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.

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National Institute of Environmental Health Sciences (NIEHS)
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Chemical Pathology Study Section (CPA)
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Lankenau Institute for Medical Research
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Stamato, T D; Richardson, E; Perez, M L (1995) UV-light induces delayed mutations in Chinese hamster cells. Mutat Res 328:175-81
Stamato, T; Guerriero, S; Denko, N (1993) Two methods for assaying DNA double-strand break repair in mammalian cells by asymmetric field inversion gel electrophoresis. Radiat Res 133:60-6
Elia, M C; Motyka, L E; Stamato, T D (1991) Electrotransfer of [32P]NAD allows labeling of ADP-ribosylated proteins in intact Chinese hamster ovary cells. Anal Biochem 192:329-33
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Stamato, T D; Denko, N (1990) Asymmetric field inversion gel electrophoresis: a new method for detecting DNA double-strand breaks in mammalian cells. Radiat Res 121:196-205
Giaccia, A J; Denko, N; MacLaren, R et al. (1990) Human chromosome 5 complements the DNA double-strand break-repair deficiency and gamma-ray sensitivity of the XR-1 hamster variant. Am J Hum Genet 47:459-69
Giaccia, A J; Richardson, E; Denko, N et al. (1989) Genetic analysis of XR-1 mutation in hamster and human hybrids. Somat Cell Mol Genet 15:71-7
Stamato, T D; Richardson, E; Ianacone, J et al. (1989) Isolation and characterization of glucose-6-phosphate dehydrogenase-deficient Chinese hamster cells derived from pure mutant colonies. Mutagenesis 4:259-64
Denko, N; Giaccia, A; Peters, B et al. (1989) An asymmetric field inversion gel electrophoresis method for the separation of large DNA molecules. Anal Biochem 178:172-6
Stamato, T D; Dipatri, A; Giaccia, A (1988) Cell-cycle-dependent repair of potentially lethal damage in the XR-1 gamma-ray-sensitive Chinese hamster ovary cell. Radiat Res 115:325-33

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