MECHANISM OF MUTATION INDUCED IN MAMMALIAN GENE

哺乳动物基因突变的机制

• 批准号: 3178652
• 负责人: DEZIDER GRUNBERGER
• 金额: $ 14.25万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 1988-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3178652
• 关键词: DNA; carcinogen testing; chromosome translocation; clone cells; complementary DNA; dihydrofolate reductase; genetic mapping; mutagen testing; point mutation; transposon /insertion element

The proposal describes approaches to define the spectrum of lesions induced by mutagens in a mammalian gene at the DNA sequence level. Mutations occurring in an endogenous eukaryotic gene differ qualitatively. We will examine carcinogen-induced mutations in the Chinese hamster ovary locus encoding dihydrofolate reductase (DHFR). In this system, we can characterize point mutations, as well as chromosomal events (translocation, deletion, sequence duplication, and insertion). DHFR-deficient cells have been isolated that are either hemozygous (dhfr+/d) or deleted for the locus (dhfr(d/d)). DHFR- mutants are triple auxotrophs for glycine, thymidine, and hypoxanthine. Mutants will be selected starting with the hemizygote. The gene structure has been well characterized. Southern blot analysis of the induced dhfr-/d genomic DNAs will show changes arising from chromosomal events. Using dhfr-specific cosmid vectors, we will clone the locus from DHFR-cells carrying point mutations. We will map these mutations by marker rescue relying on in vivo or in vitro recombination of the dhfr- cosmid with dhfr+ fragments to correct the lesion. After transfection of the mutant and wild-type DNAs into dhfr(d/d) cells, prototrophic clones will be selected. Upon further mapping, we will sequence regions containing the mutations. Another approach for studying the nature of induced mutations in animal cells similar to the Ames assay is described. We will construct by site-directed mutagenesis 4 different minigenes (full length cDNA fused to the dhfr promoter). The changes to be introduced will create transition, transversion, +1, and -1 frameshift mutations. We will transfect the minigenes into dhfr(d/d) cells selecting for another marker (XGPT). These clones will then be mutagenized to induce reversion to DHFR+. If we can induce reversion, this system will be useful for screening the mutational potential of drugs and chemicals. Also, other parameters influencing the spontaneous and induced frequencies of reversion such as chromosomal location and DNA excision repair will be investigated.

该提案描述了定义病变范围的方法 通过诱变者在DNA序列水平上的哺乳动物基因中。 突变 发生在内源性真核基因中，定性不同。 我们将 检查中国仓鼠卵巢基因座的致癌物诱导的突变 编码二氢叶酸还原酶（DHFR）。 在这个系统中，我们可以 表征点突变以及染色体事件（易位， 删除，序列重复和插入）。 DHFR缺陷型细胞具有 被隔离为血质（DHFR+/d）或删除的基因座 （DHFR（D/D））。 DHFR突变体是甘氨酸，胸苷，胸苷的三重合子， 和低黄嘌呤。 将从半合子开始选择突变体。 基因结构的表征很好。 南部印迹分析 诱导的DHFR-/D基因组DNA将显示由染色体引起的变化 事件。 使用DHFR特异性宇宙向量，我们将从 携带点突变的DHFR细胞。 我们将通过标记绘制这些突变 依靠体内或DHFR-宇宙的体外重组的救援 使用DHFR+片段以纠正病变。 转染后 突变体和野生型DNA进入DHFR（D/D）细胞，原养克隆将是 选定。 在进一步的映射后，我们将序列区域包含 突变。 研究诱导突变的性质的另一种方法 在类似于AMES分析的动物细胞中。 我们将构建 通过位置定向诱变4不同的微型烯（全长cDNA融合 到DHFR启动子）。 要引入的更改将创建 过渡，转移，+1和-1移码突变。 我们将 将微型元素转染到选择另一个标记的DHFR（D/D）细胞 （XGPT）。 然后，这些克隆将被诱变，以诱导归还 DHFR+。 如果我们能诱导归还，该系统将对 筛选药物和化学物质的突变潜力。 另外，其他 影响自发和诱导的恢复频率的参数 将研究诸如染色体位置和DNA切除修复。