SPECTRUM OF SPONTANEOUS MISSENSE MUTATIONS CAUSING CYCLIC AMP-RESISTANCE PHENOTYPES IN CULTURED S49 MOUSE LYMPHOMA-CELLS DIFFERS MARKEDLY FROM THOSE OF MUTATIONS INDUCED BY ALKYLATING MUTAGENS

Mutants of S49 mouse lymphoma cells resistant to cytolysis by analogs of cyclic AMP (cAMP) generally have missense mutations in the gene enc oding the regulatory subunit of cAMP-dependent protein kinase. We have compared the mutations in 95 spontaneous isolates with those in 60 mu tagen-induced isolates by sequence analysis of amplified cDNAs. Twenty -nine single basepair substitutions in 19 codons produced selectable p henotypes. The spontaneous mutant spectrum was dominated by a CpG tran sition hotspot in the codon for Arg334. This and other nearby CpG site s were found to be methylated in genomic S49 cell DNA by restriction e nzyme analyses. Most of the remaining spontaneous mutants had either G -C --> C-G or T-A --> G-C transversions, which have been associated wi th damage caused by oxygen radicals. In contrast, the majority of muta nts induced with the alkylating mutagens ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine had G-C --> A-T mutations at non-CpG sites; in addition, EMS induced several A-T --> G-C, A-T --> T -A, and G-C --> T-A substitutions. A single ICR191-induced mutant anal yzed had a unique A-T --> G-C lesion. A number of spontaneous and muta gen-induced isolates had closely linked double or triple substitutions , and two isolates had tandem triple substitutions.