10/9/2018

Introduction
 Any sudden change occurring in
hereditary material is called as mutation.
 They may be harmful, beneficial or
neutral.
 In multi-cellular organism, two broad
categories of mutations:
 Somatic mutations &
 Germ line mutations

Somatic mutations Germ line mutations

 Arise in the somatic cells
 Passed on to other cells through the
process of mitosis
 Effect of these mutations depends on the
type of the cell in which they occur & the
developmental stage of the organism
 If occurs early in development, larger the
clone of the mutated cells
 They occur in the cells that produce
gametes
 Passed on to future generations
 In multi-cellular organisms, the term
mutation is generally used for germ
line mutations

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Some Facts
 Term mutation was given by Devries in 1901
Definition
while studying evening primerose Oenothera
lamarckiana
 Most of these were chromosomal variations  DNA is a highly stable molecule that
 Some were point variations replicates with amazing accuracy
 Originally the term mutation was given to
 Some errors of replication do occur
both chromosomal as well as point
mutations.  A mutation is defined as an
 Recently chromosomal mutations are studied inherited change in genetic
separately. information
 The term mutation is now given only to point
mutations

Types of gene mutation Base substitution:

Number of ways to classify gene mutations:  Simplest type of gene mutation
 Involves the alteration of a single nucleotide in the DNA
 On the basis of the molecular nature of the  A base substitution usually leads to base pair substitution
defect
 On the nature of the phenotypic effect-- GGG AGT GTA GAT CGT
amino acid sequence of the protein is altered CCC TCA CAT CTA GCA
or not
 On the basis of the causative agent of the GGG AGT GCA GAT CGT A base substitution
mutation
CCC TCA CAT CTA GCA
Number of ways to classify gene mutations:
First cycle of DNA replication

 Base substitution
GGG AGT GCA GAT CGT CCC TCA CAT CTA GCA
 Insertions & deletions
CCC TCA CGT CTA GCA GGG AGT GTA GAT CGT

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Base substitution is of two types: Transversions:

A purine is replaced by a pyrimidine
Transition:
Purine is replaced with a purine

Pyrimidine is replaced with a pyrimidine or a pyrimidine is replaced by a purine

Insertions & deletions:

 2nd major class of gene mutation

 Addition or the removal, respectively, of one or more
nucleotide pair
 Usually changes the reading frame, altering all amino
acids encoded by codons following the mutation
 Also called as frame shift mutations
 Additions or deletions in the multiples of three
nucleotides will lead to addition or deletion of one
or more amino acids
 These mutations are called in-frame insertions and
deletions, respectively.

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Mutations on the basis of the Phenotypic effects of

mutations: Missense mutation: a base is substituted that
alters a codon in the mRNA resulting in a
 Most common phenotype in natural populations of
different amino acid in the protein product
the organism is called as wild type phenotype
 The effect of mutation is considered with reference to TCA TTA
wild type phenotype AGT AAT

Forward mutation:
UCA UUA
 A mutation that alters the wild type phenotype
Reverse mutation (reversion):
Ser Leu
 A mutation that changes a mutant phenotype back in
to the wild type

Nonsense mutation: changes a sense codon into a

nonsense codon. Nonsense mutation early in the mRNA Silent mutation: alters a codon but due to
sequence produces a greatly shortened & usually degeneracy of the codon, same amino acid is
nonfunctional protein specified

TCA TGA TCA TCG

AGT ACT AGT AGC

UCA UGA Stop codon UCA UCG

Ser Ser
Ser

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Neutral mutation: mutation that alters the amino Loss of function mutations:
 Complete or partial loss of the normal function
acid sequence of the protein but does not change
 Structure of protein is so altered that it no longer
its function as replaced amino acid is chemically works correctly
similar or the affected aa has little influence on  Mutation can occur in regulatory region that
protein function. affects transcription , translation or splicing of the
CTT protein
ATT
GAA TAA
 Frequently recessive

CUU Gain of function mutations:

AUU
 Produces an entirely new trait
Leu Ile
 Causes a trait to appear in inappropriate tissues
or at inappropriate times in development
 Frequently dominant

