CELL CULTURE MODELS OF MITOCHONDRIAL ENCEPHALOMYOPATHIES

线粒体脑肌病是一种临床上，形态上， 以及生化多样性的一组疾病。在过去几年中， 已发现特定的线粒体DNA(MtDNA)突变导致 在几种人类疾病中。不幸的是，很少或根本没有 线粒体DNA突变的鉴定与 推测的病因，以及这些疾病的发病机制。 这项建议的目标是分析病理后果和 几种特定线粒体DNA突变引起的分子遗传学原因 人类疾病。这项分析将利用一种独特的细胞培养 一种允许分析中性核中线粒体DNA突变的系统 背景资料。该系统建立在分离人类细胞系的基础上 完全缺乏线粒体DNA(p/o细胞系)和重新繁殖的能力 这些细胞带有外源线粒体，因此也就是线粒体DNA。这个系统 将应用于MERRF(肌阵挛癫痫)疾病的分析 和粗糙的红纤维病)。两个点突变，都在tRNA/Lys中 已知线粒体DNA是导致这种疾病的原因。通过检查 这两种病毒的生化、形态和遗传后果 突变，并比较结果，应该可以确定 发病的确切分子机制。以类似的方式， 线粒体DNA编码的tRNA基因的其他点突变与 将对疾病进行研究。只有在具体的缺陷及其原因是 已知，是否有可能为患者开发合理的治疗方法 遭受着这些疾病的折磨。这个体外系统将允许精确的 呼吸链功能受损细胞的代谢需求 待定。 此外，不同生长条件或处理对玉米生长发育的影响 可以检测到突变的和野生型的mtDNA。如果一个基因组可以 在其复制过程中优先受损或被抑制，它可能是 有可能设计出治疗这些目前无法治愈的疾病的方法，而且通常 致命的疾病。除了这些正在研究的疾病之外， 该模型体系也可应用于其他领域的研究 已知或怀疑线粒体参与的疾病，以及 拟议的分析将为这些未来提供基础 刻画。

The mitochondrial encephalomyopathies are a clinically, morphologically, and biochemically diverse group of disorders. In the past several years, specific mitochondrial DNA (mtDNA) mutations have been found to result in several human diseases. Unfortunately, there has been little or no correlation between the identification of the mtDNA mutations, the presumed etiology, and the pathogenesis of these disorders. The goal of this proposal is to analyze the pathological consequences and the molecular genetic causes of several specific mtDNA mutations causing human disease. This analysis will take advantage of a unique cell culture system that permits the analysis of mtDNA mutations in a neutral nuclear background. This system is based upon the isolation of human cell lines that completely lack mtDNA (p/o cell lines) and the ability to repopulate these cells with exogenous mitochondria, and thus, mtDNA. This system will be applied to the analysis of the disease MERRF (myoclonic epilepsy and ragged-red fiber disease). Two point mutations, both in tRNA/LYS of the mtDNA, are known to result in this disease. By examining the biochemical, morphological and genetic consequences of these two mutations, and comparing the results, it should be possible to determine the precise molecular mechanism of pathogenesis. In a similar fashion, other point mutations of the mtDNA-encoded tRNA genes associated with disease will be studied. Only after specific defects and their causes are known, will it be possible to develop rational therapies for patients suffering from these diseases. This in vitro system will permit the exact metabolic requirements for cells with impaired respiratory chain function to be determined. In addition, the effects of different growth conditions or treatments on the mutated and wild-type mtDNAs can be examined. If one genome can be preferentially damaged or inhibited in its replication, it may be possible to devise treatments for these currently incurable, and often fatal diseases. In addition to those diseases being studied in this proposal, this model system can also be applied to the study of other diseases where mitochondrial involvement is known or suspected, and the proposed analyses will provide a foundation for these future characterizations.