MUTATION. A mutagen is a physical or chemical agent that causes mutation. Mutagenesis is a process of producing mutations.

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1 MUTATION Dr.H.B.Mahesha, Yuvaraja s College, University of Mysore. Mutation is an abrupt qualitative or quantitative change in the genetic material or any change in the nucleotide sequence of DNA which give rise to a mutant genotype. The organism which undergoes mutation is known as mutant. The earliest record of point mutation dates back to 1791, when Seth Wright noticed a lamb with exceptionally short legs in his flock of sheep. The term mutation was used by Hugo De Vris in 1900 AD to describe the heritable phenotypic changes of the evening primrose Oenothera lamarckina. The first scientific study of mutation started in 1910, when Morgan started his work on fruit fly Drosophila melanogaster after he observed white eyed male individuals among red eyed male individuals. A mutagen is a physical or chemical agent that causes mutation. Mutagenesis is a process of producing mutations. Classification: According to the mode of origin flowing two kinds of mutations have been recognized. (i) Spontaneous mutations occur suddenly in the nature and their origin is unknown. They are also called as background mutations and have been reported in many organisms such as Oenothera, Maize, Drosophila, Mice, Man etc., The frequency of spontaneous or natural mutations is usually low ranging from 10-7 to per organism. The rate detectable mutations in average gene is 1 in 10 6 eg., tautomerism (the ability of a molecule to exist in more than one chemical form is called tautomerism). All the four common bases of DNA (adenine, guanine, cytosine and thymine) have unusual tautomeric forms, which are however rare. A tautomeric shift is believed to occur when an amino (NH 2 ) form of adenine is changed to an imino (NH) form. 1

2 (ii) Similarly a tautomeric shift may occur in thymine changingg from keto (C=O) form to rare enol (COH) form. Natural base pairing in DNA is A=T and G C. The taustomeric forms are however capable of unusual (forbidden) base pairing like T G, G T, C=A and A=C. This unusual base pairing results in mis replication of the DNA strand giving rise to mutants in some of the progeny. (iii) Induced Mutations: Besides naturally occurring spontaneous mutations the mutation can be induced artificially in the living organisms by exposing them to abnormal environment such as radiations, certain physical conditions (i.e. temperature) and chemicals. The substances or agents which induce mutations are called mutagens or mutagenic agents. According to their occurrence in somatic and germinal cells following types of mutations have been recognized. (i) (ii) Somatic mutations: The mutations occurring in non reproductive body cells are known as somatic mutations and related with the malignant (cancerous) growth. These do not have any genetic significance. Gametic mutations: these mutations occurring in gametic cells (eg., sperm and ova) are called gametic mutations. These mutations are heritable and of immense genetical significance. The gametic mutations only form the raw material for natural selection. Again mutations can be classified in to two types depending upon the size or quantity, they are (i) (ii) Gross mutations or chromosomal mutation or chromosomal aberrations: When change involving more than one nucleotide pair or entire gene, then such mutations are called gross mutations. They are deficiency, duplication, translocation and inversion. Point Mutations: when heritable alteration occur in a very small segment of DNA molecule i.e., a single nucleotide or nucleotidee pair, then this type of mutations are called point mutations. According to their mode of direction following types of mutations have been recognized. 2

3 (i) (ii) Forward mutation: in an organism when mutation create a change from wild type to new/abnormal phenotype, then that type of mutations are known as forward mutations. Most of the mutations are forward type. Reverse or back mutations: The forward mutations are often corrected by error correcting mechanism, so that an abnormal phenotype changes in to wild type phenotype. It is commonly called as reversion. A reverse mutant is called as revertant. Based on the type of chromosomes in which mutation occur, following types have been identified. (i) (ii) (iii) (iv) (v) (vi) Sex chromosomal mutation: if it occurs in sex chromosomes. Autosomal mutation: If it occurs in autosomes. Non sense mutation or chain termination mutation: it causes premature termination of polypeptide chain. Mis sense mutation: causes substitution of amino acid in polypeptide chain. Leaky mutation: causes amino acid substitution, eventually it reduces the activity of an enzyme. Silent mutation: this has no detectable effect of phenotype. Mutagens or mutagenic agents: Mutagens are of three types: (i) Physical mutagens, (ii) Chemical mutagens and (iii) Biological mutagens. (1) Physical mutagens are temperature and radiations. Radiations are of two categories; one type is ionizing radiations such as x-rays, gamma rays; alpha and beeta rays, electrons, neutrons, protons and other fast moving particles. The second type is non ionizing radiations such as ultraviolet and visible light. Temperature: the rate of all most all biochemical reactions are influenced by temperature eg., an increase of every 10 C temperature increases the mutation rate two or three fold. Temperature probably affects both thermal stability of DNA and the rate of reaction of other substances with DNA. (2) Chemical mutagens: Many chemical substances have been responsible to increase the mutability of genes. The ability of chemicals to induce mutations was first of all demonstrated by Auerbach and Robson in 1947 using mustard gas and related compounds. Chemical mutagens can be classified according to the way in which they bring mutations. 3

