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Microbial Genetics - Formation of Mutants | 12th Microbiology : Chapter 12 : Microbial Genetics

Chapter: 12th Microbiology : Chapter 12 : Microbial Genetics

Formation of Mutants

The term mutant refers to an organism in which either the base sequence of DNA or the phenotype has been changed.

Formation of Mutants

The term mutant refers to an organism in which either the base sequence of DNA or the phenotype has been changed. A mutant is an organism whose genotype differs from that found in nature. The process of formation of mutant organism is called mutagenesis. In nature and in the laboratory, mutations sometimes arise spontaneously, without any help from the experimenter. This is called spontaneous mutagenesis. The two mechanisms that are most important for spontaneous mutagenesis are

1. Errors occurring during replication and

2. Spontaneous alteration of bases.

Mutations can also be induced experimentally by application of mutagens. Mutagens are agents that cause mutations.


Mutagens and their Mode of Action

Physical Mutagens

UV radiation: UV light causes mutations because the purine and pyrimidine bases in DNA absorb light strongly in the ultraviolet range (254 to 260 nm). At this wavelength, UV light induces point mutations primarily by causing photochemical changes in the DNA. One of the effects of UV radiation on DNA is the formation of abnormal chemical bonds between adjacent pyrimidine molecules in the same strand, or between pyrimidines on the opposite strands, of the double helix. This bonding is induced mostly between adjacent thymines, forming what are called thymine dimers (Figure 12.10), usually designated TT. This unusual pairing produces a bulge in the DNA strand and disrupts the normal pairing of T’s (thymines) with corresponding A’s(adenines) on the opposite strand. If UV induced genetic damage is not repaired, mutations or cell death may result


Chemical Mutagens

Chemical mutagens include both naturally occurring chemicals and synthetic substances. These mutagens can be grouped into different classes on the basis of their mechanism of action. They are

i. Base analogs are bases that are similar to the bases normally found in DNA. E.g. 5 – bromouracil (5-BU). TA to CG (Figure 12.11).

Not all base analogs are mutagens.For example, AZT (Azidothymidine), one of the approved drugs given to patients with AIDS, is an analog of thymidine, but it is not a mutagen, because it does not cause base pair changes.

ii. Base Modifying Agents are chemical that act as mutagens by modifying the chemical structure and properties of bases. The three types of mutagens that work in this way are

1. A deaminating agent e.g.: Nitrous acid removes amino groups (- NH2) from the bases guanine, cytosine, and adenine.

2. Hydroxylamine (NH2 OH) is a hydroxylating mutagen that react specifically with cytosine, modifying it by adding a hydroxyl group (OH) so that it can pair solely with adenine instead of with guanine.

3. Alkylating agents like methymethane sulfonate (MMS) introduces alkyl groups onto the bases at a number of location.

iii. Intercalating agents

Acridine, proflavin, ethidium bromide are a few examples of intercalating agents. These insert (intercalate) themselves between adjacent bases in one or both strands of the DNA double helix. Intercalating agents can cause either additions or deletions


Isolation and detection of Mutants

Once mutations are induced, then, they must be detected if they are to be studied. Selection and screening procedures historically have helped geneticists isolate mutants of interest from a heterogenous mixture in a mutagenized population. When isolating mutants of a particular organism, one must know the normal or wild type characteristics so as to recognize an altered phenotype. A suitable detection system for the mutant phenotype under study also is needed. Detection systems in bacteria and other haploid organisms are straightforward because any new allele should be seen immediately, even if it is recessive mutation. The detection of mutants can be direct and complex. For example, the replica plating technique is used to detect auxotrophic mutants (mutants which are deficient in synthesizing a particular biochemical compound). Replica plating technique distinguishes between mutant and wild type strain based on their ability to grow in the absence of a particular biosynthetic end product Figure below. A lysine auxotroph, for instance, will grow on lysine supplemented media but not on a medium lacking an adequate supply of lysine because it cannot synthesize this amino acid

The Ames Test: A Screen for Potential Carcinogens

Everyday we are exposed to a wide variety of chemicals in our environment, such as drugs,cosmetics,foodadditives,pesticides, and industrial compounds. Many of these chemicals can have mutagenic effects, including genetic diseases and cancer. Some banned chemical warfare agents (e.g. mustard gas) also are mutagens.

A number of chemicals (subclass of mutagens) induce mutations that result in tumorous or cancerous growth. These carcinogens Directly testing the chemicals for their ability to cause tumors in animal is time consuming and expensive however mutagens led bruce ames to develop a simple general ames test is an indicator ofwhether assavs the ability of chemicals to rever mutant strains of the bacterium salmonella styphrium is auxotrophic to histidine his that is requires histidine its growth and cannot grow in the absence of histidine. The mutant strain is grown in a histidine deficient medium containing the chemical to be tested. A control plate is also set up which does not contain the chemical. After incubation the control plates may have few colonies resulting from spontaneous reversion of the his- strain. Compared to the control plates if there are increased number of colonies on test plate, it indicates that the chemical has reverted the mutant strain back to wild type. This chemical is likely to be a carcinogen. Figure 12.14 shows steps in Ames test.



DNA Repair

Both prokaryotes and eukaryotes have a number of repair systems that deal with different kinds of DNA damage. All the systems use enzymes to make correction. Without this repair systems lesions would accumulate and be lethal to the cell or organism. Not all lesions are repaired, and mutations do appear, but at low frequencies. At high doses of mutagens, repair systems are unable to correct all of the damage, and cell death may result. We can group repair systems into different categories on the basis of the way they operate. Some systems correct damaged areas by reversing the damage. This type of repair is called direct correction or direct reversal. Other systems excise the damaged areas and then repair the gap by new DNA synthesis of the DNA repair. Some systems are

 • Mismatch repair by DNA polymerase proofreading

• Repair of UV induced pyrimidine dimers- Photo reactivation or Light repair

• Base excision repair   

•  Nucleotide excision repair

With recombinant DNA technology it is possible to mutate a gene at specific posi-tions in the test tube by SITE SPECIFIC MUTAGENESIS and then introduce the mutated gene back into the cell and investigate the pheno-typic changes produced by the mutation in vivo. Such techniques enable geneti-cists to study, for example, genes with un-known function and specific sequences involved in regulating a gene’s expression.

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