DELAYED MUTATION & SECTORING IN MAMMALIAN CELLS

我们发现诱变剂对单只中国仓鼠的处理 卵巢细胞产生突变的马赛克(扇形)克隆，其中包含 葡萄糖-6-磷酸脱氢酶(G6PD)缺陷型 和野生型细胞的相对比例 1/2.1/8..1/32.1/256..1/1000.这表明突变 事件不仅发生在前2个细胞分裂后 诱变剂处理，但在至少8到10个细胞上产生 诱变剂暴露后的分裂。这个项目的目标是 以确定延迟的机制和共性 突变现象。延迟突变是否有可能 是一个依赖时间或复制的过程，将由 比较生长过程中突变的时间分布 停滞不前的文化，而这种文化在 诱变剂治疗。如果延迟突变是复制依赖的， 将检验两个假说：1)由不稳定的DNA引起 在以后的细胞世代中引起突变的重排 通过产生二次重排，或者2)通过持久性 导致碱基错配错误的突变DNA损伤 更晚的细胞世代。关于DNA损伤是否 产生突变的基因是线性传播的(改变的碱基) 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。使用人类 早期和晚期G6PD突变体和女儿“野生型”的cDNA探针 类型“细胞将在DNA水平上被描述为 DNA结构。类似的分析将使用 小鼠细胞株中含有单一整合的大肠杆菌gpt 可回收用于DNA测序的基因，其中 可以观察到扇区划分。如果诱变损伤是线性的 传播(非复制)，06-乙基鸟嘌呤的参与 将通过比较EMS诱发的 缺陷和精通的细胞系的后来突变 切除这一损伤。紫外光诱导的介入 损伤将通过比较延迟的频率进行检查 切除熟练细胞系和缺陷细胞系的突变事件。如果 延迟突变是时间依赖的(复制抑制)， 诱变过程的可能性或时间依赖性 将检查化学变化。延迟突变将是 在人类和小鼠细胞系中进行检查，以研究 这种现象的概括性。

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.