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.
We have used the polymerase chain reaction (PCR) to speed the DNA sequence analysis of revertants of Salmonella typhimurium TA98. Briefly, a crude DNA extract from a single colony was prepared and used in an asymmetric PCR to amplify a 328-bp fragment containing the hisD3052 mutation approximately in the center. Following ultrafiltration, the ssDNA was sequenced using an end-labeled probe and dideoxy sequencing. The most frequent mutation among the revertants was a -2 deletion of GC or CG within the sequence CGCGCGCG, which is upstream of the hisD3052 mutation. This deletion occurred in 38% (6/16) of the spontaneous (-S9) revertants and in 94% (15/16) of a set of 1-nitropyrene-induced revertants. Other mutations, mostly deletions but also some complex mutations (i.e., single mutational events involving a combination of insertions, deletions, and substitutions), occurred within quasipalindromic regions of DNA. Possible mutational mechanisms are discussed, and the results with 1-NP are compared to those obtained in other systems.
Bell
D A
DA
Genetic Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711.
Levine
J G
JG
DeMarini
D M
DM
D001483:Base Sequence; D002872:Chromosome Deletion; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests; D016133:Polymerase Chain Reaction; D011721:Pyrenes / Q000009:adverse effects*; D012486:Salmonella typhimurium / Q000235:genetics*
D011721:Pyrenes; C044707:1-nitrosopyrene; D004247:DNA; C032668:1-nitropyrene