15BIS101F2013MutatLect15

109 strain c cells c h2o minimal medium without

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Unformatted text preview: type by a second Reversion spontaneous or induced mutation in the same position. Unlike intragenic or extragenic suppression, reversion involves a back mutation that restores the back original base pair in the wild type strain or cell. Transversion Forward mutations can be Forward induced with well-characterized mutagens at a frequency characteristic for that mutagen. characteristic Back mutations occur Back spontaneously or they can be induced by a mutagen. induced UAC UAC UAC Back Mutation (reversion) Forward Mutation UAG UAG BIS101­001, Spring 2013—Genes and Gene Expression R.L. Rodriguez ©2013 BIS101­001, Spring 2013—Genes and Gene Expression R.L. Rodriguez 57 Reversion Analysis If, for example, a his- mutant called strain "C" was generated with hydroxylamine If, his (i.e., promotes GC to AT transitions), it is possible to deduce the nature of an unknown mutagen by exposing this mutant to it. If the unknown compound increases the reversion rate of mutant C from 10-9 to 10-6, then it must be a increases then mutagen of the type that causes AT to GC transitions, which is the only way to reverse the GC to AT transition caused by hydroxylamine. 109 Strain “C” cells “C” H2O Minimal Medium without histidine X Minimal Medium without histidine BIS101­001, Spring 2013—Genes and Gene Expression R.L. Rodriguez ©2013 BIS101­001, Spring 2013—Genes and Gene Expression R.L. Rodriguez 58 Components of the Ames Test A collection of defined his- Salmonella mutants with collection his Salmonella known spontaneous reversion rates: known ­ q q q his- mutant A (GC to CG transversion, 10-8 ) his- mutant B (single bp deletion, 10-10 ) his- mutant C (GC to AT transition, 10-9 ) Salmonella cells made permeable to various Salmonella compounds by a "permeability" mutation. compounds ­Introduction of a mutation that prevents repair of Introduction mutations into Salmonella cells. Salmonella ­Growth medium that incorporates rat liver Growth microsomes to help modify some compounds into their mutagenic forms. ­ BIS101­001, Spring 2013—Genes and Gene Expression R.L. Rodriguez ©2013 BIS101­001, Spring 2013—Genes and Gene Expression R.L. Rodriguez 59 Mammalian Metabolism of Benzo(a)pryrene Shown at the left is a Shown sequence modifications of benzo(a)pryrene by liver enzymes. BP is a component of car exhaust. Note the product (3) is Note reactive with guanine residues in DNA. Product number (4) is not reactive. This demonstrates how This potential genotoxic compounds can be created by liver metabolism and subsequently detoxified by other enzymes of metabolism. metabolism. BIS101­001, Spring 2013—Genes and Gene Expression R.L. Rodriguez ©2013 BIS101­001, Spring 2013—Genes and Gene Expression R.L. Rodriguez 60 Ames Test Protocol Spread each of six histidine minimal agar plates Spread (lacking histidine) with 109 Salmonella cells of his(lacking Salmonella mutants A, B and C, respectively. Make a sterile solution of unknown compound "X"...
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