ANALYSIS OF MTDNA REARRANGEMENTS IN POST MITOTIC CELLS

人类线粒体肌病和脑肌病 包括临床上、形态上和生化上的多样性 这些疾病现在开始被描述为遗传性疾病。 以来 1988年，特定的线粒体DNA（mtDNA）突变已经被发现。 发现导致疾病。 这类疾病通常会影响 分化的（有丝分裂后）组织，如肌肉和某些 神经组织最为严重 我们的目标是分析肌肉细胞 培养的表型和分子遗传后果 导致肌病的mtDNA突变， 脑肌病 此分析将利用细胞 培养系统，开发用于分析线粒体DNA突变， 有丝分裂后的细胞该系统选择性地消除了内源性 细胞的线粒体补体，并重新填充细胞 含有mtDNA的外源线粒体，包括突变的 线粒体DNA 特定的mtDNA突变可以被引入到 成肌细胞可以分化成多核肌管， 能与大鼠脊髓神经元共同神经支配， 非常成熟和功能活跃的肌纤维 因为 可以将特定比例的特定突变引入 这些文化，是可行的，系统地研究和下 控制条件下mtDNA的表型结果 突变并确定其分子遗传原因。 的 这项建议的具体目标是通过以下方式调查机制： 哪些线粒体DNA重排会阻碍蛋白质合成， 呼吸链活动的神经和神经支配的肌肉文化。 此外，线粒体在核结构域中的相互作用 单个成肌细胞在成熟肌管中的遗传 具有非重叠的线粒体互补 将研究mtDNA的缺失。 如果病原体 具体缺陷的机制可以阐明，这将有助于 为人类患者开发合理的治疗方法。 在这 在体外系统中，肌肉细胞的确切代谢要求 可以确定呼吸链功能受损。 在 此外，不同生长条件或处理对 可以检测突变的和野生型的mtDNA。 如果 突变的基因组可以优先受损或抑制其 复制，在未来可能会设计治疗方法， 这些疾病目前无法治愈，而且往往是致命的。

The human mitochondrial myopathies and encephalomyopathies comprise clinically, morphologically, and biochemically diverse disorders that are now beginning to be described genetically. Since 1988, specific mitochondrial DNA (mtDNA) mutations have been found to result in disease. This group of diseases generally affects differentiated (post-mitotic) tissues, such as muscle and certain neural tissues most severely. Our goal is to analyze in muscle cell cultures the phenotypic and molecular genetic consequences of mutations of mtDNA that result in myopathies and encephalomyopathies. This analysis will take advantage of a cell culture system that was developed to analyze mtDNA mutations in post-mitotic cells. The system selectively eliminates the endogenous mitochondrial complement of cells and repopulates the cells with exogenous mitochondria that contain mtDNA, including mutated mtDNA. Specific mtDNA mutations can be introduced into myoblasts that can differentiate into multinucleated myotubes and can be innervated with rat spinal cord neurons to form long-lived, remarkably mature, and functionally active myofibers. Because defined proportions of a specific mutation can be introduced into these cultures, it is feasible to study systematically and under controlled conditions the phenotypic consequences of mtDNA mutations and determine their molecular genetic causes. The specific aims of this proposal are to investigate the mechanisms by which mtDNA rearrangements impede protein synthesis and respiratory chain activity in aneural and innervated muscle cultures. In addition, the interactions of mitochondria in the nuclear domains of individual myoblasts in mature myotubes and the genetic complementation of mitochondria possessing non-overlapping deletions of mtDNA will be investigated. If the pathogenetic mechanisms of specific defects can be elucidated, it will facilitate the development of a rational therapy for human patients. In this in vitro system, the exact metabolic requirements for muscle cells with impaired respiratory chain function can be determined. In addition, the effects of different growth conditions or treatments on the mutated and wild-type mtDNAs can be examined. If the mutated genome can be preferentially damaged or inhibited in its replication, it may be possible in the future to devise treatments for these currently incurable, and often fatal diseases.