Creation of a test plasmid for detecting G-C-to-T-A transversions by changing serine to arginine in the active site of beta-lactamase.
Oligonucleotide-directed mutagenesis of the beta-lactamase gene, bla, on pBR322 was used to change the codon for the active-site serine 70, AGC, to CGC, coding for arginine. Escherichia coli cells carrying the mutant plasmid, pGD104, were sensitive to ampicillin, indicating that the arginine-containing enzyme is inactive. We characterized the reversion of the mutant bla gene by a number of mutagens and in different genetic backgrounds and demonstrated that full ampicillin resistance can be restored only by a G-C-to-T-A transversion occurring at the first base of the codon. Thus, reversion of the mutant bla gene is diagnostic for G-C-to-T-A transversions, and bacteria carrying pGD104 can be used as test strains to detect the occurrence of this mutation.
D000348:Aflatoxins; D000595:Amino Acid Sequence; D001120:Arginine; D001665:Binding Sites; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D009154:Mutation; D010957:Plasmids; D012694:Serine; D014466:Ultraviolet Rays; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D000348:Aflatoxins; D008769:Methylnitronitrosoguanidine; D012694:Serine; D001120:Arginine; D008741:Methyl Methanesulfonate; D001618:beta-Lactamases