Methods of Inducing Mutations and C L B Technique: Mutagens

Methods of inducing mutations and C l B technique
Mutagens
Mutations can be induced by anumber of agents; the agents capable of
inducing mutations are called mutagens. Mutagen is a natural or human-made
agent (physical or chemical) which can alter the structure or sequence of DNA. The
different mutagenic agents may be classified into the following two broad groups:
1) Physical mutagens
2) Chemical mutagens
Physical mutagens
The different types of radiations having mutagenic properties are known as
physical mutagens. The radiations may be a part of the electromagnetic spectrum
having shorter wavelength and higher energy than visible light (eg: uv rays, X rays,
gamma rays and cosmic rays) or may be particulate radiations produced by the
decay of radio isotopes.
Radiations
Radiation was the first mutagenic agent known; its effects on genes were
first reported in the 1920's. Radiation itself was discovered in 1890's: Roentgen
discovered X-rays in 1895, Becquerel discovered radioactivity in 1896, and Marie
and Pierre Curie discovered radioactive elements in 1898. These three discoveries
and others led to the birth of atomic physics and our understanding of
electromagnetic radiation.
Radiations are grouped into two classes depending on the kind of effects they
have on the atoms in their path:
1. ionizing and
2. non – ionizing radiations
Non – ionizing radiations
Ultraviolet rays are the only non ionizing radiation with mutagenic properties.
The wave length ranges from 100 – 3900 Ao And they are specifically absorbed by
puriines and pyrimidines present in DNA. The maximum absorption of UV rays by
DNA as well as by pyrimidines, particularly thymine occurs at the wavelength of
254 nm, which is also the most mutagenic wavelength of UV.
The mutagenic action of uv is the consequence of both its direct and indirect
effects on DNA. The direct effect of uv on DNA is of two types: formation of (1)
pyrimidine dimmers and pyrimidine hydrates.
Ionizing radiations
Ionizing radiations are so called because they cause ionization in the atoms
present in their path. There are two types of ionizing radiations: (1) particulate and
(2) non particulate radiations. Particulate radiations consist of high energy atomin
particles generated due to radioactive decay. The non particulate ionizing radiations
are represented by X rays and gamma rays which are high energy radiations
composed of photons.
The genetic effects of radiations may be (1) direct or (2) indirect. The direct
effect of radiations is produced due to ionizations directly in the DNA molecule,
while their indirect effect is produced through ionizations ini molecules other than
DNA and is believed to be mediated by free radical formation.
Sources of radiation
Natural sources of radiation produce so-called background radiation. These
include cosmic rays from the sun and outer space, radioactive elements in soil and
terrestrial products (wood, stone) and in the atmosphere (radon). One's exposure
due to background radiation varies with geographic location.
In addition, humans have created artificial sources of radiation which
contribute to our radiation exposure. Among these are medical testing (diagnostic
X-rays and other procedures), nuclear testing and power plants, and various other
products (TV's, smoke detectors, airport X-rays).
Chemical mutagens
The first report of mutagenic action of a chemical was in 1942 by Charlotte
Auerbach, who showed that nitrogen mustard (component of poisonous mustard
gas used in World Wars I and II) could cause mutations in cells. Since that time,
many other mutagenic chemicals have been identified and there is a huge industry
and government bureaucracy dedicated to finding them in food additives, industrial
wastes, etc. It is possible to distinguish chemical mutagens by their modes of
action; some of these cause mutations by mechanisms similar to those which arise
spontaneously while others are more like radiation in their effects.
1. Base analogs
These chemicals structurally resemble purines and pyrimidines and may be
incorporated into DNA in place of the normal bases during DNA replication:
• bromouracil (BU)--artificially created compound extensively used in
research. Resembles thymine because it has Br in the 5 position instead of
methyl group and has the same effect on its base pairing behavior as that of
–CH 3 in the same position and therefore 5 BU behaves like thymine and
usually pairs with adenine.
• aminopurine --adenine analog which can pair with T or (less well) with C;
causes A:T to G:C or G:C to A:T transitions. Base analogs cause transitions,
as do spontaneous tautomerization events.
2. Chemicals which alter structure and pairing properties of bases
There are many such mutagens; some well-known examples are:
• nitrous acid--formed by digestion of nitrites (preservatives) in foods. It
causes C to U, meC to T, and A to hypoxanthine deaminations. Hypoxanthine
in DNA pairs with C and causes transitions. Deamination by nitrous acid, like
spontaneous deamination, causes transitions.
• nitrosoguanidine, methyl methanesulfonate, ethyl methanesulfonate-
chemical mutagens that react with bases and add methyl or ethyl groups.
Depending on the affected atom, the alkylated base may then degrade to
yield a baseless site, which is mutagenic and recombinogenic, or mispair to
result in mutations upon DNA replication.
3. Intercalating agents
Acridine orange, proflavin, ethidium bromide (used in labs as dyes and
mutagens). All are flat, multiple ring molecules which interact with bases of DNA
and insert between them. This insertion causes a "stretching" of the DNA duplex
and the DNA polymerase is "fooled" into inserting an extra base opposite an
intercalated molecule. The result is that intercalating agents cause frameshifts.
4. Agents altering DNA structure
This is used as a "catch-all" category which includes a variety of different kinds of
agents. These may be:
• --large molecules which bind to bases in DNA and cause them to be
noncoding--we refer to these as "bulky" lesions (eg. NAAAF)
• --agents causing intra- and inter-strand crosslinks (eg. psoralens--found in
some vegetables and used in treatments of some skin conditions)
• --chemicals causing DNA strand breaks (eg. peroxides)
What these agents have in common is that they probably cause mutations not
directly but by induction of mutagenic repair processes.
Detection of mutation
The occurrence of mutational event at the gene level is detected by the

