A new Salmonella tester strain (TA102) with A X T base pairs at the site of mutation detects oxidative mutagens.

Abstract

A new tester strain, TA102, is described as an addition to the set of strains for the Salmonella/microsome mutagenicity test. This strain contains A-T base pairs at the site of the mutation (determined by DNA sequence analysis) in contrast to the other Salmonella tester strains that detect mutagens damaging G-C base pairs. This strain differs from previous tester strains in that the mutation has been introduced into a multicopy plasmid, so that -30 copies of the mutant gene are available for back mutation. The new strain detects a variety of oxidative mutagens, including x-rays, bleomycin, hydrogen peroxide and other hydroperoxides, streptonigrin and other quinones, and phenylhydrazine; a variety of aldehydes, including formaldehyde, glyoxal, kethoxal, glutaraldehyde, and malondialdehyde; a number of psoralens (in the presence of near-UV light), mitomycin C, neocarzinostatin, and UVlight. Some ofthese mutagens have been previously shown to damage thymine in DNA. Several auxiliary tester strains also are described, including TA96, a frameshift tester strain with a hot spot for mutation at a run of five A-T base pairs with a specificity similar to that of TA102. The importance of oxidative mutagens is discussed.Damage to DNA is likely to be a major cause ofcancer and other diseases (1, 2). The Salmonella mutagenicity test (3), along with other short-term assays (4), is being extensively used to survey a variety of substances in our environment for mutagenic activity. The test measures back-mutation in several specially constructed mutants of Salmonella. A homogenate of rat liver (or other mammalian tissue) is added to the bacterial suspension as an approximation ofmammalian metabolism (3). By using this system, over 80% of the organic carcinogens tested have been detected as mutagens (5-7).All of the histidine-requiring mutants in the standard set of Salmonella tester strains have G-C base pairs at the critical site for reversion: -C-C-C-in the base-pair substitution strain TA100,* -C-C-C-C-C-C-in the frameshift strain TA97 (8), and -C-G-C-G-C-G-C-G-in the frameshift tester strain TA98 (9). The present study describes tester strains that have A-T base pairs at the critical site for reversion. These strains detect a variety of oxidants and other agents as mutagens which were not detected in the standard tester strains. Cloning and Sequence Analysis of hisG428. The ochre mutation, hisG428 (11), was cloned by in vivo recombination into a derivative of phage M13Hol76 carrying the hisD6610 mutation (8). M13Hol76 contains the histidine operator, G, D, and part of the C genes from Salmonella. The male derivative of hisG428, TA2894, was infected with M13Hol76hisD6610 and recombinant phage were selected by their ability to complement the hisA(D)2121 deletion host, TA2891, and the inability to complement the hisA(G)8476 deletion host, TA2892. Singlestranded DNA from this HisD+ HisG-recombinant phage was isolated (12) and subjected to sequence analysis (13). MATERIALS AND METHODSPlasmid pAQl. M13Hol76hisG428 DNA was...

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