A REVERSE MUTAGENICITY ASSAY FOR ALKYLATING AGENTS BASED ON A POINT MUTATION IN THE BETA-LACTAMASE GENE AT THE ACTIVE-SITE SERINE CODON

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 o f beta-lactamase activity. This plasmid was used to transform Salmonel la typhimurium TA1535. When the new strain JK947 was treated with a mu tagen such as N-methyl-N'-nitro-nitrosoguanidine (MNNG), the bacteria could be recovered as they became ampicillin resistant. The sequence o f the active-site serine codon in these revertants was mutated from GG C to AGC. Based upon these findings, we developed a model reverse muta genicity assay. In this procedure, we treated JK947 with a test chemic al, such as N-methyl-nitrosourea (MNU), dimethyl sulphate (DMS) or met hylmethane sulphonate (MMS), for 30 min, and then scored the revertant s on agar plates containing 50 mu g/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 n umbers in our test than in the Ames test. However, our test was less s ensitive to DMS and MMS than the Ames test.