Various tests have been devised for detecting spontaneous mutations in a population. Some of them are: 1. Replica Plating 2. Mutation Rates in Maize Endosperm and Drosophila 3. Detection of Mutations in Micro-Organisms.
Test # 1. Replica Plating:
In 1946 Luria and Delbrueck noticed one of the first cases of spontaneous mutation in a bacterial population. When E. coli cells were infected with the bacteriophage T1 most of the cells lysed releasing progenies of newly synthesised viruses. However, a very small proportion of bacterial cells (about 1 in 108) did not lyse but could multiply and form colonies.
These bacteria as well as their descendants obtained by sub-culturing the colonies were found to be resistant to T1. It appeared that some of the originally infected cells had become mutant forms resistant to phage. The replica plating technique devised by Lederberg in 1952 demonstrated that E. coli cells had undergone spontaneous mutation and become resistant to T1.
The technique is as follows. E. coli cells are grown on a master plate of nutrient agar until they form a continuous lawn of cells. A sample of cells is transferred by pressing on it a sterile velvet surface supported on a circular block (replicator) to a fresh plate of agar medium already coated with T1 phage particles. The method produces an identical pattern of colonies on the second plate. Some mutant phage resistant colonies also grew on this plate.
The question arises about the origin of these mutant colonies. If the mutant colonies had originated from the master plate, then it would be possible to obtain a culture of such cells by inoculating from locations on the master plate corresponding to positions of resistant colonies on the second phage-containing plate.
Lederberg therefore repeated the procedure by preparing a second master plate and replica plating on to a phage coated plate. This was done 3 to 4 times, each time plating cells thinly. In this way single resistant colonies could be identified (Fig. 20.1) and a true breeding phage resistant strain would be developed. The experiment proves that resistant cells arose by mutation on the first master plate and were not induced by the phage to become resistant.
Test # 2. Mutation Rates in Maize Endosperm and Drosophila:
L.J. Stadler studied mutation frequency in maize population affecting endosperm characteristics. It was found that mutation frequency varied with eight different genes. The colour gene R mutated about 492 times in a million gametes; the gene for full endosperm (sh+) mutated to sh for shrunken endosperm about 3 times in 2.5 million gametes; the gene for normal endosperm did not mutate to the gene for waxy conditions in 1.5 million gametes.
From studies of sex-linked lethals in Drosophila, Muller has estimated that one out of 20 flies at some stage of life undergoes spontaneous mutation of some gene. He has also devised a method of estimating mutation rate from matings of attached X females.
When an attached X female is fertilised by a normal male, it produces 4 types of offsprings:
XXX females and YY males-both inviable; the other two types are attached X females and XY males where the males inherit their Y chromosome from the mother and X from the father. In such F1 males all the sex-linked recessive traits carried by their father are expressed and easily detected.
Test # 3. Detection of Mutations in Micro-Organisms:
In 1946 Beadle and Tatum discovered a method of detecting nutritional mutants in the bread mold Neurospora crassa. Wild type Neurospora grows on a minimal medium containing inorganic acids, sugar, nitrogen, a few salts and a vitamin biotin. Beadle and Tatum found nutritional mutants of Neurospora which failed to grow on minimal medium in absence of a particular nutrient.
The exact requirements of nutritional mutants can be determined by adding a different growth substance to each of a series of different cultures of minimal media. For instance, a mutant strain of Neurospora deficient in the enzyme tryptophan synthetase would be able to grow on the minimal medium only if tryptophan is added to the medium. The technique has turned out to be applicable to many micro-organisms.
Mutants of the kind described above are known as auxotrophs, and their growth requirement may be for a vitamin, an amino acid, a nucleoside or a nucleic acid base. Auxotrophic mutants have lost the ability to synthesise for themselves the substance which they require. The substance has to be supplied to the medium.
In 1949 B. D. Davis found an elegant way of detecting auxotrophs in E. coli by a penicillin selection method. Certain antibiotics like penicillin inhibit cell division only in growing, actively dividing bacteria. Thus when penicillin is added to a minimal medium containing bacteria the wild type cells (also called prototrophs) which are growing are killed.
The auxotrophs which are not growing are not acted upon by penicillin and survive. After a certain time period the auxotrophs are separated from the medium by centrifugation, washed free of penicillin and planted on fresh minimal medium supplemented by the necessary growth substance. The auxotroph will divide and form a more or less pure colony.
Auxotrophs themselves belong to a class of mutants broadly known as conditional mutants; their growth is dependent on the presence of a particular environmental condition. The temperature-sensitive mutants fail to grow normally in a certain temperature range. Usually higher temperatures affect growth because of defects in thermo ability of specific proteins.
Temperature sensitive mutants in bacteria can be detected by the technique of replica plating. The replica plate is incubated at or above the maximum temperature which permits growth of the wild type (for E. coli 42°C).
If the replica colony fails to grow under these conditions it indicates that it is probably a temperature sensitive mutant. In yeast some mutants become manifest at elevated temperatures through deficiencies affecting growth.