Mutation-characteristics of mutation – micro and

macro mutation – ClB technique – molecular basis

of mutation – Transition and transversion; major
physical and chemical mutagens
Mutations
Heritable changes that occur in genetic material are known as mutations. The genetic materials
of living organisms (either DNA or RNA) are always subjected to changes. Cells have highly
developed mechanisms to rectify these errors in a process we call as ‘proofreading’. Nevertheless,
sometimes these errors that escaped proofreading are inherited from the parents, leading to changes
in the offspring. Such individuals exhibiting a modified phenotype due to mutations are known as
‘mutants’. One thing to remember is that, even though the structural and numerical chromosomal
changes are also a type mutations, this term is now commonly used to refer to the DNA base
changes. Chromosomal changes are simply referred as chromosomal aberrations.

Characteristics of mutations
Nature of Change
Mutations are more or less permanent and causes heritable changes in the phenotype of an
individual. Such changes occur due to alteration in number, kind or sequence of nucleotides of
genetic material, i.e., DNA in most of the cases.

Frequency
In nature, spontaneous mutations occur at a very low frequency. However, the mutation rate can be
enhanced many fold by the use of physical and chemical mutagens. The frequency of mutation for a
gene is calculated as follows:
Frequency of gene mutation = M / M + N
where, M = number of individuals expressing mutation for a gene, and N = number of normal
individuals in a population.

Mutation Rate
Mutation rate varies from gene to gene. Some genes exhibit high mutation rate than others. Such
genes are known as mutable genes, e.g., white eye in Drosophila.
In some genomes, some genes enhance the natural mutation rate of other genes. Such genes are
termed as mutator genes. The example of mutator gene is dotted gene in maize.
In some cases, some genes decrease the frequency of spontaneous mutations of other genes in the
same genome, which are referred to as anti-mutator genes. Such gene has been reported in bacteria
and bacteriophages.
Direction of Change
Mutations usually occur from dominant to recessive allele or wild type to mutant allele. However,
reverse mutations are also known, e.g., notch wing and bar eye in Drosophila.

Effects
Mutations are generally harmful to the organism. In other words, most of the mutations have
deleterious effects. Only about 0.1% of the induced mutations are useful in crop improvement. In
majority of cases, mutant alleles have pleiotropic effects. Mutations give rise to multiple alleles of a
gene.

Site of Mutation
The sub-division of a gene where mutation occurs is termed as ‘muton’. An average gene contains
500 to 1000 mutational sites. Within a gene some sites are highly mutable than others. These are
generally referred to as hot spots. Mutations may occur in any tissue of an organism, i.e., somatic or
gametic but only the gametic mutations are passed on to the offspring.

Type of Event
Mutations are random events. They may occur in any gene (nuclear or cytoplasmic), in any cell
(somatic or reproductive) and at any stage of development of an individual.

Recurrence
The same type of mutation may occur repeatedly or again and again in different individuals of the
same population. Thus, mutations are of recurrent nature.

Micromutations
Mutations that have very little or no effect on a phenotype are termed as micromutations. Single
base changes that did not change the amino acid sequence (silent mutations), mutations in intron
regions or mutations in quantitative characters tend to have no or very little phenotypic effects.
These mutations are usually limited to a localized region of a DNA, that is a gene.

Macromutations
Mutations that have large phenotypic effects are termed as macromutations. Macromutations are
caused by large scale deletions and insertions, inversions or other types of mutations. They cause
serious phenotypic changes due to alteration of amino acids, mis-sense or non-sense codons etc.
Mutations that occur in qualitative traits usually have large phenotypic effects.

Transition
We know that the DNA is made of two kinds of nitrogenous bases: Purines and Pyrimidines.
Adenine and guanine make up the purines, and thymines and cytosines make up the pyrimidines.
In a DNA, during mutation, if one purine is replaced by an another purine (or) if one pyrimidine is
replaced by an another pyrimidine, it is known as transition mutation.

Transversion
If a purine is replaced by a pyramidine and vice versa, it is known as transversion mutations.

...GAATTCTTCAAATTGCCG… ...GAATTCTTCAAATTGCCG…

...GAATTCTTCAGATCGCCG… ...GAATTCTTCACATAGCCG…
Transitions Transversions

Types of mutations
Somatic mutations
Mutations that occur in somatic cells are known as somatic mutations. Somatic mutations are not
transmitted through the gametes to the progeny.

Germinal mutations
Mutations that occur in germ-line cells are known as germinal mutations. Germinal mutations are
readily transmitted to the progenies.

Substitutions
Here, one base of a DNA is replaced by an another base. There is no change in the length of the
DNA. This substitution causes change in the triplet codon which leads to change in an amino acid.
Nevertheless substitution mutations can be lethal as in the case of sickle cell disease. Here, the
condition of sickle cell disease is caused by the substitution of GAG to GTG. This results in the
change of amino acid from Glu to Val.

