Adaptive reversion of a +1 frameshift mutation in Escherichia coli, which requires homologous recombination functions, is shown here to occur by -1 deletions in regions of small mononucleotide repeats. This pattern makes improbable recombinational mechanisms for adaptive mutation in which blocks of sequences are transferred into the mutating gene, and it supports mechanisms that use DNA polymerase errors. The pattern appears similar to that of mutations found in yeast cells and in hereditary colon cancer cells that are deficient in mismatch repair. These results suggest a recombinational mechanism for adaptive mutation that functions through polymerase errors that persist as a result of a deficiency in post-synthesis mismatch repair.

A deion and caon system for mutagens has been developed that identifi the six possible base-pair subtio mutat. A set of six Salmonella typhimunum (TA7001 to TA7006) strains has been constructed, each of which carries a unique missense mutation In the hs e biosynthetic operon. ]. addition to the his mutation, these strains carry different auxil features that enhance the mutability ofthe target his mutation. These include the R factor pKM101, which has the SOS-inducible mcAD system; a deletion of the The previous Salmonella mutagenicity test (2) has been used extensively over the past two decades to measure the mutagenic potential of many compounds. These strains have point mutations in the histidine biosynthetic operon that render them unable to grow in the absence of histidine; however, they are not diagnostic for the type of base-pair substitution caused by the mutagen.Each of the six strains described here, either with (TA7001-TA7006) or without (TA7041-TA7046) the rfa mutation, reverts by only one specific base-pair substitution out of the six possible changes. Reversion of the target mutation in a gene for histidine biosynthesis restores the mutant his gene to the wild type so that the cell can grow and form a colony without histidine. The number of colonies formed is a direct measure of the mutagenic potential of the test compound. The spontaneous reversion rates of the strains described here are considerably lower than that of the previous Salmonella tester strains (2), and their sensitivity to reversion by mutagens is comparable. In addition, the strains described here have added the ability to determine the spectrum of base substitutions.Two other systems that detect all six possible base substitutions without further genetic or biochemical analysis have been reported. The Saccharomyces cerevisiae system (3) is based on an essential Cys-22 residue in iso-icytochrome c encoded by the CYCI gene. The Escherichia coli system (4) has a mutational target on a plasmid in an active site glutamate residue in the (3-galactosidase gene.Since the point mutation is extrachromosomal, it can be transferred into various backgrounds such as those differing in mismatch repair. MATERIALS AND METHODSTarget Mutation (A-T --GaC) in Set 1. The mutation hisG177S (5) was recombined with a bacteriophage clone, M13mp9::his4 (6), deleted for part of the hisG gene (covering the hisG1775 mutation) and the hisD gene. Recombinant M13 phage were selected with an active hisD gene product that allowed growth on histidinol and were plaque-purified to prepare single-stranded DNA templates for dideoxynucleotide sequencing. The hisG1775 mutation was identified as a G-C -* APT transition in which the wild-type Gly-153 (GGT) was replaced by the mutant . No other mutations were found in hisG. The hisGl775 mutation is the basis of the TA7041 and TA7001 strains in set 1 (see Table 1).In Vitro Mu n . Target mutations for sets 2-5 were synthesized in DNA oligomers. These oligomers were used as primers to extend single-stranded M13 DNA temp...

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