QUANTIFICATION AND MOLECULAR CHARACTERIZATION OF HPRT MUTANTS OF HUMAN T-LYMPHOCYTES

Somatic mutations have been implicated as critical early events in car cinogenesis. Point mutations, deletions, and translocation events have been shown to activate oncogenes or inactivate suppressor oncogenes. In human population monitoring, quantitative analysis of mutation even ts that affect gene function is limited to those genes whose cellular phenotypes can be identified by selection procedures and to those tiss ues (like blood) that are accessible for analysis. In an effort to det ermine the frequency and types of mutations that can be detected at th e hypoxanthine guanine phosphoribosyltransferase (hprt) gene, we have used the T-cell cloning assay and have developed a strategy to propaga te mutants and screen for point mutations and breakage events. Early i n the clonal expansion of mutants, 1-2 x 10(4) cells are prepared as a crude cell lysate, and a sample is analyzed using the multiplex polym erase chain reaction (PCR). Those mutants that yield altered DNA fragm ents are then expanded for Southern blot hybridization, PCR, flanking probe isolation, and I)NA sequencing. To date we have found presumed p oint mutations, intragenic deletions, and deletions that extend outsid e of the hprt gene. By analyzing mutations in selectable, nonessential gene markers, it should be possible to understand mechanisms of both spontaneous and induced genetic damage. An association of these specif ic genetic events with human diseases and the evaluation of the abilit y of environmental chemicals to induce these specific types of mutatio ns will lead to a rational basis for evaluating risks from various che mical exposures.