Conditional mutations:
 Expressed only under certain conditions
On the basis of Causative agent of mutation:
Spontaneous:
Lethal mutations:  Mutations that result from natural changes in
 Cause the death of the organism DNA
Suppressor mutation: Induced:
 Suppresses the effect of other mutation  Results from changes caused By
 Occurs at a site different from the site of original
mutation
environmental chemicals & radiations
 Organism with a suppressor mutation is a double  Any environmental agent that increases the
mutant but exhibits the phenotype of un mutated wild rate of mutation above the spontaneous is
type called a mutagen such as chemicals &
 Different from reverse mutation in which mutated site is radiations
reverted back into the wild type sequence

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Chemical Mutagens:
 First discovery of a chemical mutagen was
5-bromouracil
made by Charlotte Auerbach an analog of thymine

O O
Base Analogs:
 Chemicals with structures similar to that of 4 4
N3 5 Br N3 5 CH₃
any of the four standard bases of DNA 5BU T
 DNA polymerases cannot distinguish these 2
6
2
6
O 1 O 1
analogs N N
 They may be incorporated into newly
synthesized DNA molecules

T TRANISITION
O OH A T C
5dBU A G
4 4
5dBU
N3 5 Br N3 5 Br A
5BU 5BU
2 2
6 6
O 1 O 1 5dBU
N N G

Keto Enol C
pairs with A mispair with G G

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3’ 5’ 3’ 5’
GAC G
GAC CTG TRANISITION
3’ 5’ 5’ 3’ C
GAC G A
3’ 5’ CBG 5dBU
GAC
5’ 3’ CBG 5’
CBG
3’ C T
3’ 5’ Incorporated error
5’ 3’
GGC
G
GAC Strand 3’ 5’ 5dBU
CTG seperation 3’ 5’
5’ 3’ 5’ 3’
CBG GGC
5dBU
3’ 5’ A
GAC 3’ 5’ 3’ 5’
CTG CTG GAC GGC
5’ 3’ 5’ 3’ CBG CCG
replication 5’ 3’ 5’ 3’ A
T

T.A C.G
Incorporated error
3’ 5’ 3’ 5’
2-amino purine (P) 3’ 5’ 3’ 5’ GTC GTC
GTC GTC CAG
3’ 5’
CPG 5’ 3’
 Base analog of adenine GTC
CAG
Strand 5’ 3’ CPG 5’ 3’
CPG
separation 3’ 5’
 Normally pairs with thymine 5’ 3’ GTC
5’ 3’
GCC
CAG 3’ 5’
 May mispair with cytosine 5’ 3’
5’
CAG
3’ 5’ 3’ 3’
 Causes a transition mutation replication
CPG GCC
5’

3’ 5’ 3’ 5’
GTC GCC
CAG CGG
5’ 3’ 5’ 3’

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T TRANISITION
A C TRANISITION
T C G
2AP C T
A G 2AP
T G A
2AP C
2AP
C
2AP T
2AP
C
G T
A

 Both base analogs produce transition
mutations
 Mutations by base analogs can be
reversed by treatment with the same
analog or different analog
Alkylating agents:
 Chemicals that donate alkyl groups e.g.
Ethylmethanesulfonate (EMS)
 It adds an ethyl group to guanine and produces 6-
ethylguanine, which pairs with thymine and leads to
CG:TA transitions
 Also adds an ethyl group to thymine to produce 4-
ethylthymine, which then pairs with guanine, leading
to a TA:CG transition
 Mutations produced by EMS can be reversed by
additional treatment with EMS.
 Mustard gas is another alkylating agent.

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Nitrous acid: causes deamination

Cytosine Uracil
C T
G A NH2
EMS o
EMS

T 4ET 4 4
6EG G N 3 5 N 3 5

HNo2
2 2
6 6
T C O 1 O 1
N N
A G H H
CYTOSINE URACIL

Adenine changes into Hypoxanthin which then pairs with

5’ 3’
C Cytosine
HNO2
G 5’ 3’ 5’ 3’
3’ 5’ 5’ 3’ U A
5’ 3’ U A T HNO2 5’ 3’
U 3’ 5’ 3’ 5’ 5’ 3’ H
G 5’ 3’ 3’ 5’ 5’ 3’ H C
3’ 5’ H
G U A 3’ 5’
3’ 5’ T 5’ 3’ 3’ 5’
3’ 5’
5’ 3’ 5’ 3’ 3’ 5’
T H C
3’ 5’
C U T
G A A 5’ 3’ 5’ 3’ 3’ 5’
3’ 5’ 3’ 5’ 5’ 3’ T H C
A C G
3’ 5’ 3’ 5’ 5’ 3’
C.G TA
A.T G.C

