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QUANTIFICATION AND MOLECULAR CHARACTERIZATION OF HRPT MUTANTS OF HUMAN T-LYMPHOCYTES
Citation:
Moore, M., K. Harrington-Brock, L. Zimmerman, L. Burnette, T. Smith, R. Everson, J. O'Neill, AND J.C. Fuscoe. QUANTIFICATION AND MOLECULAR CHARACTERIZATION OF HRPT MUTANTS OF HUMAN T-LYMPHOCYTES. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/J-94/207.
Description:
Somatic mutations have been implicated as critical early events in carcinogenesis. oint mutations, deletion and translocation events have been shown to activate oncogenes or inactivate suppressor oncogenes. n human population monitoring, quantitative analysis of mutation events that affect gene function is limited to those genes whose cellular phenotypes can be identified by selection procedures, and to those tissues (like blood) that are accessible for analysis. n an effort to determine the frequency and types of mutations that can be detected at the hypoxanthine guanine phosphoribosyltransferase (hprt) gene, we have utilized the T-cell cloning assay and have developed a strategy to propagate mutants and screen for point mutations and breakage events. arly in 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 PCR reaction. hose mutants which yield altered DNA fragments are then expanded for Southern blot hybridization, PCR, flanking probe isolation and DNA sequencing. o date we have found presumed point mutations, intragenic deletions, and deletions that extend outside of the hprt gene. y analyzing mutations in selectable non-essential gene markers, it should be possible to understand mechanisms of both spontaneous and induced genetic damage. n association of these specific genetic events with human diseases and the evaluation of the ability of environmental chemicals to induce these specific types of mutations will lead to a rational basis for evaluating risks from various chemical exposures.