EVIDENCE FOR NONTARGETED MUTAGENESIS IN A MONKEY KIDNEY-CELL LINE ANDANALYSIS OF ITS SEQUENCE SPECIFICITY USING A SHUTTLE-VECTOR PLASMID

Intact pZ189 DNA was allowed to replicate in monkey kidney vero cells that had been pretreated with N-methyl-N'-nitro;N-nitrosoguanidine (MN NG). The E. coli MBM7070 was transfected with replicated plasmid, and those with mutations in the supF gene were identified. The frequency o f mutants that did not contain recognizable changes in the electrophor etic mobility of the plasmid DNA was scored. The frequency of such mut ants was 12.2 x 10(-4) (43/35376) and 6.2 x 10(-4) (22/35712) in mutan ts derived from cells pretreated with 0.2 mu moles/l and 2 mu moles/l MNNG respectively; these values represent an increase of 5.8- and 2.9- fold over the spontaneous mutation frequency of 2.1x10(-4) (10/47741) (p < 0.01). Sequence analysis of the supF genes of these mutants showe d that 89% (24/27) of base substitutions occurred at G.C base pairs; 5 9% of the base substitutions (16/27) were transversions, and 41% (11/2 7) were transitions. The types of base substitutions were predominantl y G.C --> T.A and G.C --> A.T. 48% of base substitutions occurred at 6 sites of the supF gene; 4 of these sites consist of 5'-TTNN where N i s G or C. Base substitutions never previously reported were found, nam ely, T --> C at 61, G --> T at 70, G --> T at 99, and G --> C at 103 w ere found; these have never been reported up to now. In addition, 2 of the 5 frameshifts occurred in the region 99-105 of the supF gene (GGT GGGG), suggesting that this region is a hot spot for nontargeted frame shifts. These results strongly suggest that nontargeted mutagenesis ca n occur in mammalian cells and shows that the spectrum of mutations in duced differs from that of spontaneous and targeted mutations.