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Guanine changes into Xanthin which pairs with Cytosine. Nitrous acid
Xanthin can also pair with Thymine  Nitrous acid produces exclusively transition
5’ 3’ mutations
G  Both C.G T.A & T.A C.G transitions are
C HNO2 5’ 3’
3’ 5’ 5’ 3’ X produced
5’ 3’ X T
X 3’ 5’  Thus mutations can be reversed with the nitrous acid
C 5’ 3’ 3’ 5’ Hydroxyl amine
3’ 5’
C X T
3’ 5’  Specific base modifying mutagen which adds a
5’ 3’ 5’ 3’ 3’ 5’
hydroxyl group to cytosine producing hydroxylamine
G X T cytosine which pairs with adenine instead of guanine
C T A  This Leads to C.G T.A transitions
3’ 5’ 3’ 5’ 5’ 3’

G.C A.T  Acts only on cytosine thus can not revert the
mutation produced

Cytosine changes into hydroxylamine Cytosine which

pairs with Adenine instead of Guanine Oxidative reactions:
5’ 3’  Reactive forms of oxygen like superoxide
C
G
NH₂OH 5’ 3’ radicals, hydrogen peroxide and hydroxyl
3’ 5’ hC
5’ 3’
radicals produced in the course of normal
5’ 3’ hC A
hC 3’ 5’ aerobic metabolism or by radiation, ozone,
G
3’ 5’ 5’ 3’ 3’ 5’ peroxides, and certain drugs Cause damage to
G hC A
3’ 5’ DNA & induce mutations by chemical changes
5’ 3’ 5’ 3’ 3’ 5’  Oxidation converts guanine into 8-oxy-7,8-
C hC A
dihydrodeoxyguanine which mispairs with
G A T
3’ 5’ 3’ 5’ 5’ 3’ adenine leading to G.C T.A transversion
C.G T.A

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Intercalating agents
 Proflavin, acridine orange, ethidium bromide, and
dioxin
 They are about the same size as a nucleotide
 They produce mutations by sandwiching themselves
(intercalating) between adjacent bases in DNA
 They distort the three-dimensional structure of the
helix and cause single-nucleotide insertions and
deletions in replication
 These insertions and deletions frequently produce
frame-shift mutations

Radiations:
Ionizing radiations:
Mutagenesis
 In 1927, Herman Muller demonstrated that mutations
could be induced by X-rays.
 Mutagenesis (the creation or formation
of a mutation) can be used as a
 X-rays, gamma rays, and cosmic rays are all capable of
penetrating tissues and damaging DNA.
powerful genetic tool.
 They remove electrons from the atoms that they  By inducing mutations in specific ways
encounter, changing stable molecules into free and then observing the phenotype of
radicals and reactive ions which then alter the the organism the function of genes and
structures of bases and break phosphodiester bonds even individual nucleotides can be
in DNA. determined.
 Ionizing radiation also frequently results in double-
strand breaks in DNA.

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Some types of Mutagenesis Directed Mutagenesis

• Directed Mutagenesis
• Site-directed / Site-specific Mutagenesis
• Mismatched Mutagenesis
• A largely discredited hypothesis
proposing that organisms can
respond to environmental stresses
through directing mutations to
certain genes or areas of the
genome.

Image of Site-directed Mutagenesis

Site-directed Mutagenesis
• Where a specific site in a cloned DNA
needs to be altered in a precise,
pre-determined way

• Can be designed to create specific

nucleotide substitutions, deletions, and
so on

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Mismatched Mutagenesis
• Can create a desired point mutation at a
unique predetermined site within a
cloned DNA molecule

• At the intended mutation site it bears a

base that is complementary

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