MEASUREMENT OF SOMATIC MUTATIONS IN MITOCHONDRIAL DNA

线粒体DNA(MtDNA)的体细胞突变被认为与 伴随哺乳动物衰老而丧失的功能。然而，没有 目前很容易测量罕见的mtDNA点突变的方法(频率和lt； 10-5)在野生型分子的背景下。将开发一种方法 用于对点突变、小缺失和小插入进行量化 线粒体DNA。这项技术被称为翻译逮捕分析(TAAssay)-- 将检测到将终止密码子引入目标序列的突变 或者改变它的阅读框架。线粒体DNA中一段合适的限制性内切酶片段 样本将与编码β-LacZ基因的氨基末端融合。 半乳糖苷酶，这样一个功能性的mtDNA/lacZ融合蛋白将被 在携带重组质粒的转化大肠杆菌中产生。 引入终止密码子或改变阅读框架的突变将 中断翻译，导致乳胶克隆。通过比较 幼虫mtDNA TA分析产生的乳酸菌落的频率 老年大鼠，线粒体DNA体细胞突变率的估计将是 获得。中性位点的突变率将与 和mtDNA突变率将在 不同的组织(分割与不分割)。总的来说，这些 实验将提供衰老过程中线粒体DNA序列保真度的测量 哺乳动物，从而关键地测试“核外体细胞突变” 衰老理论。

Somatic mutations in mitochondrial DNA (mtDNA) have been implicated in the loss of function that accompanies mammalian aging. However, there is no way at present to easily measure rare mtDNA point mutations (frequency < 10-5) in a background of wild-type molecules. A method will be developed for quantifying point mutations, small deletions, and small insertions in mtDNA. The technique -- called Translational Arrest Assay (TAAssay) -- will detect mutations which introduce stop codons into the target sequence or change its reading frame. A suitable restriction fragment from mtDNA samples will be fused to the amino-terminus of the lacZ gene encoding beta- galactosidase, such that a functional mtDNA/lacZ fusion protein will be produced in transformed E. coli carrying the recombinant plasmid. Mutations which introduce stop codons or shift the reading frame will interrupt translation, resulting in lac colonies. By comparing the frequency of lac colonies resulting from TAAssay of mtDNA from young versus old rats, an estimate of the rate of mtDNA somatic mutation will be obtained. The rate of mutation at neutral sites will be compared with that at replacement sites, and the rate of mtDNA mutation will be compared in different tissues (dividing versus nondividing). Overall, these experiments will provide a measure of mtDNA sequence fidelity in the aging mammal, thereby critically testing the "extranuclear somatic mutation" theory of aging.