4 (i) (ii) (iii) Base analogues: which are incorporated in to DNA instead of normal bases eg., 5-bromourasil, 2-aminopurine etc., Agents modifying purine and pyrimidine bases: eg., Nitrous acid (HNO2), Hydroxylamine (HN2OH), etc., Agents producing distortions in DNA: eg. Proflavin and acridine orange. The agents in categories 1 and 3 requires replication for their action while the agents in category 2 can modify even in non replicating DNA. (3) Biological mutagens: They may be viral or bacterial. Viral mutagens may use a number of different complex mechanisms to cause a cell to become cancerous. Viruses that causes cancer include the human papiloma virus (implicated in cervical cancer) the human T-cell lymphocytic virus Iimplicated in lymphoma), the hepatitis B virus (implicated in liver cancer). Known bacterial mutagens are Helicobacter pylori, implicated in stomach cancer. Point Mutation or Gene Mutation The earliest record of point mutations dates back to 1791, when Seth Wright noticed a lamb with exceptionally short legs in his flock of sheep. Visualizing the economic significance he produced a flock of sheep with short legs. The short legged breed of sheep was known as Ancon breed. Since then several cases of mutations have been reported in a variety of organisms viz., E. coli, neurospora, pea, maize, rodents, fowls, man etc., The point mutations may occur due to the following types of sub nucleotide changes in the DNA and RNA. Frame shift mutation: The frame shifts occur due to the addition or deletion of one or a few nucleotides or a mutation in which there is deletion or insertion of one or a few nucleotides is called frame shift mutation. There are two types of frame shifts; they are i. Deletion: removal of one or few bases from a nucleotide chain is called a deletion. ii. Insertion: this is due to the addition of one or more (few) nucleotides to the DNA. iii. Substitution mutation: a point mutation in which a nitrogenous base of a codon is replaced by another base is called substitution mutation. They may be of following types. Transition: when a purine base of a triplet codon is substituted by another purine base or a pyrimidine base is substituted by another pyrimidine, then such kind of substitution is called transition. The transitional substitution mutations occur due to tautomerization (purine A>G or G > A) (Pyrimidine C>T or T>C) Transversion: The substitution mutation when involves the substitution or replacement of a purine with a pyrimidine or vice versa, then such type of substitution mutation is called transversion mutation. We have poor information 4

5 about the mechanism of induction, identification and characterization of the transversion mutations. However, it is extremely difficult to recognize transversion mutations genetically. But they can be recognized by the analysis of amino acid substitution in proteins. High temperature and low ph value are known to cause depurination or loss of purine bases. The removal of purine leaves a gap at that point. At the time of replication it would be possible for any of the 4 bases to insert in the newly formed strand. Central dogma of molecular biology showing Different types of frame shift mutations Reading frame CHEMICAL MUTAGENESIS Test for mutagenic effects of chemical agents are almost as old as modern genetics. In 1934, Morgan tried to produce mutations in Drosophila by treating with alcohol and ether, but without success. After a number of attempts by many workers the search for chemical agents met with only during Second World War. The mutagenic activity of chemical agents cannot be guessed on the basis of chemical structure. Some agents cause mutations only in certain organisms and not in others. And some other agents cause mutations only during specific stages of development. Eg. Formaldehyde causess mutations during spermatogenesis in drosophila and it will not cause any effect in adult or female larva. Chemically induced mutations are of different kinds they are 5

6 (i) Copy errors by base analogue: These base analogues have the molecular structure similar to thatt of nucleic acid bases, which are incorporated in to SNA without destroying its capacity for replication. However, because the analogue differs from the normal base in the distribution of hydrogen atoms, it has greater tendency for improper pairing (also in 5-BU as much as bromine has same Vander Wall s radius as methyl group of thymine). One of the most powerful base analogues is 5-bromouracil, an analogue of thymine. If 5-bu in enol state is incorporated in to DNA, in the next generation it may be converted in to keto state. During incorporation in enol state it pairs with guanine and in the next generation in keto state it pairs with adenine. Thus the G C pair is replaced by A=T pair. This process occurs at replication and hence called replication errors. 6

