Revertant screening

The Ames salmonella-microsome test is a principal sensitive mutagen screening test. Compounds are tested on the mutants of Salmonella typhimurium for reversion from a histidine requirement back to prototrophy. A positive result is seen by the growth of revertant bacteria (which do not require an external histidine source). A microsomal activation system should be included in this assay. The use of five different bacterial test strains are generally required. 

Three primary tests are incorporated in the health effects area. The microbial mutagenesis assay is based on the property of selected Salmonella typhimurium mutants to revert from a histidine requiring state to prototrophy due to exposre to various classes of mutagens. The test can detect nanogram quantities of mutagens and has been adapted to mimic some mammalian metabolic processes by the addition of a mammalian liver microsomal fraction. The test is used as a primary screen to determine the mutagenic activity of complex mixtures or component fractions. 

The test is commonly employed as an initial screen for genotoxic activity and, in particular, for point mutation-inducing activity. It detects point mutations, which involve substimtion, addition or deletion of one or a few DNA base pairs. The reverse mutation test in either Salmonella typhimurium or Escherichia coli detects mutation in an amino acid requiring strain (histidine or tryptophan, respectively) to produce a strain independent of an outside supply of amino acid. The principle of the test is that it detects mutations, which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain. 

Kato-Stankiewicz, J., Hakimi, I., Zhi, G., et al. (2002) Inhibitors of Ras-Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells. Proc. Nat. Acad. Sci. USA 99, 14,398-14,403. 

Biotinidase deficiency is an autosomal recessive disorder with an estimated incidence of 1 in 72,000-126,0(X). Many newborn-screening programs of genetic diseases include testing for this enzyme. Prompt treatment with oral biotin administration of 5-20 mg/d in affected infants will prevent clinical consequences. If the treatment is delayed, neurological manifestations (e.g., hearing loss and optic atrophy) and developmental delay occur and may not revert to normal. 

Phenotype-based screens ar e essential to assay small synthetic molecules and more complex substances for biological activity, such as the enhancement or suppression of a particular phenotype. Tlie different states may be morphologically identifiable, such as the spindly patliologic morphology of cells transformed with oncogenes v-ras and v-src. Tlie patliologic state is reverted by depudecin into the wild-type state. 

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