ANALYSIS OF MTDNA REARRANGEMENTS IN POST MITOTIC CELLS

人类线粒体肌病与脑肌病 包括临床上、形态上和生化上的多样性 现在开始从基因上描述这种疾病。自.以来 1988年，特定的线粒体DNA(MtDNA)突变 被发现会导致疾病。这组疾病通常会影响 分化的(有丝分裂后)组织，如肌肉和某些 最严重的是神经组织。我们的目标是在肌肉细胞中进行分析 培养的表型和分子遗传学后果 线粒体DNA突变导致肌病和 脑肌病。这一分析将利用细胞 为分析线粒体DNA突变而开发的培养系统 有丝分裂后细胞。该系统选择性地消除了内源性 线粒体补充细胞，并使细胞重新填充 含有线粒体DNA的外源线粒体，包括突变的 线粒体DNA。特定的线粒体DNA突变可以被引入到 成肌细胞可以分化为多核肌管和 可以与大鼠脊髓神经元一起神经支配，形成长期存活的， 非常成熟，功能活跃的肌纤维。因为 特定突变的特定比例可以被引入到 对这些文化进行系统的研究是可行的。 受控条件下线粒体DNA的表型结果 突变并确定其分子遗传原因。这个 这项提案的具体目标是通过以下方式调查机制 哪些线粒体DNA重排阻碍蛋白质合成和 神经和神经肌肉培养中呼吸链的活性。 此外，核域中线粒体的相互作用 成熟肌管中的单个成肌细胞及其基因 线粒体具有互不重叠的互补性 将对线粒体DNA的缺失进行调查。如果病原体 具体缺陷的产生机理可以得到阐明，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 呼吸链功能受损的患者可以确定。在……里面 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 突变的基因组可以优先在其 复制，未来可能会设计出治疗方法 这些疾病目前是无法治愈的，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.