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1. MOLECULAR BASIS OF
MUTATION
Prof. A. B. Das
Dept. of Botany
Utkal University, Vini Vihar
Bhubaneswar -751004
E-mail: abdas.uubot@gmail.com

2. What is mutation?
Mutation is the permanent hereditary changes of
genetic make up of an individual which is caused by
structural and compositional changes of genes.
Mutation is of gene mutation or point mutations
and chromosomal mutation

3. Hugo DeVries propose the
‘Mutation Theory’
&
the term mutation during his
rediscovery of Mendel's laws of
Inheritance.

4. Mutations are of two type
i) Somatic mutation
ii) Germinal mutation.
Somatic mutation – occur in the somatic cells
that leads to phenotypic changes of the organs
or structures.
Somatic mutations are nonheritable and thus not
carried forward to the next generation.
In plants the mutation can be passed on to the
progeny by vegetative reproduction like budding
and grafting like in cacti.

5. Mutations are of two type
i) Somatic mutation
ii) Germinal mutation.
Germinal mutations - occur in the
germ cells or gametes. These are
heritable and are expressed in the
next generation.

6. Mutable genes - Each gene is a potential site
for mutation, but some genes mutate more
frequently than others and these genes are
called mutable genes.
These are mainly structural genes which on
mutation produce a changed phenotype.
For example, somatic mutations giving rise to
leaf variegation are due to mutable genes.
Example: R-gene in maize (that controls
anthocyanin production) mutates more
frequently at the rate of 01 out of 1,00,000
gamets (discovered by Emerson).

7. Mutator genes – the gene which influence the rate of
mutation is called mutator gene.
Ex: Dt gene influences gene a1 (of the locus a1 - a1
for green colour) to mutate to a1 so that the green
leaves of maize become variegated (streaked with
purple) and seeds are speckled with purple dots
(Rhoades,1938).
Haldane estimated that a human gene could remain
without any mutation for 2,500,000 yrs.
As a result Mc Kausick could trace out 1,364 heritable
diseases in man.
Till date 2,811 heritable diseases have been
discovered.

8. Reverse mutation: Most mutant
events consist of a change from
normal or wild type to a new
genotype (recessive or dominant).
Such mutation events are called
forward mutations, while if the same
mutant genotype changes back to the
wild type, it is back or reverse
mutation.

9. Paramutations: In heterozygotes, alleles are
unaffected by the presence of other alleles. The locus
R in corn bears a number of alleles, concerned with
the formation of anthocyanin pigment.
Normally Rr gene in single dosage produces dark
mottling and two or three produce full colour.
But when Rr alleles extracted from heterozygous
condition with Rsf or Rmb alleles from other localities
produces considerable lighter pigment than before.
This change in Rr is heritable and Brink has
described this effect as paramutation and Rsf and
Rmb are paramutagenic.

10. Detection of mutations
1.ClB method in Drosophila (Muller ClB method of induced mutation)
C = represent a long inversion of
chromosomes that prevents
crossing over within the inverted
segments. Gene l and B are
located in this segment.
l = recessive lethal mutant present
on X chromosome. Female
Drosophila homozygous for gene
l/l and male hemizygous l/Y are
non viable.
B= Sex-linked dominant mutant
producing bar-shaped eyes.
Parents – ClB heterozygous
female X irradiated wild type male.
Mutagen - X-ray
Result- Bar eyed female but no
males. X2-progeny consists of only
females and 50% surviving male if
mutation is not lethal

11. 2. Muller – 5 method in Drosophila for detection for
lethals in X-chromosomes.
Gene for apricot eye and bar eye are used as
genetic markers.
Gene Wa – Apricot eye (a recessive mutant of
the red colour)
Gene B – Bar eye character (a dominant
mutant of the normal eye)
Locations of genes: These genes are located
on the X-chromosome which has a long
inversion in the area of these two genes. The
inversion eliminates crossing over in this area.
Parents: Homozygous apricot bar female
(WaB) and wild male (irradiated to induced
mutations with X ray).
Result: The r2 generation 50% male will
receive X-chromosomes with WaB genes and
other 50% with irradiated X chromosomes.
50% R2 male will not survive if mutation is
lethal. The 50% male with WaB will be apricot
bar i.e. R2 offspring female and male are
produced in 2:1 ratio. Of the females 50% are
bar –eyed and 50% normal. Of the male 50%
will survive and are apricot-bar while 50% do
not survive because the irradiated X-
chromosomes has lethal mutation.

