DELAYED MUTATION & SECTORING IN MAMMALIAN CELLS

我们已经发现，诱变剂处理单个中国仓鼠， 卵巢细胞产生突变嵌合体（扇形）集落， 葡萄糖-6-磷酸脱氢酶（G6 PD）缺陷突变体 和野生型细胞的相对比例为 1/2.1/8.. 1/32.. 1/256.. 1/1000. 这表明突变 事件不仅发生在前两次细胞分裂中， 诱变剂处理，但在至少8至10个细胞中产生 暴露于诱变剂后的分裂。 这个项目的目标是 以确定延迟的机制和一般性 突变现象 延迟突变是否 是一个时间或复制依赖的过程将被检查， 比较生长中突变的时间分布 一个被逮捕的文化与一个被允许增长后， 诱变剂处理 如果延迟突变是复制依赖的， 两个假设将被检验：1）这是由不稳定的DNA引起的 在以后的细胞世代中诱导突变的重排 通过产生二级重排，或2）通过持续存在 产生碱基错配错误的致突变DNA损伤 后来的细胞世代。 DNA损伤是否 产生突变的基因是线性传递的（碱基改变） 或与细胞群体一起复制（不稳定的DNA排列） 将通过跟踪马赛克中的单个细胞进行研究 单个或多个突变事件的菌落。 使用人 cDNA探针，早期和晚期G6 PD突变体和子代“野生型”， 将在DNA水平上表征“型”细胞的变化， DNA结构。 类似的分析将使用 小鼠细胞系含有单一整合的E.大肠杆菌谷丙转氨酶 可以回收用于DNA测序的基因，并且其中 可以观察到扇区化。 如果致突变损伤是线性的 传递（非复制），06-乙基鸟嘌呤的参与 将通过比较EMS引起的频率进行检查 后来的突变细胞系，缺乏和精通， 切除这个病灶 紫外光诱导的参与 将通过比较延迟的 切除熟练和缺陷细胞系中的突变事件。 如果 延迟突变是时间依赖性的（复制依赖）， 诱导性诱变过程或时间依赖性的可能性 将检查化学变化。 延迟突变将是 在人类和小鼠细胞系中检查，以研究 这种现象的普遍性。

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.