MOLECULAR ANALYSIS OF POINT MUTATIONS IN CHINESE HAMSTER OVARY CELLS

点突变被认为是重要的病因。 一些人类遗传性疾病的因素，包括几个 在血液病、成骨不全以及在 一些癌基因的激活。然而，人们对此知之甚少。 这种突变发生在哺乳动物身上的机制 细胞。用产生法确定机械路径 利用DNA序列分析的突变谱证明 在细菌系统中极其强大。类似的研究在 由于技术问题，哺乳动物细胞一直受到限制。 与快速分离目的基因序列有关 哺乳动物细胞。我们正在分析这两个问题 中国仓鼠自发和诱发的点突变 卵巢(CHO)细胞系，AS52，携带单个拷贝的 细菌gpt基因的导入及功能整合 CHO基因组。Gpt基因与哺乳动物相似。 Hgprt基因和任一基因座的突变可通过以下方法分离 对嘌呤类似物6-硫鸟嘌呤的抗性筛选 (6TG)。Gpt基因的小尺寸(456个碱基对)提供了 为了方便地将突变gpt序列从 用于DNA序列分析的CHO基因组。我们的数据表明 在gpt整合部位的基因组序列可以 影响克隆实验中的序列稳定性。我们加入了 利用克隆技术进行克隆实验的过程 已知的替代大肠杆菌宿主可以稳定基因组插入。在……里面 此外，我们已经开始用 聚合酶链式反应(PCR)技术。这种方法 省去了克隆每个突变基因的要求， 提供足够的DNA，无需进行直接序列分析 进一步的亚克隆。聚合酶链式反应技术的初步研究 表明整个456个碱基对的gpt结构基因 从1克基因组DNA中扩增。利用这些扩增的DNA 我们将进行DNA序列分析并确定 聚合酶链式反应技术在点突变中的应用 哺乳动物细胞的光谱。

Point mutations have been implicated as important etiological factors in a number of human genetic disorders including several of the haemopathies, osteogenesis imperfecta, as well as in the activation of some oncogenes. Little is known, however, about the mechanisms by which such mutations occur in mammalian cells. Determination of mechanistic pathways by generating mutational spectra using DNA sequence analysis has proven extremely powerful in bacterial systems. Similar studies in mammalian cells have been limited due to technical problems associated with the rapid isolation of target gene sequences from mammalian cells. We are in the process of analyzing both spontaneous and induced point mutations in a Chinese hamster ovary (CHO) cell line, AS52, that carries a single copy of the bacterial gpt gene transfected and functionally integrated into the CHO genome. The gpt gene is analogous to the mammalian hgprt gene and mutations at either locus can be isolated by selecting for resistance to the purine analog, 6-thioguanine (6TG). The small size of the gpt gene (456 base pairs) provides for the convenient rescue of mutant gpt sequences from the CHO genome for DNA sequence analyses. Our data suggest that genomic sequences at the site of the gpt integration may influence sequence stability in cloning experiments. We are in the process of performing cloning experiments utilizing alternative E. coli hosts known to stabilize genomic inserts. In addition, we have begun to amplify the gpt gene sequences using the polymerase chain reaction (PCR) technique. This approach bypasses the requirement of cloning each mutant gene and provides enough DNA to allow direct sequence analysis without further subcloning. Initial studies with the PCR technique indicate the entire 456 base pair gpt structural gene can be amplified from 1 g of genomic DNA. Using these amplified DNA sequences we will perform DNA sequence analysis and determine the utility of the PCR technique in generating point mutational spectra from mammalian cells.