MOLECULAR ANALYSIS OF POINT MUTATIONS IN CHINESE HAMSTER OVARY CELLS

我们使用中国仓鼠卵巢(CHO)细胞系(AS52) 功能性细菌gpt转基因稳定整合到基因组中 研究哺乳动物细胞的点突变变化。突变体出现在 GPT基因座为6-硫代鸟嘌呤抗性(6TGr)菌落，其特征是 携带假定的点突变或使用 聚合酶链式反应(PCR)。然后是点突变光谱 使用DNA序列分析生成。我们最近实施了 简化突变分子分析的实验室方法 在哺乳动物细胞中。聚合酶链式反应片段中突变位置可以是 在DNA测序前使用热(羟胺或 四氧化铯)修饰协议。这种方法限制了 需要的DNA测序量，并允许序列 更多的哺乳动物突变体的特征。 我们评价了抗癌药物碱基类似物5-氮胞苷(5AC)， U73,975和人类致癌物质Treosulphan以及嫌疑人 海藻硫丹的活性代谢产物二环氧丁烷在AS52中的诱变作用 细胞。所有这些都是有效的诱变剂，并且已经产生了突变谱。 在5AC诱发的点突变中，存在着很大的偏差 (&gt；90%)用于CG&gt；GC转换。这些数据表明了5AC的直接作用 作为直接作用的诱变剂，最有可能通过产生5AC：C 复制过程中出现配对错误。药物U73,975是毒性较低的类似物 被广泛研究的抗肿瘤药物CC-1065。两种药物的体外研究 揭示了首选的DNA结合序列基序。125个U73,975- 分析诱变，44个(35%)是推定的点突变，81个(65%) 经聚合酶链式反应分析均为缺失。在点突变中，一个热点是 在gpt基因的一个包含3-重叠的位置观察到突变。 一致的药物结合序列。Treosulphan和DEB导致两种缺失 突变(70%)和点突变(30%)。这些数据表明 Treosulphan和DEB可以诱导哺乳动物细胞至少通过 两条机械式的道路。DNA序列分析正在进行中，以比较 海藻硫丹或DEB处理引起的点突变光谱。全 这些研究的目的是提供关于机制的洞察力 这些因素会影响基因组的完整性。

We use the Chinese hamster ovary (CHO) cell line (AS52) with a single functional bacterial gpt transgene stably integrated into the genome to study point mutational changes in mammalian cells. Mutants arise at the gpt locus as 6-thioguanine resistant (6TGr) colonies and are characterized as carrying putative point mutations or deletion mutations using the polymerase chain reaction (PCR). Point mutational spectra are then generated using DNA sequence analysis. We have recently implemented methods in the laboratory to simplify the molecular analysis of mutations in mammalian cells. The site of a mutation in a PCR fragment can be quickly identified prior to DNA sequencing using the HOT (hydroxylamine or osmium tetroxide) modification protocol. Such an approach limits the amount of DNA sequencing necessary and allows for the sequence characterization of a substantially greater number of mammalian mutants. We have evaluated the base analog 5-Azacytidine (5AC), the anticancer drug, U73,975 and the human carcinogen, Treosulphan, as well as the suspect active metabolite of treosulphan, diepoxy butane (DEB), as mutagens in AS52 cells. All are potent mutagens and mutational spectra have been generated. Among the point mutations induced by 5AC, there is a substantial bias (>90%) for CG>GC transversions. These data suggest a direct role for 5AC as a direct acting mutagen, most likely through the generation of a 5AC:C mispairing during replication. The drug U73,975 is a less toxic analog of the widely studied antitumor drug, CC-1065. In vitro studies of both drugs have revealed preferred DNA binding sequence motifs. Of 125 U73,975- induced mutants analyzed, 44 (35%) are putative point mutants and 81 (65%) are deletions as analyzed by PCR. Among the point mutants, a hot spot for mutations is observed at a site in the gpt gene containing 3-overlapping consensus drug binding sequences. Treosulphan and DEB induce both deletion mutations (70%) and point mutations (30%). These data suggest that Treosulphan and DEB can induce mutations in mammalian cells via at least two mechanistic pathways. DNA sequence analysis is underway to compare point mutational spectra resulting from treosulphan or DEB treatment. All of these studies are designed to provide insight regarding the mechanisms by which these agents affect genomic integrity.