Recently developed molecular genetic techniques will be used to analyze the nature of mutagenic events occurring at the tk and hprt loci in mouse L5178Y cells exposed to chemical mutagens. Because mutation assays measure phenotype and not genotype, the possibility exists that scored mutations could result from other mechanisms. Mutant sequence analysis will determine whether the mutation assays provide true measures of mutagenic events by correlating sequence changes with mutant phenotype. Mutant molecular analyses will also allow determination of the kinds of sequence changes associated with exposure to specific chemicals and whether such changes are similar to changes known to be important in tumorigenesis. Mutants will be analyzed by Southern blotting for the presence of major deletions and/or rearrangements. Polymerase chain reaction (PCR) amplification of cDNA sequences from mutants will allow preparation of specific sequencing template without the complication of screening clone banks or preparing single-stranded sequencing template. Chemical mismatch cleavage of PCR products will be tested to further speed sequence change analysis. These streamlined techniques will allow analysis of sufficient mutants to identify trends in mutation type associated with particular chemicals. Detection of such trends will influence the specific methods used to implement Phase II, which will generally involve developing methods to further streamline mutant analysis, and to apply these methods to a test panel of mutagens. As techniques and protocols become sufficiently refined, they will be made available to other investigators as a commercial service and/or as a reagent kit.
