Enhanced restriction site mutation (RSM) analysis of 1,2-dimethylhydrazine induced mutations, using endogenous p53 intron sequences.

The restriction site mutation (RSM) assay was used to study the mutational sensitivities of three target regions of the murine p53 gene. The non-coding intron 6 target region was compared with the coding regions exon 4 and exon 5 with respect to their relative sensitivity to the induction of mutations by 1,2-dimethylhydrazine (DMH). Our results demonstrated that the majority of induced mutations detected were in the intron 6 gene region. A total of 15 enzyme-resistant restriction sites were detected in DMH treated mice, nine of these in the intron 6 region, four in the exon 4 region and two in the exon 5 region. The elevated sensitivity of the intron 6 region was exemplified by our detection of spontaneous mutations in this region; two resistant restriction sites were detected in untreated animals. No spontaneous mutations were detected in either of the exon sequences studied here, nor have any been detected in exon targets in our previous in vivo RSM analyses. The mutations induced by DMH were mostly GC-->AT transitions, as were the spontaneous mutations identified. The mutation frequencies calculated by the inclusion of a mutant internal standard (MIS) in the RSM method, revealed that the non-coding intron 6 region and the exon 4 region had a 10-fold higher mutation frequency than the exon 5 region. This heterogenous distribution of mutations and their differential mutation frequencies, were probably a consequence of the greater selection in the coding regions in p53 function. However, the actual mechanism of differential mutation induction is, as yet, to be defined.