DELETIONS IN MITOCHONDRIAL DNA WITH AGING

最近的人类研究证据强烈表明，衰老- 帕金森氏病以及衰老过程本身 可能是由于细胞能量的积累造成的损失， 线粒体DNA分子中含有大量的缺失突变。 失踪 缺失分子中的基因都是线粒体的亚基， 能源生产系统和他们的缺席是特别重要的， 大脑功能 导致删除大的 片段（高达8 kb）mtDNA可能是直接错配之间滑动错配 线粒体DNA单链片段的重复序列 复制的 哺乳动物线粒体含有一个核编码的单 链结合蛋白（称为“P16”），其抑制复性， 可能保护复制中间体免受意外伤害 在正常情况下会出现错误配对。 这个的目标 建议是（1.）以确定删除的广泛发生 mtDNA突变事件;（2.）来衡量 部分缺失的mtDNA作为衰老的函数，和（3.）研究 P16和删除过程的机制之间的联系。 由于我们 P16的过去研究（包括氨基酸序列数据），进行了 在大鼠组织中，衰老模型也将在大鼠中建立。 胎儿、新生儿、成人和新生儿的几种组织（脑、心脏和肝脏） 将通过Southern印迹分析和聚合酶筛选衰老大鼠 链反应（PCR）扩增mtDNA的发生， 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PCR引物，用于扩增NH 2-末端cDNA 来自λ噬菌体文库的P16序列。 完整的cDNA序列 将从锚定PCR实验或从完整的 通过用经确认的PCR探针筛选鉴定cDNA克隆， 连接到已知的P16的NH 2末端。 推导的完整氨基酸 序列将被计算机分析用于预测结构基序 和功能相关性。 P16在产生 将在合适的大鼠细胞中研究mtDNA的缺失突变 使用针对P16 mRNA的反义寡核苷酸的培养体系， 特异性抑制P16生物合成。 的长期影响 不足的P16然后将通过筛选进行检查， 具有实质性缺失的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.