MOLECULAR ANALYSIS OF MUTATION IN MAMMALIAN CELLS

哺乳动物细胞中突变的分子分析受到了 哺乳动物基因组的复杂性和缺乏快速方法， 诱导突变的序列分析。 大多数哺乳动物基因座可用于 研究哺乳动物细胞中的诱变（hgprt、tk、oua-r、dpt-r等）， 不容易进行分子分析， 基因处于其正常基因组状态，例如，在hgprt的管型中为35 - 40 kb，或 由于缺乏克隆的序列和/或关于 导致耐药表型的突变的性质。 那里 然而，有三个值得注意的例外，它们在他们的 在分子水平上分析基因组靶标中突变的能力 基因 其中两个是哺乳动物细胞中的aprt和tk基因座。 这些 已克隆的基因座被测序，并且足够小以允许 应用快速克隆技术分析诱导突变。 第三种系统允许更大范围的应用， 细菌gpt基因的单拷贝（456个碱基对）连接到一个 抗生素抗性基因，氨苄青霉素（Apr）或新霉素（neo-r）， 整合到哺乳动物基因组中。 使用SV40早期启动子或 逆转录病毒LTR，gpt基因表达嘌呤补救途径酶， HGPRT（HGPRT的细菌等效物）。 在这个系统中，标准 用于分离HGPRT突变的技术可应用于 在整合的GPT序列上分离突变。

Molecular analyses of mutation in mammalian cells have been hindered by the complexity of the mammalian genome and the lack of rapid methods for the sequence analysis of induced mutations. Most mammalian loci useful for studying mutagenesis in mammalian cells (hgprt, tk, oua-r, dpt-r, etc.), are not readily amenable to molecular analyses due to the large size of the gene in its normal genomic state, e.g., 35-40 kb in the cast of hgprt, or due to the lack of cloned sequences and/or sequence information concerning the nature of mutations that will lead to the resistant phenotype. There are three notable exceptions, however, which appear promising in their ability to analyze mutations at the molecular level in a genomic target gene. Two of these are the aprt and tk loci in mammalian cells. These loci have been cloned are sequenced and are sufficiently small to allow the application of rapid cloning techniques for analysis of induced mutations. A third system, allowing somewhat greater range of applications, utilizes a single copy of the bacterial gpt gene (456 basepairs) linked to an antibiotic resistance gene, Ampicillin (Apr) or Neomycin (neo-r), integrated into the mammalian genome. Using the SV40 early promotor or a retroviral LTR, the gpt gene expresses the purine salvage pathway enzyme, HGPRT (the bacterial equivalent of HGPRT). In this system, standard techniques used for the isolation of mutations at hgprt can be applied to the isolation of mutations at the integrated gpt sequences.