A reverse mutagenicity assay for alkylating agents based on a point mutation in the beta-lactamase gene at the active site serine codon.

Abstract

The serine at the active site of beta-lactamase is responsible for the ester link to the acyl group of beta-lactam during hydrolysis of the substrate to its acid derivatives. A construct was made from a plasmid in which the active-site serine of beta-lactamase was substituted by glycine by site-directed mutation. This mutation results in the loss of beta-lactamase activity. This plasmid was used to transform Salmonella typhimurium TA1535. When the new strain JK947 was treated with a mutagen such as N-methyl-N'-nitro-nitrosoguanidine (MNNG), the bacteria could be recovered as they became ampicillin resistant. The sequence of the active-site serine codon in these revertants was mutated from GGC to AGC. Based upon these findings, we developed a model reverse mutagenicity assay. In this procedure, we treated JK947 with a test chemical, such as N-methyl-nitrosourea (MNU), dimethyl sulphate (DMS) or methylmethane sulphonate (MMS), for 30 min, and then scored the revertants on agar plates containing 50 micrograms/ml ampicillin after incubation at 37 degrees C for 16 h. MNU and MNNG were more potent than DMS and MMS in this assay. Treatment with MNU and MNNG resulted in larger colony numbers in our test than in the Ames test. However, our test was less sensitive to DMS and MMS than the Ames test.