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

突变光谱测定——技术开发及其在人类细胞系中的应用

• 批准号: 5211086
• 负责人: WILLIAM G THILLY
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5211086
• 关键词: DNA; DNA repair; cell cycle; clone cells; denaturing gradient gel electrophoresis; gene mutation; human subject; method development; mitochondrial DNA; mutant; point mutation; polymerase chain reaction; ribosomal RNA; technology /technique

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.

几个重要的结果改变了我们对过程的看法 主要负责点突变。在此，我们设想， 大多数突变可能由环境诱变剂引起。新证据 表明自发机制的作用更大。首先我们 发现85%的人的第12个密码子的G到A的转换是特异性的， 甲基亚硝基脲诱导的大鼠乳腺癌， 化学物质，但出现在未经处理的动物， 在第一次发情期或接近第一次发情期时自发突变。其次包括 该项目的技术进步使我们能够直接观察到 人类组织DNA的突变谱的比较中 在线粒体序列的光谱中，我们发现突变的集合 在原始实验室条件下TK-6人细胞系中， 基本上与结肠上皮细胞和骨骼肌中所见相同， 作为手术样本获得的肌肉组织。最后，本文研究了 项目已经向我们表明， 我们的方法揭示了所有突变体中的一小部分（如 有时争论），但一套包括超过50%，也许更多 超过90%的点突变出现在人类细胞的HPRT基因中 线 我们现在建议将突变光谱技术 在整个计划，使它也可以用来研究 酵母和细菌自发突变的生化遗传学。我们 进一步建议扩展技术以测量突变谱， 核单拷贝人类基因。重点关注癌症“守门人” 基因.我们建议继续研究自发突变， 人类细胞中的光谱以确定DNA突变的影响 复制和修复基因。