12. Molecular basis of gene mutations
The process of replication of DNA
sometimes shows inaccuracy which
may occur within the DNA due to
some internal or external, natural or
artificial factors.
Such inaccuracy at any one of
these levels introduces changes in
the arrangement of nucleotides in a
polynucleotide chain of a DNA
molecule.

13. Molecular basis of gene mutations
The smallest change may involve the addition,
deletion or substitution of a single nucleotide
pair in the DNA molecule, which may change
the reading of genetic code and ultimately may
be manifested into an altered phenotype.
If mutation produced, codes for one of the
terminator or nonsense codon (UAA, UAG or
UGA), the reading of RNA would stop at that
point.
This would prevent a complete polypeptide
chain from being formed and could result in a
lethal mutation

14. Gene Mutation
Substitution Mutations
1.Deletion Mutations
2. Insertion Mutations
1.Transition
2. Transversion
Frame-Shift Mutations

15. 1. Transition
Replacement of a purine or pyrimidine
by another purine or pyrimidine in a
polynucleotide chain, caused by:
(a)Tautomerisation
(a) Ionization
(c) Base analoges
(d) Deamination

16. 2. Transversion
Replacement of purine by a pyrimidine
or vice versa is caused by :
(a) Alkylating agents
(b) Depurination

17. Since these mutations include very
limited segment of DNA, these are
called point mutations. They are
of following types :
1.Substitution mutations
2. Frame-shift mutations

18. I. Substitution mutations
In a substitution mutation, a nitrogenous base of a triplet codon of DNA is
replaced by another nitrogenous base or some derivative of the nitrogen
base, changing the codon. The altered codon may code for a different
amino acid and may result in the formation of a protein molecule with single
amino acid substitution, whose effect may be seen in altered phenotype.
The substitution mutations may be of the following two types :
1. Transitions
Transitions are changes that involve replacement of one purine in a
polynucleotide chain by another purine and correspondingly in the
complementary chain the replacement of one pyrimidine by another
pyrimidine. This is called copy error mutation. They are of following four
types.

19. a.Tautomerisation: In a normal molecule of DNA, the purine-
adenine (A) is linked to the pyrimidine-thymine (T) by two bonds, while
the purine-guanine (G) is linked to pyrimidine-cytosine (C) by three
bonds.
However, all these four nitrogenous exist in alternate states. These states
are called tautomers and are formed by the 'rearrangement in the
distribution of hydrogen atoms (tautomeric shifts).
Due to tautomerisation the amino (- NH2) group of cytosine and
adenine is converted into imino (-NH) group and likewise keto (C=O) of
thymine and guanine is converted to enol group (-OH).
In its tautomeric state, a nitrogenous base can not pair to its normal partner.
Rather a tautomeric adenine, pairs with the normal cytosine and
tautomeric guanine with thymine.
Similarly, tautomeric thymine pairs with normal guanine and cytosine with
adenine.
Such pairs of nitrogenous bases are known as 'forbidden base pairs' or
'unusual base pairs'.

20. a.Tautomerisation:
The rare bases can introduce mutations during DNA replication.
If for example, adenine in the chain of a parent DNA is in
rare state, the complementary new chain formed from this
chain will contain cytosine.
At the time of next replication this cytosine would pair with
guanine.
This will produce a substitution of A = T base pair by G ≡ C pair.
Similarly, a substitution of G ≡ C by A = T pair can be
produced if cytosine is in tautomeric state.
This situation is know as copy error and is not stable because
at the next replication the tautomaric base returns to
common state and pair with thymine.
Common bases of DNA and their tautomers. Keto (C=O)
form on nitrogenous base changes to enol (-OH) and
amino (-NH2) form to imino (-NH) form.

