Southern-blot analyses of human T-lymphocyte mutants induced in vitro by γ-irradiation

Abstract

G 0 phase cultures of human peripheral blood T-lymphocytes from a single individual were exposed to 300 rad of γ-irradiation from a 137Cs source and cultured in vitro for 8 days to allow phenotypic expression. Thioguanine-resistant (TG r) mutants were isolated by a cell cloning assay in microtiter plates. These mutants were studied by Southern blot analysis to define the gross structural alterations in the hypoxanthine-guanine phosphoribosyl transferase ( hprt) gene by use of an hprt cDNA probe. A similar analysis of the T-cell receptor (TCR) gene rearrangement patterns was employed to define the independent nature of each mutant colony by use of TCR β and γ cDNA probes. 74 mutants were isolated in 5 separate experiments. TCR gene rearrangement analysis showed these to represent 24 independent mutations, of which 18 contained hprt structural alterations. These alterations included simple deletions ( 10 18 ) as well as more complex rearrangements resulting in molecular weight changes of restriction fragments representing both the 5′ and 3′ regions of the hprt gene ( 4 18 and 4 18 , respectively). These results demonstrate that γ-irradiation primarily induces TG r mutations through gross structural alterations in the hprt gene and that these alterations are randomly distributed across the gene. This approach to mutation analysis will provide information on the types of alterations induced by this irradiation, especially the extent of deletions involving the hprt gene. These results also demonstrate the feasibility of employing in vitro exposure of human T-lymphocytes to a single mutagenic agent as an aid to understanding the mechanisms of mutations occurring in vivo in humans.