracil (not in DNA)
and
o Adenine to Hypoxanthine (not in DNA)
 These modified bases do not pair with the appropriate nucleotides in
the daughter strand during DNA replication.
 After replication
Template strand

H NH2 H
O H N H
N
H N HNO2
N
N H N
N
Sugar
O N N
Sugar
Sugar O H
These mispairings create
Cytosine Uracil Adenine mutations in the newly
replicated strand

H
H N NH2 H N H H
O N
HNO2
N N N
N N H H
Sugar Sugar
N N N
H H O Sugar
Adenine Hypoxanthine Cytosine

Mispairing of modified bases

• Alkylating agents = disrupt the appropriate pairing between
nucleotides by alkylating bases within the DNA.

[E.g., Ethylmethane sulfonate (EMS)]

Tautomerization
2. Intercalating agents
 Intercalating agents contain flat planar structures
that intercalate themselves into the double helix.

 This distorts the helical structure.

 When DNA containing these mutagens is replicated, the

daughter strands may contain single-nucleotide
additions and/or deletions resulting in frameshifts.

45
Intercalating agents:
Acridine dyes - increase rate of frameshift mutations
e.g., Ethidium Bromide
Chemical Frameshift Mutagens Intercalate into DNA

Carboplatin
(anti-cancer drug)
Benzpyrene in
cigarette smoke
Aflatoxin from
Aspergillus
fungus growing
on corn
Daunarubicin
ATGC ATGC (anti-cancer drug)
ATGC CGTA
TAGC TAGC GCC
CG CG G

Bleomycin (anti-
cancer drug produced
by Streptomyces)
3. Base analogues
 Base analogues become incorporated into daughter
strands during DNA replication thymine analogue
For example, 5-bromouracil can be incorporated into DNA instead
of thymine.

H N H
Br O H O
N H
Br O H N
N
N H N
N
N H N Sugar
Sugar N N
N N Sugar O H N
Sugar O H
H
5-bromouracil Adenine 5-bromouracil Guanine
(keto form) (enol form)

Mispairing
This tautomeric shift
Normal pairing occurs at a relatively
high rate

Base pairing of 5BU with adenine or guanine

How 5BU causes a mutation in a base pair during
DNA replication

5′ 3′

A T 5′ 3′
5′ 3′
DNA
replication
3′ 5′ G C
A 5BU 5′ 3′
DNA
replication
3′ 5′
3′ 5′ G 5BU 5′ 3′

3′ 5′ G or A 5BU

3′ 5′

In this way, 5-bromouracil can promote a change of an AT base pair

into a GC base pair

49
 Physical mutagens
 Physical mutagens come into two main types
1. Ionizing radiation
2. Nonionizing radiation

Ionizing radiation
 Includes X-rays and gamma rays
 Has short wavelength and high energy
 Can penetrate deeply into biological molecules
 Creates chemically reactive molecules termed free radicals
 Can cause
o Base deletions
o Oxidized bases
o Single nicks in DNA strands
o Cross-linking
o Chromosomal breaks
Nonionizing radiation
H
 Includes UV light O
O N O
O P O CH2
O
 Has less energy O–
H H
N
CH3
H H

 Cannot penetrate deeply H

H
Thymine

into biological molecules O

CH3
H O
 Causes the formation of O P O CH2
O
O– N N
cross-linked thymine H
H H
H
O
H

dimers H Thymine

Ultraviolet
 Thymine dimers may cause light
H
O
mutations when that DNA O P O CH2
O N O
O
strand is replicated O– N
H H CH3
H H
H

O CH3
H O
O P O CH2
O
O– N N
H H H
H H
O
H Thymine dimer
UV light Radiation

UV light- Thymine dimers

– No repair can be mutagenic
X-rays and Gamma Rays Cause Breaks in DNA
• DNA polymerase proofreads and removes
incorrect bases, mismatch repair enzymes fix
incorrectly paired bases, nucleotide excision
repair cuts out damaged DNA, telomeres are
repeated segments at end of chromosome so
repeated DNA replication does not erode genes
as chromosomes shorten

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Mismatch repair: mistake in replication, leaving a mismatch; parental
strand is methylated, thus telling the glycosylase/ endonuclease which
base to remove.

Base excision repair, and

nucleotide excision repair.
BER:

 Mismatch ---
glycosylase--AP site
-5'AP endonuclease

 Fill in/ligate (note

multiple roles for PolI,
ligase!)
Nucleotide excision repair:
• -cut out the
damaged/mismatched
bases with
endonucleases and
exonucleases;
• - fill in and
• -ligate.
Summary of Mutation Types

Run-on mutation
Stop codon lost
so protein is
extra long

(can also
produce nonsense
and run-ons)
Genes in Prokaryotes Are Grouped Together and Regulated Together

Inducible operon of genes Repressible operon of genes (normally

(normally “off”) “on”)
Used to regulate genes that are used
Used in “rainy day” gene sets
all the time, like amino acid making
like for lactose utilization genes