A Mutation occurs when a DNA gene is damaged or changed in such a way as to alter the genetic message carried by that gene.
Characteristics of Mutations:
Mutations have several characteristic features.Some of the important characteristics of mutations are briefly presented below:
i. Nature of Change:
Mutations are more or less permanent and 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.
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.
iii. 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.
iv. 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.
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.
vi. Site of Mutation:
Muton which is a sub-division of gene is the site of mutation. 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.
vii. 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.
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.
Classification of Mutations:
Mutations can be classified in various ways. A brief classification of mutations on the basis of:
(6) Character, and
(7) Visibility is presented
Types of Mutants:
The product of a mutation is known as mutant. It may be a genotype or an individual or a cell or a polypeptide.
There are four main classes of identifiable mutants, viz:
(iii) Conditional and
These are briefly described below:
Morphological mutants refer to change in form, i.e., shape, size and colour. Albino spores in Neurospora, curly wings in Drosophila, dwarf peas, short legged sheep are some examples of morphological mutants.
In this class, the new allele is recognized by its mortal or lethal effect on the organism. When the mutant allele is lethal all individuals carrying such allele will die; but when it is semi-lethal or sub-vital some of the individuals will survive.
iii. Conditional Lethal:
Some alleles produce a mutant phenotype under specific environmental conditions. Such mutants are called restrictive mutants. Under other conditions they produce normal phenotype and are called permissive. Such mutants can be grown under permissive conditions and then be shifted to restrictive conditions for evaluation.
iv. Biochemical Mutant:
Some mutants are identified by the loss of a biochemical function of the cell. The cell can assume normal function, if the medium is supplemented with appropriate nutrients. For example, adenine auxotroph’s can be grown only if adenine is supplied, whereas wild type does not require adenine supplement.
Agents of Mutations:
Mutagens refer to physical or chemical agents which greatly enhance the frequency of mutations. Various radiations and chemicals are used as mutagens. Radiations come under physical mutagens. A brief description of various physical and chemical mutagens is presented below:
Physical mutagens include various types of radiations, viz. X-rays, gamma rays, alpha particles, beta particles, fast and thermal (slow) neutrons and ultra violet rays
X-rays were first discovered by Roentgen in 1895. The wavelengths of X-rays vary from 10-11 to 10-7. They are sparsely ionizing and highly penetrating. They are generated in X-rays machines. X-rays can break chromosomes and produce all types of mutations in nucleotides, viz., addition, deletion, inversion, transposition, transitions and trans-versions.
These changes are brought out by adding oxygen to deoxyribose, removing amino or hydroxyl group and forming peroxides. X-rays were first used by Muller in 1927 for induction of mutations in Drosophila.
In plants, Stadler in 1928 first used X-rays for induction of mutations in barley. Now X-rays are commonly used for induction of mutations in various crop plants. X-rays induce mutations by forming free radicals and ions.
ii. Gamma Rays:
Gamma rays are identical to X-rays in most of the physical properties and biological effects. But gamma rays have shorter wave length than X-rays and are more penetrating than X-rays. They are generated from radioactive decay of some elements like 14C, 60C, radium etc.
Of these, cobalt 60 is commonly used for the production of Gamma rays. Gamma rays cause chromosomal and gene mutations like X-rays by ejecting electrons from the atoms of tissues through which they pass. Now a days, gamma rays are also widely used for induction of mutations in various crop plants.
iii. Alpha Particles:
Alpha rays are composed of alpha particles. They are made of two protons and two neutrons and thus have double positive charge. They are densely ionizing, but lesser penetrating than beta rays and neutrons. Alpha particles are emitted by the isotopes of heavier elements.
They have positive charge and hence they are slowed down by negative charge of tissues resulting in low penetrating power. Alpha particles lead to both ionization and excitation resulting in chromosomal mutations.
iv. Beta Particles:
Beta rays are composed of beta particles. They are sparsely ionizing but more penetrating than alpha rays. Beta particles are generated from radioactive decay of heavier elements such as 3H, 32P, 35S etc. They are negatively charged, therefore, their action is reduced by positive charge of tissues. Beta particles also act by way of ionization and excitation like alpha particles and result in both chromosomal and gene mutations.
v. Fast and Thermal Neutrons:
These are densely ionizing and highly penetrating particles. Since they are electrically neutral particles, their action is not slowed down by charged (negative or positive) particles of tissues. They are generated from radioactive decay of heavier elements in atomic reactors or cyclotrons. Because of high velocity, these particles are called as fast neutrons.
