SEQUENCE ANALYSIS OF TK AND HPRT GENE MUTATIONS

RECENTLY DEVELOPED MOLECULAR TECHNIQUES WILL BE USED TO ANALYZE MUTATIONS AT THE TK LOCUS IN MOUSE L5178Y CELLS EXPOSED TO CHEMICAL MUTAGENS. BECAUSE MUTATION ASSAYS MEASURE PHENOTYPE, NOT GENOTYPE, SCORED MUTATIONS COULD RESULT FROM OTHER MECHANISMS. SEQUENCE ANALYSIS WILL DETERMINE WHETHER THE MUTATION ASSAYS TRULY MEASURES OF MUTAGENIC EVENTS BY CORRELATING SEQUENCE CHANGES WITH MUTANT PHENOTYPE. MOLECULAR ANALYSES WILL ALLOW DETERMINATION OF THE KINDS OF SEQUENCE CHANGES ASSOCIATED WITH EXPOSURE TO SPECIFIC CHEMICALS AND WHETHER SUCH CHANGES ARE SIMILAR TO CHANGES IMPORTANT IN TUMORIGENESIS. MUTANTS WILL BE ANALYZED BY SOUTHERN BLOTTING FOR MAJOR DELETIONS AND/OR REARRANGEMENTS. POLYMERASE CHAIN REACTION (PCR) AMPLIFICATION OF CDNA WILL ALLOW PREPARATION OF SPECIFIC SEQUENCING TEMPLATE WITHOUT SCREENING CLONE BANKS OR PREPARING SINGLE-STRANDED DNA. CHEMICAL MISMATCH CLEAVAGE AND SINGLE STRAND CONFORMATIONAL POLYMORPHISM ANALYSIS OF PCR PRODUCTS WILL BE TESTED TO SPEED SEQUENCE ANALYSIS. THESE STREAMLINED TECHNIQUES WILL ALLOW ANALYSIS OF SUFFICIENT MUTANTS TO IDENTIFY TRENDS IN MUTATION TYPE ASSOCIATED WITH PARTICULAR CHEMICALS. THE RESULTS WILL INFLUENCE THE SPECIFIC METHODS USED IN PHASE II, WHICH WILL INVOLVE DEVELOPING METHODS TO FURTHER STREAMLINE MUTANT ANALYSIS, AND APPLYING THEM TO A TEST PANEL OF MUTAGENS. REFINED TECHNIQUES AND PROTOCOLS WILL BE MADE AVAILABLE TO OTHER INVESTIGATORS AS A SERVICE AND/OR AS KIT.