Mutation

Mutation

Any gene can undergo mutation. Because a mutated gene has an altered nucleotide sequence, the protein for which it codes may have an altered amino acid sequence. A cell or an organism carrying a mutated gene is called a mutant, whereas the parent organism with a normal (nonmuted) gene, is called the wild type. By studying mutations, scientists can learn more about such things as cell biochemistry, genetics disorders such as Huntington's chorea and achondroplasia, and development of drug resistance in microbial pathogens.

Mutations can occur in all living organisms. Now and then an albino cat appears in a black litter, or a yellow pea grows among many green peas. The same phenomenon can occur among microorganisms. In nature, however, mutations are relatively rare events can occur at random without any apparent cause. In bacteria, such spontaneous mutations usually in a population of several million bacterial cultures are like looking for the proverbial needle in a haystack.

Microbiologists have developed some helpful techniques to find, or select, these rare mutants. For example, antibiotics can be added to a growth medium to select for mutants that are antibiotic resistant. Under these conditions, the wild-type cells will be killed and only the mutant cells will survive and multiply. Similarly, a virus that attacks bacteria can be introduced into a growing bacterial culture to find bacterial mutants that are resistant to the virus. It is important to realize that such mutations have occurred before exposure to the antibiotic or the virus, not as a result of such exposure. The antibiotic or virus merely selects for a mutant that is already present.

Some mutants require more complicated selection methods. For instances, a mutant cell of Escherichia coli may be unable to make an essential amino acid or vitamin normally made by the wild type. An ingenious procedure is used to increase the proportion of such mutants in a population, greatly enhancing their isolation:

1. A culture of e. Coli containing the wildtype cells and a few mutant cell is inoculated into a medium lacking the nutrient that the mutant cannot make (eg. The amino acid tryptophan). The wildtype cells can reproduce but the mutant cells cannot.
2. Penicillin is added to the culture. This antibiotic kills only cells that are reproducing. Thus the wildtype cells are killed, whereas the mutant cells are not killed because they are not reproducing.
3. The enzyme penicillinase is added to destroy the penicillin, or the cells can simply be centrifuged out of the penicillin-containing the medium.
4. The cells are plated onto an agar medium containing tryptophan. The mutant cells (and any remaining viable wild-type cells) multiply and form colonies.

The replica plating technique, a selective isolation procedure developed in 1952, can now be used to identify which of the colonies on the agar plate are mutants, this procedure uses a velvet disk replicator, a cylinder covered with sterile velveteen cloth. When the cloth covered end of the replicator is pressed onto the agar plate, the velveteen fibers pick up bacteria from the colonies. The pattern of imprinting corresponds to the arrangement of the colonies on the plate. The replicator is then pressed onto two sterile agar plates, one containing tryptophan and the other without tryptophan. The fibers inoculate bacteria onto these new plates in the same pattern as that of the colonies on the original plate. Mutant bacteria will be able to grow only on the tryptophan-containing the medium, whereas wildtype bacteria will grow on both media. By comparing the two plates after incubation, you can identify the mutants.

Other techniques for selecting specific mutants include the use of filtration systems and use of viruses that infect specific bacteria. Many of the techniques traditionally used by microbiologists for mutant induction and selection have been adapted to the study of animal and plant cells grown in laboratory cultures. Automated techniques have been developed for the detection and isolation of mutant cells.

Mutations can be classified into various types based on the kinds of changes they produce in a gene. Two common types are point mutations and frameshift mutations. A point mutation is one that results from the substitution of one nucleotide for another in a gene. In one form of the point mutation, called neutral mutation, the altered codon continues to code for the same amino acid as before and thus the same protein is synthesized. For example, if the mRNA codon AAU became AAC, it would still code for the amino acid asparagine because more than one codon codes for this amino acid. In the form of the point mutation, called missense mutation, the altered codon codes for a different amino acid. For example. If the codon AAU became AAG, it would code for lysine instead of asparagine. This might alter the properties of the protein, or even make it non-functional.

A good example of a missense mutation in humans is the disease sickle cell anemia. A single base substitution in the codon for the sixth amino acid or normal hemoglobin A changes this amino acid from glutamic acid (codon=GAG) to valine (codon=GUG). (Hemoglobin is the protein in red blood cells that carries oxygen). The result is the abnormal hemoglobin, hemoglobin S, that characterizes sickle cell anemia. At low oxygen concentrations in the blood, the hemoglobin S molecules become stacked into the crystal, distorting red blood cells into sickle shapes. These abnormally shaped cells cause a variety of health problems.

In the third type of point mutation, called nonsense mutation, the nucleotide substitution produces a chain terminating codon (for example. A change of UAU to UAA). This results in the premature halting of protein synthesis during translation. The product is an incomplete protein that is probably non-functional.

Frameshift mutations occur with either the addition or the loss of one or more nucleotides in a gene and are termed insertion mutations or deletion mutations, respectively. Since mRNA is read in consecutive blocks of three bases (codons), a frameshift mutation causes a shift of what is called the reading frame of the gene. This usually leads to the formation of non-functional protein, a situation analogous to adding or deleting one or more letters in a sentence.

References

Arvind, Keshari K. and Kamal K Adhikari. A Textbook of Biology. Vidyarthi Pustak Bhander.

Michael J.Pleczar JR, Chan E.C.S. and Noel R. Krieg. Microbiology. Tata Mc GrawHill, 1993.

Powar. and Daginawala. General Microbiology.

Rangaswami and Bagyaraj D.J. Agricultural Microbiology.