It is proposed to determine DNA sequence changes induced by specific mutagens in the E. coli gpt gene, of which a single copy has been stably integrated into the genome of a line of CHO cells. The product of the gpt gene in this cell line, AS52, is functionally equivalent to the product of the mammalian hprt gene, which has been deleted. Thus, forward and back mutations in gpt can be assayed in AS52 cells by the well-tested methods developed to assay mutations in hprt in CHO cells. The gpt gene is only 456 base pairs in size, so it is much easier to sequence than the hprt gene (about 32 kilobases). An active Apr gene is adjacent to the gpt gene, which will greatly facilitate recovery of the mutated (inactive) gpt gene from the AS52 genome by plasmid rescue. Finally, it is possible to determine sequence changes induced by the same agents in the same gene in E. coli cells, and obtain comparative data in both mammalian and bacterial cells. Mutagenesis by ultraviolet light and by spontaneous processes will be studied first. The next agents will probably be prototype compounds: ICR-191, an acridine derivative which acts as an intercalator in bacterial cells; MNNG (N-methyl-N1-nitro-N-nitrosoguanidine; an alkylating agent; bromouracil, a base analog; EMS (ethylmethane sulfonate); etc.