Insertions
Addition of one or more base pairs to the nucleic acids. Here, the length of the DNA increases.
Example: Huntington’s disease in humans are caused by insertion mutations.

Deletions
Loss of one to many base pairs of DNA is known as deletions. Deletions cause reduction in the
length of DNA. Example: in humans, deletion in chromosome 22 known as 22q11.2 cause a
syndrome resulting in autoimmune disorders.

Silent mutations
We know that the DNA is read into proteins by triplet codons. Even though 64 triplet codons are
possible in a DNA, only 20 amino acids are found. This shows that more than one codon must be
coding for a same amino acid. Mutations in which an altered base does not result in a new amino
acid is known as silent mutations. E.g. UUA → UUC both results in the same amino acid Leucine.

Mis-sense mutations
Mutations that result in a modified amino acid is known as mis-sense mutations. E.g. UUC → UUA
will change the amino acid Phenylalanine (Phe) to Leucine (Leu).

Non-sense mutations
Sometimes, a single base change in the DNA may result in the appearance of Stop codon
(termination) prematurely. It is known as non-sense mutations. E.g. UAC → UAA will result in the
amino acid Tyrosine (Tyr) to termination (STOP). Usually protein synthesis stops at this point.

Frameshift mutations
In case of indel mutations (insertions and deletions), the codon reading order of genes is changed.
This may result in modified amino acid sequences, known as frameshift mutations.
E.g. AUG UUC CAA GCC GGG → AUG UUC CGC CGG
Met Phe Gln Aln Gly Met Phe Arg Arg

Induced mutations
In 1927, Hermann J. Muller discovered and showed that mutations can be artificially induced in
genomes of Drosophila by subjecting them to X-ray treatment. This is one of the greatest
discoveries of all time and for this, he received the Nobel prize in 1946. Muller demonstrated this
effect by a simple technique known as ClB technique.

ClB technique (‘C’rossover suppressor-recessive ‘l’ethal-‘B’ar

eye)
Muller developed a special group of female Drosophila flies that were having a normal X
chromosome and an abnormal X chromosome with genes known as ClB: ‘C’-crossover suppressor;
‘l’-recessive lethals and ‘B’-bar eye.
(1) Crossover suppression prevented recombination between the two X chromosomes, hence all the
offspring would be of parental types.
(2) The recessive lethal gene killed all hemizygous male offspring and homozygous female
offspring.
(3) The bar eyed gene produced flies with bar-shaped compound eyes instead of the normal
globular shape.
The basic idea is that either the female flies carrying homozygous ClB chromosomes or the male
flies with hemizygous ClB chromosomes will die due to the lethality. And, the heterozygous female
flies can be identified by the presence of Bar eyes.
The demonstration:
1. To study if X rays cause mutations, Muller irradiated the reproductive cells of a population
of male Drosophila flies with X rays. The X-rays are now expected to be mutated.
2. He crossed these irradiated males to ClB heterozygote females (one ClB X chromosome and
one normal X chromosome; denoted as ClB/+).
3. The result of the cross was like this: (i) Females with one normal X chromosome from
mother and one irradiated X chromosome from father; (ii) females with one ClB X
chromosome from mother and one irradiated X chromosome from father—they have bar
eyes; (iii) Males with one normal X chromosome from mother; (iv) males with one ClB X
chromosome from mothers—these would die due to the expression of lethality in
hemizygous condition.
4. Muller chose only the ‘bar-eyed’ daughters and crossed them individually with normal
males. Based on the results, he was able to confirm the mutagenic ability of the X-rays
◦ If the X-rays cause lethal mutations, the population will be of only females. Because, all
the males with either a ClB chromosome or a mutated X chromosome will die.
◦ If the X-rays do not cause lethal mutations, the population will have females and males
in 2:1 ratio. Because, only the males receiving a ClB chromosome would die.
5. Muller observed 150 fold increase in death of male flies which showed that X-rays are
mutagenic.

Outcome:
• This is a simple technique which helps in identification of X-linked lethal mutations
• The ability of X-rays to cause mutations are proved from this experiment
Types of mutagens
Agents that can cause mutations in living organisms are termed as mutagens. Apart from being
used in genetic studies, mutagens are important in plant breeding and crop improvement. Mutagens
are of different types, broadly classified into physical and chemical mutagens.