altercation it brings about in the phenotypic expression of one or more traits of the
concerned organism. Therefore the efficiency of detection of mutations will depend
largely on the availability of techniques for an easy and rapid scoring of the mutant
phenotypes in very large populations. Scoring of some types of mutations in certain
organisms is relatively easy. For example, mutations for antibiotic resistance in
bacteria are simply detected by plating the bacterial cells on a medium containing a
lethal concentration of the concerned antibiotic (selective medium); the colonies
that develop on such a medium will be produced by cells resistant to the antibiotic.
The medium lacking the antibiotic is called the non selective medium.
Number of colonies on the selective medium
Frequency of mutant cells (%) = --------------------------------------------------X 100
Number of colonies on the non selective medium
Detection of morphological mutations in eukaryotes requires examination of
each individual of the population for the mutant phenotype; this is not only tedious
requiring time, but is also a source of errors in the data. Therefore, elaborate
procedures for mutation detection have been developed in some eukaryotes . eg;
Drosophila, maize etc. These procedures employ specific markers to facilitate the
identification of chromosomes from the treated or irradiated individuals. Clearly
these techniques detect only germinal mutations. In drosophila, several specila
genetic stocks have been constructed for the detection of lethal and visible
mutations in X chromosomes and in autosomes; the two genetic stocks most
commonly used for mutation detection in X chromosome are (1) CIB and (2)
Attached X stocks.
CIB Technique
This method was invented by Muller and used for the unequivocal
demonstration of mutagenic action of X rays. In this method, females containing
one normal X-chromosome and another X-chromosome (CIB) containing extra 3
genes are used for the analysis. Out of the 3 extra genes, one gene suppresses
crossover (c), the other is a recessive lethal (L) in heterozygous condition, and the
last gene is semidominant marker, Bar (B) gene.
Females containing CIB chromosome are called as CIB stock drosophila. The
normal males are exposed to mutagenic source for a fixed period and then mated
to the CIB stock drosophila. Males containing CIB chromosome will die due to the
effect of lethal genes, whereas norm ill males and females both normal and with
CIB will survive.
Females with CIB chromosomes and identified by barred phenotype are
selected and crossed to normal males. In this next generation 50% of males (which
have received the CIB gene) will die.
If mutation has occurred in normal X chromosome then even the normal
male (without CIB gene) will die. If no mutation has occurred all the other 50% of
males will survive. The frequency of lethal mutations can be accurately scored in
large samples. This technique is simple, rapid and there is little chance of an error
in scoring. However, it is suitable for the scoring of sex linked recessive lethal only.
The attached X chromosome technique
This technique is based on attached – X females (X- XY) and is designed to
study visible sex linked mutations in Drosophila. Mutagen treated males are mated
with attached X females. The X- XX (super female) and YY progeny produced from
such crosses do not survive; only X- XY (female) and XY (male) progeny are
recovered. All male (XY) progeny receive their Y chromosomes from their attached
X female parent, while their X chromosomes is contributed by their mutagen
treated male parent. If a visible mutation was induced in the X chromosomes of any
sperm produced by the mutagen treated male, it will be expressed in the male
progeny. Therefore all the male progeny obtained from the cross are scored for
visible mutations. The frequency of a visible mutation is expressed as the ratio
between the number of progeny males showing a mutation and the total number of
males in the progeny.
Detection of mutations in plants
Techniques for the detection of mutations are relatively poorly developed for
plant species. The following two approaches are generally adopted for this purpose.
(1) In some species, eg; maize, strains homozygous for several recessive genes as
well as for dominant alleles of these genes are available. In such cases seeds or
plants of a strain homozygous for several dominant genes are treated with a
mutagen. The plants (M1 generation) are crossed with a strain having the recessive
forms of the traits governed by the concerned genes are counted and the frequency
of mutation for a gene is estimated as follows:
Number of plants having the recessive
form of trait governed by the gene
Mutation frequency (%) for a gene =-------------------------------------------------- X 100
Total number of plants in the progeny
The plants showing the recessive form of such a trait will receive one
recessive allele from the tester parent with the recessive traits, while the other
recessive allele would have been produced due to mutation in the mutagen treated
parent.

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