Their velocity can be reduced by the use of graphite or heavy water to produce slow neutrons or thermal neutrons. Fast and thermal neutrons result in both chromosomal breakage and gene mutation. Since they are heavy particles, they move in straight line. Fast and thermal neutrons are effectively used for induction of mutations especially in asexually reproducing crop species.
vi. Ultraviolet Rays:
UV rays are non-ionizing radiations, which are produced from mercury vapour lamps or tubes. They are also present in solar radiation. UV rays can penetrate one or two cell layers. Because of low penetrating capacity, they are commonly used for radiation of micro-organisms like bacteria and viruses.
In higher organisms, their use is generally limited to irradiation of pollen in plants and eggs in Drosophila UV rays can also break chromosomes. They have two main chemical effects on pyrimidine’s.
The first effect is the addition of a water molecule which weakens the H bonding with its purine complement and permits localized separation of DNA strands. The second effect is to join pyrimidines to make a pyrimidine dimer.
This dimerization can produce TT, CC, UU and mixed pyrimidine dimers like CT. Dimerization interferes with DNA and RNA synthesis. Inter-strand dimers cross link nucleic acid chains, inhibiting strand separation and distribution.
There is a long list of chemicals which are used as mutagens. Detailed treatment of such chemicals is beyond the scope of this discussion.
The chemical mutagens can be divided into four groups, viz:
(a) Alkylating agents,
(b) Base analogues,
(c) Acridine dyes, and
A brief description of some commonly used chemicals of these groups is presented below.
a. Alkylating Agents:
This is the most powerful group of mutagens. They induce mutations especially transitions and transversions by adding an alkyl group (either ethyl or methyl) at various positions in DNA. Alkylation produces mutation by changing hydrogen bonding in various ways.
The alkylating agents include ethyl methane sulphonate (EMS), methyl methane sulphonate (MMS), ethylene imines (EI), sulphur mustard, nitrogen mustard, etc.
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. Alkylating agents can cause various large and small deformations of base structure resulting in base pair transitions and transversions.
Transversions can occur either because a purine has been so reduced in size that it can accept another purine for its complement, or because a pyrimidine has been so increased in size that it can accept another pyrimidine for its complement. In both cases, diameter of the mutant base pair is close to that of a normal base pair.
b. Base Analogues:
Base analogues refer to chemical compounds which are very similar to DNA bases. Such chemicals sometimes are incorporated in DNA in place of normal base during replication. Thus, they can cause mutation by wrong base pairing. An incorrect base pairing results in transitions or transversions after DNA replication. The most commonly used base analogues are 5 bromo uracil (5BU) and 2 amino purine (2AP).
5 bromo uracil is similar to thymine, but it has bromine at the C5 position, whereas thymine has CH3 group at C5 position. The presence of bromine in 5BU enhances its tautomeric shift from keto form to the enol form. The keto form is a usual and more stable form, while enol form is a rare and less stable or short lived form. Tautomeric change takes place in all the four DNA bases, but at a very low frequency.
The change or shift of hydrogen atoms from one position to another either in a purine or in a pyrimidine base is known as tautomeric shift and such process is known as tautomerization.
The base which is produced as a result of tautomerization is known as tautomeric form or tautomer. As a result of tautomerization, the amino group (-NH2) of cytosine and adenine is converted into imino group (-NH). Similarly keto group (C = 0) of thymine and guanine is changed to enol group (-OH).
5BU is similar to thymine, therefore, it pairs with adenine (in place of thymine). A tautomer of 5BU will pair with guanine rather than with adenine. Since the tautomeric form is short-lived, it will change to keto form at the time of DNA replication which will pair with adenine in place of guanine.
In this way it results in AT GC and GC —> AT transitions. The mutagen 2AP acts in a similar way and causes AT <-> GC transitions. This is an analogue of adenine.
c. 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.
d. Other Mutagens:
Other important chemical mutagens are nitrous acid and hydroxy amine. Their role in induction of mutation is briefly described here. Nitrous acid is a powerful mutagen which 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.