This research is designed to study mechanisms of radiation mutagenesis in human cells. X-rays induce different mutational classes which can be distinguished from one another by several cellular and molecular techniques. It is hypothesized that these different classes arise from different types of DNA damage. Radioprotective conditions are known to affect the relative yields of certain DNA lesions, and therefore it is anticipated that they will alter the spectrum of mutations observed. The spectrum for X-rays will be determined and compared with spectra obtained following irradiation with radioprotectors which modulate the yield of induced mutants. Mutational spectra will be defined in several ways. The cellular component will consist of dose-response data. The X-linked hprt locus will be compared with the autosomal tk locus; measurement of a class of slow-growing mutants which occur only at tk will allow the differentiation of larger-scale chromosomal mutations from gene mutations. Conditions to be studied include X-rays alone, or in the presence of cysteamine, dimethylsulfoxide, or hypoxia, or combinations of these radioprotectors. The molecular aspect of the spectra will differentiate among classes of gene mutations by utilizing Southern blot, Northern blot and DNA sequencing methods to compare the types and positions of mutation within the hprt gene. Sets of mutants will be isolated after treatment with X-rays alone or in the presence of the various radioprotectors. All mutants will be analyzed by Southern blot to determine the fraction of gene mutations which arise from large- scale changes. Each mutant which contains hprt DNA will be analyzed by Northern blot to see which of four classes they fall into (normal RNA, reduced levels, reduced size, no RNA). Mutants which synthesize hprt mRNA will be cloned and sequenced, using a cDNA cloning/dideoxy sequencing technique. This research will provide information on the nature of X-ray- induced mutations in an endogenous human gene. It will determine whether radioprotectors differentially affect specific classes of induced mutations. Comparison with published work on the effects of radioprotectors on DNA change will allow an assessment of which sorts of lesions lead to particular classes of mutations.