7 2-aminopurine is a purine analogue. It can be read as either adenine or guanne. It normally pairs with thymine but can also for a single hydrogen bond with cytosine. It can therefore produce A=T > G C transition. It is less effective as mutagen than 5-bu. (ii) Direct effect on DNA: Agents modifying purines and pyrimidines or agents which stabilize the base include Nitrous oxide, Hydroxylamine and alkylating agents. a. Nitrous acid (HNO 2 ): it is a very powerful mutagen becauses it acts directly on the nucleic acid, replacing amino groups (NH 2 ) by hydroxyl groups (OH). Thus it directly alters the genetic code by converting one base in to another. The HNO 2 converts adenine to hypoxanthin, cytosine to urasil and guanine to xanthin. Since NH2 groups of these bases are altered by the acid, the products thus evolved display change in pairing behavior resulting in the substitution of one base in the place of another. Guanine > Xanthin pairs with cytosine Adeninee > Hypoxanthin pairs with cytosine Cytosine > Uracil pairs with adenine b. Hydroxylamine NH 2 OH: reacts specifically with cytosine and converts it to a modified base that pairs only with adenine so that a G C pair ultimately becomes an A=T pair c. Alkalatng agents: Alkalating agents like ethyl methane sulfonate (EMS), ethyl ethane sulfonate (EES) have been used extensively in genetic engineering research. Chemical mutagens like nitrous acid and hydroxylamine which are exceedingly useful in prokaryotic system are not useful as mutage in higher eukaryotes, because the chemical condition necessary for reaction are not easily obtained. Whereas alkalating agents are highly effective. These alkalating agents 7

8 impair the normal hydrogen bonding of the bases causing mispairing of G with T, leading to transition of A=T > G C and G C to A=T. also induces recessive lethal mutations, specific locus mutation, translocations, dominant lethal and partial and complete chromosomal loss in Drosophila melanogaster. EMS causes lethal mutations, deletion, translocation, dominant lethal in silkworm Bombyx mori. (iii) Agents producing distortions in DNA: certain fluorescent acridine dyes such as proflavin and acridine orange causes mutations by insertion or deletion of bases. The acridines are planer (flat) molecules like purine bases and can be intercalated between the bases of the DNA helics. This distorts the structure of DNA and can result in deletion or insertion of bases during replication. a. Intercalation resulting in insertion of bases: Intercalation of the acridine molecule between the bases of the template strand results in the lengthening of the DNA molecule. During replication a base (x ) is inserted at random opposite the acridine molecule in the new chain. In the next replication a complementary base (x) will pair with the newly inserted base. Thus the new DNA has an additional pair. b. Insertion resulting in deletion of base: the acridine molecule may be inserted in the new chain during synthesis. This blocks the base n the emplate strand and does not permit any base to pair with it. The chain producedd is thus deficient in one base, and in the next generation produces DNA with a deficient base pair. 8

9 Intercalation resulting in deletion of the base RADIATION MUTAGENESIS The radiations which are important in mutagenesis are of two categories; one type is ionizing radiations such as X-rays, gamma rays, alpha and beet rays; neutrons, protons and electrons. The second type of non ionizing radiations such as ultraviolet rays, infrared rays and visible light. In the process of penetration, however high energy radiations also produces ions by colliding with atoms and releasing electrons, which intern collide with other atoms releasing further electrons etc., when the DNA duplex and RNA is exposed to UV light both DNA and RNA absorbs UV light causing their nitrogen containing bases to become highly reactive free radicals. The resulting unstability causes conversion of one base to another (a purine to another purine or a pyrimidine to another pyrimidine). The primary mutagenic effect of UV light is the production of dimmers. Irradiation of a bacterial culture and subsequent extraction of DNA yields three possible types of pyrimidine dimmers in DNA. (i) Thymine Thymine 50% (ii) Thymine-Cytosine 40% (iii) Cytosine-Cytosine 10%. Pyrimidine dimmers can also be formed between adjacent strands. Also, UV light causes cross linking, single strand breakage etc., Ionizing radiations causes a nick, cross linking, single strand break, base damage etc., 9

10 Some types of DNA damaged by ionizing radiations Besides ionizing radiations causes chromosome type as well as chromatid type aberrations namely terminal deletion, interstitial deletion, translocation, inversion etc., this can be seen under a compound microscope. References: 1. Internet 2. Molecular biology by David Freifelder 3. Cell Biology by C B Powar