Ninety hypoxanthine phosphoribosyltransferase-deficient mutants were isolated from lymphocytes of 31 individuals drawn from both control populations and populations exposed to low doses of ionizing radiation. Southern analysis of the DNA revealed altered hybridization patterns in 15 mutants. Of these, 14 changes consisted of deletions of 2 to 40 kilobases or more.Recent advances in T-lymphocyte cloning procedures have led to the development of methods to estimate in vivo human mutation frequencies (1,12). These techniques have been used to measure the effect of ionizing radiation on mutation frequency in groups of individuals exposed to high (10) and low (11) doses of low linear energy transfer radiation, and an effect was observed at both levels. This led to an estimate for the rate of radiation-induced mutation, a value which previously could only be guessed at by a variety of indirect measures of genetic damage. The lymphocyte cloning procedure can be further exploited since many of the mutant colonies isolated have sufficient growth potential to yield mass cultures suitable for biochemical or molecular analysis (2, 17). Therefore, the possibility exists of establishing in vivo mutational spectra for different genotoxic agents (4-6, 13, 18).In the course of our previous work, many 6-thioguanine (6-TG)-resistant mutant lymphocytes were isolated from subjects differing with respect to radiation exposure, as well as age, sex, and health status. We have begun a systematic analysis of the hypoxanthine phosphoribosyltransferase (hprt) gene structure of these mutants and present here results comparing 90 mutants from 31 individuals. These individuals included seven nuclear medicine patients (Messing, Bradley, and Seifert, Mutat. Res., in press) and 17 female technicians (11) from the nuclear medicine and radiotherapy departments of Notre-Dame Hospital. The control population consisted of one female and six male unexposed hospital workers, with an average age of 30 years. From these subjects more than 600 mutants were cloned, and of these, 250 were transferred into wells of increasing capacity in order to generate mass cultures. Of these, 90 mutants yielded sufficient DNA for analysis. Southern blots were prepared according to standard procedures (9, 16) and probed with insertions from plasmids pHPT5 (8) and pHPT31 (3), kindly provided by C. T. Caskey.

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Patterns of methylation of CpG dinucleotides in the promoter region of the thymidine kinase (TK) gene in wild-type and TK-deficient Chinese hamster cell lines were studied. Whereas wild-type cells were unmethylated, three conventionally derived TK-deficient cell lines were all almost completely methylated in the promoter region. Demethylation at a number of different CpG sites was observed upon selection for reexpression of the TK gene. Of thirteen HhaI (GCGC) or HpaII (CCGG) sites studied, the highest correlation between absence of methylation and at least partial TK activity was obtained at one HhaI site within 20 bp of the putative cap site. Silencing in the three conventionally derived mutants is therefore accompanied by hypermethylation of the promoter-associated CpG-rich island. We contrast this situation with another type of silencing event, in which TK was coordinately inactivated at a high frequency with at least one other linked allele. Methylation of the promoter region of TK was not associated with this event, but two lines of evidence suggested a role for methylation at sites other than in the promoter region of TK.

Ninety hypoxanthine phosphoribosyltransferase-deficient mutants were isolated from lymphocytes of 31 individuals drawn from both control populations and populations exposed to low doses of ionizing radiation. Southern analysis of the DNA revealed altered hybridization patterns in 15 mutants. Of these, 14 changes consisted of deletions of 2 to 40 kilobases or more.