MUTATIONAL SPECTROMETRY--TECHNICAL DEVELOPMENT AND USE IN HUMAN CELL LINES

几个重要的结果改变了我们对过程是什么的看法 主要负责点突变。到目前为止，我们一直以为 大多数突变可能是由环境诱变剂引起的。新证据 这表明自发机制的作用要大得多。首先，我们 发现85%的人中第12密码子的特异性G到A的转变 甲基亚硝脲不能诱发大鼠乳腺癌 化学物质，但在未经处理的动物中出现似乎是一种 在第一个发情期或接近发情期时自发发生突变。其次，a 项目中的技术进步使我们能够直接观察 人类组织DNA的突变谱。在比较中， 在线粒体序列中，我们发现产生的突变集 在原始实验室条件下的TK-6人类细胞系中 基本上与在结肠上皮细胞和骨骼中看到的相同 作为外科手术标本获得的肌肉组织。最后，对此进行了研究 该项目向我们表明，由 我们的方法揭示了所有突变体中的一小部分(一直以来 有时会争辩)，但包含50%以上甚至更多的集合 人类细胞中90%以上的HPRT基因点突变 排队。 我们现在建议将突变光谱分析技术 以使其也可用于研究 酵母菌和细菌自发突变的生化遗传学。我们 进一步建议将该技术扩展到测量突变光谱 核单拷贝人类基因。重点放在癌症的“看门人”身上 基因。我们建议继续对自发突变的研究 用人类细胞光谱来确定DNA突变的影响 复制和修复基因。

Several important results have changed our view of what processes are primarily responsible for point mutations. Heretofore, we imagined that most mutations might be caused by environmental mutagens. New evidence suggests a much greater role for spontaneous mechanisms. First we discovered that the specific G to A transition of the 12th codon in 85% of methyl- nitrosourea induced rat breast cancers was not induced by the chemical but arose in untreated animals in what appears to be a spontaneous burst of mutation at or near first estrus. Secondly a technologic advance within the Project has allowed us to directly observe mutational spectra in the DNA of human tissues. In a comparison of the spectra in a mitochondrial sequence, we find the sets of mutations arising in the TK-6 human cell line under pristine laboratory conditions are essentially the same as seen in colonic epithelial cells and skeletal muscle tissue obtained as surgical samples. Finally, research of this Project has shown us that the set of point mutational hotspots studied by our approach reveals not a small fraction of all mutants (as has been sometimes argued), but a set comprising more than 50% and perhaps more than 90% of the point mutations arising in the hprt gene of a human cell line. We now propose to transfer the mutational spectrometry technology throughout the Program so that it may also be used to study the biochemical genetics of spontaneous mutation in yeasts and bacteria. We further propose to extend the technology to measure mutational spectra in nuclear single-copy human genes. Emphasis is placed on cancer "gatekeeper" genes. We propose to continue our studies of spontaneous mutational spectra in human cells to determine the effects of mutations in DNA replication and repair genes.