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哪些过程的看法 主要负责点突变。迄今为止，我们想象 大多数突变可能是由环境诱变引起的。新证据 提出了自发机制的更大作用。首先 发现特定的G向第12密码子的过渡中的85％ 甲基硝基库诱导的大鼠乳腺癌并未由 化学，但在未处理的动物中产生 自发的突变突变在第一发或附近。其次是a 该项目中的技术进步使我们能够直接观察 人体组织DNA中的突变光谱。在比较 线粒体序列中的光谱，我们发现突变的集合 在原始实验室条件下的TK-6人类细胞系中 本质上与结肠上皮细胞和骨骼相同 作为手术样品获得的肌肉组织。最后，对此进行研究 项目向我们表明，点突变热点集 我们的方法表明，所有突变体的一小部分（就像 有时会争论），但一套占50％以上，也许更多 在人类细胞的HPRT基因中产生的点突变的90％ 线。 我们现在建议转移突变光谱技术 在整个程序中，也可以使用 酵母和细菌自发突变的生化遗传学。我们 进一步提议扩展技术以测量 核单拷贝人基因。重点放在癌症的“看门人”上 基因。我们建议继续对自发突变的研究 人类细胞中的光谱确定DNA突变的影响 复制和修复基因。