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

Mutat Res. 1991 Feb;252(1):35-44. doi: 10.1016/0165-1161(91)90249-8.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Chromosome Deletion
  • DNA / chemistry*
  • DNA / isolation & purification
  • Electrophoresis, Agar Gel
  • Genes, Bacterial*
  • Molecular Sequence Data
  • Mutagenicity Tests
  • Polymerase Chain Reaction
  • Pyrenes / adverse effects*
  • Salmonella typhimurium / genetics*

Substances

  • Pyrenes
  • 1-nitrosopyrene
  • DNA
  • 1-nitropyrene