A rapid method for determining the DNA sequences of Salmonella typhimurium hisD3052 revertants is presented. DNA colony hybridization was used to analyze revertants previously studied by Isono and Yourno [Proc Natl Acad Sci USA 71:1612-1617, 1974]. Synthetic oligodeoxyribonucleotide probes (18-mers) were able to distinguish sequences that differed by a single base pair. Mutant his sequences not identified by probing analysis were amplified using polymerase chain reaction (PCR) and directly sequenced. The combined use of DNA-colony hybridization and direct sequencing offers a precise and rapid means for the molecular characterization of hisD3052 revertants.

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Extragenic suppressors of the hisG46 missense mutation were mapped to the 71 and 88 min regions of the Salmonella typhimurium chromosome, positions that in Escherichia coli contain the thrV (tRNA(Thr1)) and thrT (tRNA(Thr3)) genes, respectively. The suppressor loci were identified as mutant alleles of thrV and thrT, using allele-specific colony hybridization. An oligomer, based on the conserved 5' sequence of the thrT and thrV genes in E. coli and designed to contain the putative mutant anticodon, discriminated between suppressor-containing and wild-type strains. Similarly, probes specific for the thrV[SuGGG] and thrT[SuGGG] were used to differentiate the two suppressors. To date, all extragenic suppressors of hisG46 have been identified as either thrV[SuGGG] or thrT[SuGGG]. A near equal distribution of thrV[SuGGG] and thrT[SuGGG] suppressors was found among 29 spontaneous and 43 mutagen-induced hisG46 extragenic suppressor revertants. It was concluded, therefore, that mutant alleles of thrV and thrT are predominantly, if not solely, responsible for intergenic suppression of the hisG46 mutation.

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