DELAYED MUTATION & SECTORING IN MAMMALIAN CELLS

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We have found that mutagen treatment of single Chinese hamster ovary cells produces mutant mosaic (sectored) colonies containing both glucose-6-phosphate dehydrogenase (G6PD) deficient mutant and wild-type cells in the relative proportions of 1/2.1/8..1/32..1/256..1/1000. This indicates that mutational events occur not only within the first 2 cell divisions after mutagen treatment but are produced over at least 8 to 10 cell divisions following mutagen exposure. The goal of this project is to determine the mechanism and generality of the delayed mutation phenomenon. The question of whether delayed mutation is a time- or replication-dependent process will be examined by comparing the temporal distribution of mutations in a growth arrested culture with one which was allowed to grow after mutagen treatment. If delayed mutation is replication-dependent, two hypotheses will be tested: 1) That is caused by unstable DNA rearrangements which induce mutations at later cell generations by producing secondary rearrangements, or 2) by the persistence of mutagenic DNA lesions that produce base mispairing errors at later cell generations. The question of whether the DNA lesion which produces the mutation is linearly transmitted (altered base) or replicates with the cell population (unstable DNA arrangement) will be investigated by following individual cells from a mosaic colony for single or multiple mutational events. Using a human cDNA probe, early and late G6PD mutants and daughter "wild- type" cells will be characterized at the DNA level for changes in DNa structure. A similar analysis will be performed using a mouse cell line containing a single integrated of the E. coli gpt gene which can be recovered for DNA sequencing and in which sectoring can be observed. If the mutagenic lesion is linearly transmitted (non-replicating), the involvement of 06-ethylguanine will be examined by comparing the frequency of EMS-induced later mutations in cell lines which are deficient and proficient in the removal of this lesion. The involement of UV-light induced lesions will be examined by comparing the frequency of delayed mutation events in excision proficient and deficient cell lines. If delayed mutation is time-dependent (replication indendent), the possibility of an inducible mutagenic process or time-dependent chemical change will be examined. Delayed mutation will be examined in human and mouse cell lines to investigate the generality of this phenomenon.