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RAPID DNA SEQUENCE ANALYSIS OF REVERTANTS OF THE HISD3052 ALLELE OF SALMONELLA TYPHIMURIUM TA98 USING THE POLYMERASE CHAIN REACTION AND DIRECT SEQUENCING: APPLICATION TO 1-NITROPYRENE-INDUCED REVERTANTS
Citation:
Bell, D., J. Levine, AND D.M. DeMarini. RAPID DNA SEQUENCE ANALYSIS OF REVERTANTS OF THE HISD3052 ALLELE OF SALMONELLA TYPHIMURIUM TA98 USING THE POLYMERASE CHAIN REACTION AND DIRECT SEQUENCING: APPLICATION TO 1-NITROPYRENE-INDUCED REVERTANTS. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/J-91/098 (NTIS PB91200329).
Description:
We have used the polymerase chain reaction (PCR) to speed the processing of revertants of Salmonella typhimurium TA98 for DNA sequence analysis. riefly, a crude DNA extract from a single colony was prepared and used in an asymmetric PCR to amplify a 228-bp fragment containing the hisD3052 mutation approximately in the center. ollowing ultra filtration, the ssDNA was sequenced using an end-labeled probe and dideoxy sequencing. he most frequent mutation among the revertants was a -2 deletion of GC or CC within the sequence CGCGCGCG, which is upstream of the hisD3052 mutation. his deletion occurred in 38% (6/16) of the spontaneous (-59) revertants and in 94% (15/16) of a set of 1-nitropyrene-induced revertants. isalignment of complementary DNA strands within this repeat may account for this mutation, although the possible formation of 2-DNA within this region may also play a role. ther. mutations, mostly deletions but also some complex mutations (insertions/deletions/substitutions), occurred within quasi-palindromic regions of DNA. he potential DNA secondary structures within such region may mediate the templated production of some of these mutations. he important role of DNA context and selection system on the mutational spectrum produced by an agent is strongly suggested by comparison of the spectrum produced by 1-NP at the hisD3O52 allele to that produced by 1-nitrosopyrene at the lambda cI gene.