Mutagens refer to various physical or chemical agents which greatly enhance the frequency of mutations. Various radiations and chemicals are used as mutagens.
Mutagens are of two types, viz.:
(1) Physical mutagens, and
(2) Chemical mutagens.
These are discussed as follows:
(1) Physical Mutagens:
Physical mutagens include various types of radiation, viz. X-rays, gamma rays, alpha particles, beta particles, fast and thermal (slow) neutrons and ultra violet rays (Table 25.2).
A brief description of these mutagens is presented below:
(1) X-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 and 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 transversions.
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.
(2) 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.
(3) 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.
(4) 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.
(5) 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 more in straight line. Fast and thermal neutrons are effectively used for induction of mutations especially in asexually reproducing crop species.
(6) Ultra Violet 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 pollens in plants and eggs in Drosophila. UV rays can also break chromosomes. They have two main chemical effects on pyrimidines.
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. Interstrand dimers cross link nucleic acid chains, inhibiting strand separation and distribution.
(2) Chemical Mutagens:
There is a long list of chemicals which are used as mutagens.
The chemical mutagens can be divided into four groups, viz.:
(1) Alkylating agents,
(2) Base analogues,
(3) Acridine dyes, and
(4) Others (Table 25.3).
A brief description of some commonly used chemicals of these groups is presented below:
(1) 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 the cases, diameter of the mutant base pair is close to that of a normal base pair.
(2) 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 in 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 = O) 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.
(3) Acridine Dyes:
Acridine dyes are very effective mutagens. Acridine dyes include proflavin, acridine orange, acridine yellow, acriflavin and ethidium bromide. Out of these, proflavin 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 of few base pairs when DNA replicates. Thus they cause frame-shift mutations and for this reason acridine dyes are also known as frame-shift mutagens. Proflavin is generally used for induction of mutation in bacteriophages and acriflavin in bacteria and higher organisms.
(4) Other Mutagens:
Other important chemical mutagens are nitrous acid and hydroxylamine. Their role in induction of mutation is briefly discussed here. Nitrous acid is a powerful mutagen which reacts with C6 amino groups of cytosine and adenine. It replaces the amino groups with oxygen (+H 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, GC → AT transition. All the chemical mutagens except base analogues are known as DNA modifiers.
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