23. MUTATION
Standard base-pairing arrangements
Anomalous base-pairing arrangements

24. b. Ionization:
Transition may also be introduced by ionization of a base at the time of DNA
replication. It involves the loss of the hydrogen from number 1 nitrogen of
nitrogenous base.
Example: Ionized T pair with normal G and ionized G pair with normal T .
Forbidden base pairs of thymine and guanine resulting from the ionization of
No. 1 nitrogen.

25. Two forms of 5-BU
C. Base analogues
Some of the chemical compounds have molecular structure similar to the
nitrogenous bases present in DNA necleotides. These are called base analogues.
These are usually deravatives of nitrogenous bases of DNA and occur as natural as
well as artificial base analogues.
Eg. Natural analogues - 5-methyl cytosine, 5-hydroxymethyl cytosine (in E. coli), 5-
hydroxymethyl uracil.
Artificial base analogues: 5-bromouracil (5-BU), 5-iodo uracil (5-IU)

26. Base analogues – bromouracil is base analogue of thymine
Aminopuration
Aminopurine a chemical artificial base analogue of adenine. It
can substitute adenine as well as can pair with cytosine.

27. D. Deamination:
Some chemical substances like nitrous acid (HNO2),
hydroxylamine diethyl sulphate (DES), ethylmethane
sulphonate (EMS) ethyl ethane sulphonate (EES)
changes base sequences in DNA by a series of
chemical steps. Nitrous acid and hydroxylamine
causes deamination of nitrogenous bases by
replacing amino group (-NH2) by hydroxyl group
(-OH).

28. 2. Transversions:
Certain chemicals like EMS (ethyl methane sulphate) , MMS
(methyl methane sulphate) induce substitutions by two ways:
A. Transition: by substituting a purine for purine or
pyrimidine for a pyrimidine.
B. Transversion: by substituting a purine for a pyrimidine
or pyrimidine for a purine.

29. The cause of transversions is that these chemicals alkylate the
purine nitrogenous base in the nitrogen at the 7th position in the
guanine and adenine and finally lead to its separation from the
DNA strand. This is also know as depurination

30. Mechanism of depurination caused by alkylating agent. Substitution of
A=T by C≡G as a result of transversion.
The removal of a purine from strand of DNA leaves a gap. At the time of
replication any of the four base can possibly get inserted at this place in the
complementary strand. If the nucleotide contains a pyrimidine it is a
transition and if purine then it is transversion. In the next cycle of DNA
synthesis a DNA molecule is formed which contains complete transversion
which is nonreversible.

31. 2. Frame Shift Mutations
The mutations caused by the addition or deletion of nitrogenous bases in the
DNA or mRNA are known as frame-shift mutations, because these shift the
reading frame of codons from the site of change onward.
One deletion may be neutralised by one addition or vice-versa, provided they
occur at the same place or very close to each other.
Frame-shift mutation and the changes in the reading of genetic code

32. Types of Frame-shift Mutations
The frame-shift mutations are of two types :
1. Deletion mutations : These mutations are caused due to the
loss or deletion of one or more nucleotides.
2. Insertion mutations : These mutations are caused by the
addition of one or more extra nucleotides in a DNA molecule at
one or more places.

33. Mechanism of Origin of Frame-shift Mutation
Acridine dyes have been found to cause deletion or
insertion of a single base pair.
Acridines like 5-aminoacridine and proflavin
become intercalated between two adjacent purines
and thus increase the distance between then from
3.4 Å to 6.8 Å.
At the time of DNA replication, either a nitrogenous
base pair is introduced in the gap or a nitrogenous
base pair is lost.
Frame-shift mutations caused by the deletions are
also introduced by the removal of ethylated bases.

34. Biological Significance of Point Mutations
All point mutations are not lethal.
Transition and transversions are relatively benign,
because these cause replacement of only one amino acid in
the peptide chain coded, which might not produce any
significant change.
Such mutations are known as silent mutations.
However, frame-shift (insertion and deletion) mutations
cause all the DNA beyond the point of mutation to be
misread.
Such mutations are often lethal.
If the deleted segment includes nucleotides in three or
multiples of three, there is no frame-shift error beyond the
site of mutation but protein produced may be defective.

35. Effect of some chemical mutagens