SEQUENCE ANALYSIS OF TK AND HPRT GENE MUTATIONS

最近开发的分子遗传技术将用于分析 小鼠tk和hprt基因座突变事件的性质 L5178 Y细胞暴露于化学诱变剂。 因为突变检测 测量表型而不是基因型，存在得分 突变可能由其他机制引起。 突变序列分析 将决定突变检测是否能提供 通过将序列变化与突变体表型相关联来检测突变事件。 突变体分子分析也将允许确定突变体的种类。 与接触特定化学品相关的序列变化， 这些变化是否与已知的重要变化相似， 肿瘤发生 将通过Southern印迹分析突变体， 主要缺失和/或重排的存在。 聚合酶链 来自突变体的cDNA序列的PCR扩增将允许 特异性测序模板的制备，而没有 筛选克隆库或制备单链测序模板。 将测试PCR产物的化学错配切割，以进一步 速度序列变化分析 这些精简的技术将允许 分析足够的突变体，以确定突变类型的趋势 与特定的化学物质有关。 发现这种趋势将 影响用于实施第二阶段的具体方法， 通常涉及开发方法以进一步简化突变体 分析，并将这些方法应用于诱变剂的测试面板。 作为 技术和协议变得足够完善，它们将被 作为商业服务和/或 试剂盒

Recently developed molecular genetic techniques will be used to analyze the nature of mutagenic events occurring at the tk and hprt loci in mouse L5178Y cells exposed to chemical mutagens. Because mutation assays measure phenotype and not genotype, the possibility exists that scored mutations could result from other mechanisms. Mutant sequence analysis will determine whether the mutation assays provide true measures of mutagenic events by correlating sequence changes with mutant phenotype. Mutant molecular analyses will also allow determination of the kinds of sequence changes associated with exposure to specific chemicals and whether such changes are similar to changes known to be important in tumorigenesis. Mutants will be analyzed by Southern blotting for the presence of major deletions and/or rearrangements. Polymerase chain reaction (PCR) amplification of cDNA sequences from mutants will allow preparation of specific sequencing template without the complication of screening clone banks or preparing single-stranded sequencing template. Chemical mismatch cleavage of PCR products will be tested to further speed sequence change analysis. These streamlined techniques will allow analysis of sufficient mutants to identify trends in mutation type associated with particular chemicals. Detection of such trends will influence the specific methods used to implement Phase II, which will generally involve developing methods to further streamline mutant analysis, and to apply these methods to a test panel of mutagens. As techniques and protocols become sufficiently refined, they will be made available to other investigators as a commercial service and/or as a reagent kit.