MOLECULAR ANALYSIS OF POINT MUTATIONS IN CHINESE HAMSTER OVARY CELLS

我们使用的是中国仓鼠卵巢(CHO)细胞系(AS52)，只有一个拷贝 细菌gpt基因稳定整合到基因组的研究重点 哺乳动物细胞的突变变化。突变体以6-硫代鸟嘌呤的形式出现 抗性(6TGr)克隆和突变序列使用 聚合酶链式反应(PCR)和DNA序列分析 产生突变光谱。我们已经确定了一个特定的3碱基缺失 在分析的自发AS52突变体中，约有30%是这种突变。 然而，在对大肠杆菌中gpt基因座的平行研究中，这个3碱基 尚未观察到删除。真核生物与其他生物的区别 原核生物的DNA代谢和/或染色体结构可能解释了这一点 光谱上的显著差异。基于这个3-碱基的特异性 在哺乳动物细胞中的缺失，影响DNA序列和高 这一事件的频率，我们怀疑涉及真核DNA 使用拓扑异构酶I的实验正在进行中 喜树碱抑制剂以及逆转分析和靶向基因 在AS52细胞中的转化以评估其机制基础 删除。此外，我们正在产生相对诱变的突变 使用E.Coli和AS52细胞系统进行光谱分析。突变的评估 由紫外线照射和H202诱导的病毒目前正在进行中。最后， 我们正在对AS52细胞系统进行修改，以产生 加快了表型表达时间，因此更快 用于DNA序列分析的独立突变体的产生。

We use the Chinese hamster ovary (CHO) cell line (AS52) with a single copy of the bacterial gpt gene stably integrated into the genome to study point mutational changes in mammalian cells. Mutants arise as 6-thioguanine resistant (6TGr) colonies and mutant sequences are recovered using the polymerase chain reaction (PCR) followed by DNA sequence analysis to generate mutational spectra. We have identified a specific 3-base deletion represented in approximately 30% of the spontaneous AS52 mutants analyzed. However, in parallel studies at the gpt locus in E. coli, this 3-base deletion has not been observed. Differences between eukaryotic and prokaryotic DNA metabolism and/or chromosome structure may account for this striking difference in spectrum. Based on the specificity of this 3-base deletion in mammalian cells, the DNA sequences affected and the high frequency of this event, we suspect the involvement of eukaryotic DNA topoisomerase I. Experiments are underway using the topoisomerase I inhibitor, Camptothecin, as well as reversion analyses and targeted gene conversion in AS52 cells to evaluate the mechanistic basis of this deletion. In addition, we are generating comparative induced mutational spectra using the E. coli and AS52 cell systems. Evaluation of mutations induced by both UV-irradiation and H202 are currently in progress. Finally, we are in the process of modifying the AS52 cell system to yield an accelerated phenotypic expression time and, therefore, more rapid generation of independent mutants for DNA sequence analysis.