Physical mutagens
Various types of radiations are known as physical mutagens. These include X-rays, gamma rays,
alpha particles, beta particles, fast and thermal (slow) neutrons and ultraviolet rays. Physical
mutagens are further classified into (i) ionising and, (ii) non-ionising mutagens. All radiations listed
here, except UV rays are ionising radiations.
X-rays
Roentgen discovered the X-rays in 1895 and was first used to induce mutations in Drosophila by
Muller in 1927; in crop plants it was first used by Stadler during 1928. Its wavelength ranges from
10-11 to 10-7. X-rays produce mutations by forming ions and free radicals in living cells. They can
cause all types of mutations viz., addition, deletions, inversions, transpositions, transversions and
transitions.
Gamma rays
Gamma rays have shorter wavelength and higher penetration ability than X-rays. In crop
improvement programmes gamma rays are often the most used. Cobalt 60 (Co60) is the commonly
used source of gamma rays. Gamma rays ionise the atoms in living tissues and as a results large
scale deletions and other types of mutations occur.
Alpha particles
Alpha particles are double positive charged (contains two protons and two neutrons) and ionises and
excites the atoms in living cells. Alpha particles are emitted during the decay of isotopes of heavier
elements. They cause large chromosomal mutations.
Beta particles
Beta particles are negatively charged and ionise and excites the atoms similar to alpha particles.
They are released during the decay of heavier elements. Beta particles cause both chromosomal and
gene mutations.
Fast and thermal (slow) neutrons
Neutrons are densely ionising and have high penetrating ability in living tissues. Fast and thermal
neutrons can be created in a controlled manner in atomic reactors or cyclotrons. The velocity of
neutrons can be reduced using heavy water or graphite to produce slow or thermal neutrons.
Neutrons are effectively used to induce mutations in asexually reproducing crops. They cause large
chromosomal breaks and deletions.
Ultraviolet rays
UV rays are non-ionising and have low penetration ability. UV is predominant in solar radiation, but
artificially produced from mercury vapour lamps. UV rays can cause free radicals or even
chromosomal breakage. They interact with pyrimidines of DNA to produce pyrimidine dimers like
TT, CC, UU or CT.

Chemical mutagens
Some chemicals are known to cause serious changes in the genetic material of living organisms.
These are routinely used to induce mutations in genetic research, agriculture and medical research.
The major difference between physical and chemical mutagens is that the chemical mutagens cause
more of base pair mutations (single nucleotide changes), whereas the physical mutagens cause large
chromosomal mutations.
The chemical mutagens can be divided into four groups, viz.,:
(a) Alkylating agents,
(b) Base analogues,
(c) Acridine dyes, and
(d) Others
Alkylating Agents:
As the name suggests, these chemicals add an alkyl group (either an ethyl or a methyl group) at
various positions in the DNA which results in transitions and transversions. This is the most
powerful group of mutagens.
Some of the important alkylating agents are ethyl methane sulphonate (EMS), methyl methane
sulphonate (MMS), ethylene imines (EI), sulphur mustard and nitrogen mustard. Out of these,
the first three are in common use. Since the effect of alkylating agents resembles those of ionizing
radiations, they are also known as radiomimetic chemicals.
Base Analogues
Base analogues refer to chemical compounds which are structurally very similar to the DNA bases,
i.e. A, T, G & C. For example, the two most commonly used base analogs—5 bromo uracil (5BU)
and 2 amino purine (2AP)—resemble thymine and adenine respectively. During DNA replication,
such chemicals can be sometimes are incorporated in DNA in place of normal bases, causing
mutation by wrong base pairing. An incorrect base pairing results in transitions or transversions
after DNA replication.
Thymine has a CH3 group at the C5 position whereas 5 bromo uracil has bromine at this position.
This simple structural change results in shifting of hydrogen atoms from one position to another
position known as ‘tautomeric shifts’. This tautomeric shift (or tautomerization) can occur in any
of the four bases. The base which is produced as a result of tautomerization is known as tautomeric
form or tautomer. Tautomerization converts the amino group (-NH 2 ) of cytosine and adenine into
imino group (-NH). Similarly, stable keto group (C=O) of thymine and guanine is converted into
less stable enol group (-OH). This results in mispairing which leads to AT→ GC and GC → AT
transitions.
Acridine Dyes
Acridine dyes are very effective mutagens. Acridine dyes include, pro-flavin, acridine orange,
acridine yellow, acriflavin and ethidium bromide. Out of these, pro-flavin and acriflavin are in
common use for induction of mutation. Acridine dyes get inserted between two base pairs of DNA
and lead to addition or deletion of single or few base pairs when DNA replicates. Thus, they cause
frameshift mutations and for this reason acridine dyes are also known as frameshift mutagens.
Proflavin is generally used for induction of mutation in bacteriophages and acriflavin in bacteria
and higher organisms. Ethidium bromide is commonly used as a DNA staining agent during gel
electrophoresis.
Other Mutagens
Other important chemical mutagens are nitrous acid and hydroxy amine. Nitrous acid reacts with C6
amino groups of cytosine and adenine. It replaces the amino group with oxygen (+ to – H bond). As
a result, cytosine acts like thymine and adenine like guanine.
Thus, transversions from GC → AT and AT → GC are induced. Hydroxylamine is a very useful
mutagen because it appears to be very specific and produces only one kind of change, namely, the
GC → AT transition. All the chemical mutagens except base analogues are known as DNA
modifiers.