Biochemical mutations are mutations that may not be visible or affect a specific morphological characteristic but may have a general effect on the ability to grow or proliferate.
Beadle and E.L.Tatum got Nobel Prize due to their discovery of biochemical mutations in Neurospora. They proposed a concept termed as one gene-one enzyme hypothesis. The hypothesis states that each gene controls the synthesis or activity of a single enzyme. Mutations in these genes were helpful in elucidating several biosynthetic pathways in a variety of organisms, particularly in microorganisms such as fungi and bacteria.
For the study of these biochemical mutations and mutations for resistance against phages and antibiotics, microbial systems are very useful.
Eye transplantation in Drosophila
Beadle and Ephrussi transplanted larval embryonic eye primordia from one mutant type into another. They studied the development of pigment in such transplanted eyes. When wild-type eyes are transplanted on a vermilion or cinnabar host, wild type eyes develop, indicating that phenotype of eyes in this case is independent of host. In reciprocal transplantations, when vermilion or cinnabar host is transplanted on wild type, the transplanted eyes also develop into wild-type. It is because a diffusible substance (probably an enzyme) from host helps the transplanted eyes to develop into wild-type.
Transplantation of vermilion eye on cinnabar, results into a wild type eye , indicating that a diffusible substance from cinnabar enables the transplanted vermilion eye to develop into wild-type. However, when cinnabar is transplanted on vermillion, no diffusible substance is available which would enable cinnabar to develop into wild type. This indicates that vermilion colour appears due to a block appearing earlier in biosynthetic pathway than in case of cinnabar.
Biochemical mutations in Neurospora
Neurospora, a member of Ascomycetes was helpful in the study of biochemical mutations by Beadle and Tatum.
It can grow on a culture medium which contains a few simple salts, some sugar and biotin. From these raw materials, the organism is capable of manufacturing all other compounds required for its growth. As a result of mutations, strains can be obtained which lack the ability to synthesise one or more of these compounds. While the wild strain which is capable of growing on minimal median is termed as prototroph, the nutritional deficient mutants are termed as auxotrophs.
Total isolation method
Beadle and Tatum irradiated connidia of Neurospora, mated these irradiated connidia with wild-type and subsequently studied segregation for induced mutations if any. One of the advantages of using Neurospora is that the four products of meiosis stay in same ascus and segregation can be easily studied in 8 ascospores of an ascus.
For the study of segregation, individual ascospores where first grown on complete medium (a medium having all metabolites in addition to minimal medium), so that both mutant and wild types may grow. Then they grew on minimal medium. Lack of growth on minimal medium indicates mutant nature. Each mutant culture, after identification, then grew on media containing specific metabolite in addition to minimal medium and specific nutritional requirement for which mutation took place.
Penicillin enrichment method:
Bacteria and fungi like Neurospora in proliferating stage is sensitive to penicillin. Therefore, in a suspension culture with minimal medium, addition of penicillin leads to the death of the prototrophs . The auxotrophs will survive due to lack of growth. After killing phototrophs thus, the penicillin can be removed by washing the cells on a filter. If the culture is now plated on a solid medium supplemented with a specific chemical, auxotrophs deficient in this chemical will grow and can be counted and isolated for further study.
Biochemical Mutations and Biosynthetic Pathways
A biosynthetic pathway can be worked out using biochemical mutations, since mutation in a particular gene leads to block in a particular step of biosynthetic pathway. The step which has been blocked can be known either by accumulation of an intermediate product or by recovery of trait due to supply of another intermediate product.
Arginine synthesis in Neurospora
In Neurospora, following three kinds of mutations for arginine synthesis were isolated.
- Mutations, which grow only on the supply of arginine. But do not grow with the help of either ornithine or citrulline alone.
- Mutations, which grow only on the supply of either citrulline or arginine. But not on the supply of ornithine.
- The mutations which can grow, on the supply of either ornithine or citrulline or arginine.
These mutants indicate that, in the first case mutants are incapable of utilising ornithine or citrulline. In the second case mutants are incapable of utilising ornithine. But in the last case, only the precursor cannot be utilised.
Synthesis of tryptophan in bacteria and Neurospora
In Salmonella typhimurium four kinds of mutants for tryptophan synthesis occur. These mutations include the following:
- trp-8 does not have the ability to synthesise anthranilic acid. But addition of either of the following compounds restores the growth. The compounds are anthranilic acid, indole glycerol phosphate, indole or tryptophan.
- trp-2 and trp-4 cannot grow on either the minimal medium or on the supplemented medium with anthranilic acid. But can grow on addition of either indole glycerol phosphate or indole or tryptophan.
- trp-3 can only grow on a supplemented medium with either indole or tryptophan but not on any other medium.
- trp-1,6,7,9,10,11,etc., can grow only on a supplemented medium with tryptophan and on no other medium. These different mutants indicate interruption of biochemical pathway at different steps.
In Escherichia coli also five different kinds of mutations termed as trp- A,B,C,D and E occur. These mutations are similar to those described for Salmonella.
Tryptophan synthetase in Neurospora and bacteria:
The enzyme i.e., tryptophan synthetase controls the last two step of tryptophan metabolism in Neurospora as well as in bacteria, so that even mutants lacking the ability to utilise indole do not have tryptophan synthetase enzyme. In Neurospora this enzyme consists of only one component but in E. coli and Salmonella, it has two components:
- ‘A’ component affecting penultimate step, so that trp-A mutants lacking A component can be supplemented by both indole and tryptophan and
- ’B’ component which affects last step, so that trp-B mutants must be supplemented directly by tryptophan.
The presence of two components (A and B) of tryptophan synthetase enzyme, synthesized under the control of two different genes suggests that a particular enzyme may consist of more than one polypeptides, each synthesized under the control of a different functional unit termed as cistron.