MEASUREMENT OF SOMATIC MUTATIONS IN MITOCHONDRIAL DNA

线粒体DNA（mtDNA）的体细胞突变与人类的免疫功能有关。 伴随着哺乳动物衰老的功能丧失。 但没有 目前，一种容易测量罕见mtDNA点突变的方法（频率< 10-5）。 将开发一种方法， 用于定量点突变、小缺失和小插入， 线粒体DNA 这项技术被称为翻译抑制试验（TAAssay）， 将检测将终止密码子引入靶序列的突变 或改变其阅读帧。 一个合适的mtDNA限制性片段 将样品融合到编码β-内酰胺酶的lacZ基因的氨基末端， 半乳糖苷酶，使得功能性mtDNA/lacZ融合蛋白将被 转化E.将重组质粒转入大肠杆菌中。 引入终止密码子或移位阅读框的突变将 中断翻译，导致菌落缺失。 通过比较 用TAAssay对年轻人和 老年大鼠，估计线粒体DNA体细胞突变率将是 得到了 将中性位点的突变率与 在替换位点，线粒体DNA突变率将比较， 不同的组织（分裂与非分裂）。 总体而言，这些 实验将提供一个衡量线粒体DNA序列保真度在老化 哺乳动物，从而严格测试“双核体细胞突变” 老化理论

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.