Back mutation

Back mutation (reversion) of the mutant production strain to the wild type (prototrophic form) can occur. 

Since auxotrophic mutants and regulatory mutants are widely used in the overproduction of amino adds, this can be a severe problem. In nature, mutation always takes place but this takes some time. However, in fermentation many generations are produced in a relatively short period of time and the chances of back mutation are enhanced. 

Addition of antibiotics to the fermentation broth may be used to avoid problems associated with growth revertants (eg auxotrophic back mutation) ensure that genetic material (eg plasmid DNA) is maintained within the process micro-organism. 

Use of fresh starting material for each fermentation run may avoid problems of back mutation and loss of genetic material. 

The Ames test measures the reversion from mutant to wild type form (back-mutation) in a culture of Salmonella. The test is used to screen large numbers of compounds for their potential mutagenicity. 

Professor Bruce Ames, a biochemist at the University of California at Berkeley is one of the pioneers of this type of short-term testing. The Ames Test, as it is called, is now widely used, typically as one of several short-term tests that constitute a series of tests, or battery. A battery is thought necessary because no single test is adequate to detect all types of genotoxicity. The Ames Test involves the use of mutant strains of a common bacterium. Salmonella typhimurium, that back-mutate to their normal state in the presence of a mutagenic chemical or metabolite. Many other bacterial and mammalian cell systems have been made available for this type of testing. 

Mitotic abnormalities Back mutation frequency Chromosomal aberrations... 

From the observed rate of appearance of point mutations (one mutation per 106 gene duplications), we can estimate that one mutation occurs per 109 replications at a single nucleotide site. Point mutants tend to "back mutate," often at almost the same rate as is observed for the forward mutation. That is, one in 109 times a mutation of the same nucleotide will take place to return the code to its original form. The phenomenon is easy to understand. For example, if T should be replaced by C because the latter formed a minor tautomer and paired with A, the mutation would appear in progeny duplexes as a GC pair. When this pair was replicated, there would be a finite probability that the C of the parental DNA strand would again assume the minor tautomeric structure and pair with A instead of G, leading to a back mutation. 

Although the rates of spontaneous mutation are low, they can be greatly increased by mutagenic chemicals (Chapter 27) or by irradiation. It is perfectly practical to measure the rates of both forward and back mutation. When this was done, it was found that certain chemicals, e.g., acridine dyes, induce mutations that undergo reverse mutation at a very much lower frequency than normal. It was eventually shown that these mutations resulted either from deletions of one or more nucleotides from the chain or from insertions of extra nucleotides. Deletion and insertion mutations often result from errors during genetic recombination and repair at times when the DNA chain is broken. 

BACK MUTATION Mutation of a mutated gene to its former condition. (See also REVERSION and SUPPRESSION)... 

REVERSE MUTATION Mutation that restores the wild-type phenotype or gene function in a mutant may occur either by restoration of the original DNA sequence (back mutation) or by indirect compensation for the original mutation (suppression). 

SUPPRESSION The restoration of lost genetic function in a mutant by a second mutation in a different gene (inter-genic suppression) or at a second site in the same gene (intragenic suppression). (See also BACK MUTATION and REVERSE MUTATION)... 

If certain amino acids (such as disulfide bonds or crucial amino acids in the hydrophobic core) are indispensable for protein stability, these positions can be changed by site-directed mutagenesis (Proba et al., 1998). To avoid back-mutations during the evolution process or the selection of a residual wild-type contamination, the pool is amplified after each round of ribosome display with a primer that reintroduces the destabilizing mutation. If the mutation is not close to one of the termini, the coding sequence has to be amplified in two parts, which are then reassembled by PCR. Thus, to evolve improved stabilities this strategy first removes known crucial stabilizing factors to select for compensatory mutations at different positions. 

The bacterial reverse mutation test (Ames Test) investigates the ability of chemicals and drags to induce reverse (back) mutations in bacteria, which involves base pair substitutions additions and/or deletions (frameshift mutations) of one or a few DNA base pairs. The bacterial strains used in the test system have mutations in genes coding for enzymes required for the biosynthesis of the amino acids histidine (Salmonella typhimurium) and tryptophan (Escherichia coli). If... 

If the variant is now sensitive, the mutated gene needs to be sequenced to determine whether there has been a back mutation(s) to the original amino acid(s), or whether there has been a complementary mutation suppressing the original one(s). 

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