DIRECT MUTAGENICITY TESTING IN MAN

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This research project will investigate and improve the recently described 6-thioguanine resistant (TGr) T-lymphocyte (T-Ly) clonal assay for detecting somatic gene mutations occurring in vivo in humans in order to: (i) provide assays for human in vivo mutagenicity monitoring and (ii) define the structural and molecular bases of these mutations. The clonal assay will be made more precisely quantitative and then be used to define the range of TGr T-Ly mutant frequency values for normal individuals and for persons exposed to mutagens. Wild type and TGr T-Ly colonies, recovered from clonal assays, will be characterized as to their phenotypic stability, gene product alterations, T-Ly surface antigen subclass, chromosome change, DNA structural alteration in the hprt gene, alterations in specific messenger RNA and specific configeration of the gene for the T-cell receptor. These characteristics will allow definition of the spectrum of hprt gene mutations occurring in vivo in humans, will provide minimal estimates as to the number of mutations responsible for an observed collection of mutants, and will determine if selected mutant characteristics differentiate between "spontaneous" and "induced" in vivo somatic gene mutations in humans. The older autoradiographic assay for quantitating TGr T-Ly variant frequencies in human peripheral blood will be refined, used for human studies, and results obtained using it will be compared with results obtained for similar blood samples using the clonal assay. The T-Ly clonal assay will then be used as the basis for developing a multi-locus (i.e. HLA-loss) somatic mutation detection system that will be capable of detecting alteration of autosomal genes in vivo in humans. Wild type and HLA mutant T-Ly colonies recovered from these assays will be characterized in a manner analogous to the characterization of hprt mutants.