15BIS101F2013MutatLect15

Auxotrophic these strains can grow on minimal medium

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Unformatted text preview: gens carcinogens (cancer-causing compounds) and teratogens (cause of birth defects), it is teratogens important to identify and characterize these mutagens in terms of their mode of action and potency. During the period between 1964 to 1974, During Dr. Bruce Ames, Professor of Biochemistry at UC Berkeley, developed a rapid screen for potential mutagenic compounds using a series of defined hishis mutants of Salmonella typhimurium. Salmonella With the "Ames Test", thousands of With chemicals can be quickly tested for mutagenicity. Chemicals that test positive for mutagenicity could then be screened for potential carcinogenicity in small animal models. BIS101­001, Spring 2013—Genes and Gene Expression R.L. Rodriguez ©2013 BIS101­001, Spring 2013—Genes and Gene Expression R.L. Rodriguez 54 Ames Test The Ames Test is based on the principles of mutation The analysis and bacterial genetics. analysis The test uses a common bacterial system for The reversion analysis which includes a series of wellreversion defined histidine deficient (his-) Salmonella defined (his Salmonella auxotrophic strains. auxotrophic These strains can grow on minimal medium only These only when histidine is added to the medium. Once the spontaneous reversion rate has been Once determined (e.g., 10-9 or 1 his+ revertant in 1 billion determined his revertant his- cells), one can examine the effects of his compounds of unknown toxicity on the reversion rate. BIS101­001, Spring 2013—Genes and Gene Expression R.L. Rodriguez ©2013 BIS101­001, Spring 2013—Genes and Gene Expression R.L. Rodriguez 55 Ames Test: Basic Bacterial Genetics Bacterial mutants incapable of growing on minimal medium (glucose and salts) are Bacterial incapable minimal called auxotrophs. These mutants have lost (by mutation) the ability to perform auxotrophs These biochemical functions such as the ability to utilize the milk sugar, lactose, as a carbon source or to synthesize one or more amino acids. Wild type strains, capable of performing these functions, are called prototrophs and therefore they are capable of growing on minimal media. Some Common Bacterial Genotypic Symbols Some Symbol Character of phenotype bioargmethisthi lacgalstrr strs Requires biotin for growth Requires Requires arginine for growth Requires Requires methionine for growth Requires Requires histidine for growth Requires Requires thiamin for growth Requires Unable to use lactose as a carbon source Unable Unable to use galactose as a carbon source Unable Resistant to streptomycin Sensitive to streptomycin BIS101­001, Spring 2013—Genes and Gene Expression R.L. Rodriguez ©2013 BIS101­001, Spring 2013—Genes and Gene Expression R.L. Rodriguez 56 Ames Test: Reversion Analysis Much of what we now know about the molecular mechanism of mutation was Much determined by reversion analysis of bacterial mutants (e.g., Salmonella reversion typhimurium and E. coli). typhimurium E. ). Reversion involves the reversal of a mutation back to wild...
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