DEVELOPMENT OF NEW MOLECULAR PROCEDURES FOR THE DETECTION OF GENETIC ALTERATIONS IN MAN

The Restriction Site Mutation (RSM) procedure is a DNA-based method fo r detecting mutations at any unselected locus. Mutations are identifie d as alterations of the DNA sequence at a chosen restriction site. DNA from cells exposed to mutagenic treatment is exhaustively digested wi th the restriction enzyme (RE). Sequences containing the mutated targe t site are specifically amplified using the polymerase chain reaction (PCR), whereas DNA without mutations at this site will have been cleav ed and can not therefore provide a substrate for PCR. We have develope d this procedure using both bacterial and mammalian cells. With bacter ia, in plasmid reconstruction experiments we were able to detect mutat ions at a frequency of 10(-6) at an EcoRI site in the AraA locus of Sa lmonella typhimurium. The detection limit with an RsaI site in the lad gene of Escherichia coli was 10(-5), and we were able to detect DNA d amage and repair after treatment with N-methyl-N-nitrosourea (MNU). Wi th mammalian cells, we have detected mutations induced by ethyl methan esulphonate (EMS) at a TaqI site in the aprt gene of Chinese hamster c ells. In extensive studies with normal and repair-deficient human cell s, we have detected and sequenced mutations induced by UV-C or UV-B in fibroblasts and lymphoblastoid cells from repair-deficient xeroderma pigmentosum (XP) donors. Similar results were obtained at TaqI sites i n three genes, hprt, c-Ha-ras1 and p53. These results demonstrate that the system is able to detect and analyse mutations induced at high fr equencies. In our extensive attempts to extend the work to conditions of lower mutation frequencies, we have encountered several obstacles, the most serious being false-positive mutant DNA in totally untreated cells. This appeared to be a cell-line specific phenomenon, which we h ave not been able to eliminate by altering conditions. We propose ther efore that, at present, RSM is a suitable method for studying high mut ation frequencies at different loci and could be used for mutagen test ing with repair-deficient cells. As yet, however, its sensitivity and specificity is not sufficient for population monitoring.