DELETIONS IN MITOCHONDRIAL DNA WITH AGING

最近来自人类研究的证据有力地表明，衰老- 相关综合征帕金森氏病以及衰老过程本身 可能是积聚导致的细胞能量损失的结果 携带大量缺失突变的mtDNA分子。失踪者 缺失分子中的基因都是线粒体的亚基 发电系统及其缺失在以下方面尤为关键 大脑功能。导致删除大型数据库的潜在机制 MtDNA片段(高达8 kb)可能错配 线粒体DNA移位单链片段的重复序列 复制。哺乳动物线粒体包含一个核编码的单一 链结合蛋白(称为“P16”)，抑制复性，从而 可能会保护复制中间体免受意外 在正常情况下错配时滑倒了。这样做的目的是 建议有(1.)建立了广泛存在的删除现象 线粒体DNA的突变事件；(2)要衡量……的积累程度 线粒体DNA随年龄增长而部分缺失，(3)为了研究 P16与删除过程的机制之间的联系。因为我们的 对P16进行了以往的研究(包括氨基酸序列数据) 在大鼠组织中，也将在大鼠身上建立衰老模型。 几种组织(脑、心脏和肝脏)来自胎儿、新生儿、成人和 将通过Southern印迹分析和聚合酶对衰老大鼠进行筛查 应用聚合酶链式反应(PCR)技术检测线粒体DNA 缺失突变。缺失突变将由 双脱氧终止法。基于先前的氨基酸序列数据， 合成了一对聚合酶链式反应(PCR)引物，用于扩增NH2-末端的基因 从Lambda噬菌体文库中获得P16的序列。完整的cDNAs序列 将从锚定的聚合酶链式反应实验或从完整的 用确认的聚合酶链式反应探针筛选鉴定的cDNA克隆 已知的P16的NH2末端。推导出的完整氨基酸 将对序列进行计算机分析以预测结构基序 和功能相关性。P16基因在人类胚胎发育过程中的重要性 线粒体DNA的缺失突变将在合适的大鼠细胞中进行研究。 P16基因反义寡核苷酸有序培养体系 特异性地抑制P16的生物合成。的长期影响 然后将通过筛查P16不足来检查增强 具有大量缺失的mtDNA的产生和积累 突变。

Recent evidence from human studies strongly suggest that the aging- related syndrome Parkinson's disease as well as the aging process itself may be the result of a loss of cellular energy caused by the accumulation of mtDNA molecules harboring substantial deletion mutations. The missing genes in the deleted molecules are all subunits of the mitochondrial energy-generating systems and their absence is particularly crucial in brain function. The underlying mechanism that leads to deletion of large segments (up to 8 kb) mtDNA is probably slipped mispairing between direct repeat sequences in displaced single strand segments of mtDNA during replication. Mammalian mitochondria contain a nuclear-encoded single strand binding protein (called "P16") that inhibits renaturation and thus probably protects the replicative intermediates against accidental slipped mispairing under normal circumstances. The goals of this proposal are (1.) to establish the widespread occurrence of deletion mutation events in mtDNA; (2.) to measure the extent of accumulation of partially deleted mtDNA as a function of aging, and (3.) to study the link between P16 and the mechanism of the deletion process. Since our past studies of P16 (including amino acid sequence data), were carried out in rat tissue, the aging model will be developed in rat as well. Several tissues (brain, heart, and liver) from fetal, newborn, adult and senescent rats will be screened by Southern blot analysis and polymerase chain reaction (PCR) amplification for the occurrence of mtDNA with deletion mutations. The deletion mutations will be sequenced by the dideoxy termination method. Based on previous amino acid sequence data, PCR primers were synthesized and used to amplify the NH2-terminal cDNA sequence of P16 from a lambda phage library. The complete cDNA sequence will be obtained from either anchored PCR experiments or from complete cDNA clones identified by screening with the confirmed PCR-probe directed to the known NH2-terminal end of P16. The deduced complete amino acid sequence will be computer analyzed for prediction of structural motifs and functional correlations. The importance of P16 in the generation of deletion mutations in mtDNA will be studied in a suitable rat cell culture system using antisense oligodeoxynucleotides to P16 mRNA in order to specifically inhibit P16 biosynthesis. The long term effects of insufficient P16 will then be examined by screening for enhanced production and accumulation of mtDNAs having substantial deletion mutations.