SYNTHESIS/ASSEMBLY-BRANCHED CHAIN KETOACID DEHYDROGENASE

合成/组装支链酮酸脱氢酶

• 批准号: 3237630
• 负责人: DEAN J DANNER
• 金额: $ 17.8万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-08-15 至 1994-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3237630
• 关键词: complementary DNA; enzyme biosynthesis; enzyme complex; enzyme mechanism; fibroblasts; gel electrophoresis; gene expression; gene mutation; genetic library; genetic manipulation; genetic regulatory element; genetic transcription; human subject; human tissue; immunochemistry; immunoelectrophoresis; in situ hybridization; ketoacid; maple syrup urine disease; messenger RNA; mitochondria; molecular cloning; monoclonal antibody; natural gene amplification; nucleic acid probes; oxidoreductase; tissue /cell culture

Branched chain alpha-ketoacid dehydrogenase provides an excellent model by which to study the synthesis and assembly of mitochondrial multienzyme complexes. The proteins of this complex are encoded in the nucleus on separate genes and must assemble in a specific stoichiometry within the mitochondria. Inherited mutations in humans are known to affect the function of this complex. These mutations continue to be expressed in cells cultured from the patients and therefore can be used to study mutant and wild type gene expression and protein function. The plan of this project is to isolate cDNA and genomic clones for the various protein components. The nucleotide sequence for these DNA molecules will be determined and the wild type structures compared with mutant structures. This can easily be done using the polymerase chain reaction to amplify specific regions of the genes. Cells cultured from some individuals expressing defects in the branched chain dehydrogenase complex activity have been shown to lack one or another protein component of the complex. With clones for the missing component the cells can be transfected with the cloned DNA for the missing component and return of function can be determined. In this way we can begin to define the mutations at the gene level which give rise to Maple Syrup Urine disease.

支链α-酮酸脱氢酶提供了一个很好的模型 用它研究线粒体多酶的合成和组装 复合体。这种复合体的蛋白质在细胞核中编码。 分离的基因必须在特定的化学计量比中组装在 线粒体。已知人类中的遗传突变会影响 这个综合体的功能。这些突变继续在 从患者身上培养出来的细胞，因此可以用来研究 突变型和野生型基因表达及蛋白功能。的计划 这个项目是为了分离各种不同物种的cdna和基因组克隆。 蛋白质成分。这些DNA分子的核苷酸序列将 与突变体的野生型结构比较 结构。这可以通过聚合酶链式反应很容易地完成。 来扩增基因的特定区域。从一些地方培养出来的细胞 表达支链脱氢酶缺陷的个体 复杂的活性已被证明缺乏一种或另一种蛋白质成分 整个建筑群的。有了缺失成分的克隆，细胞可以 用克隆的DNA转染缺失的成分并返回 功能可以确定。通过这种方式，我们可以开始定义 导致枫糖尿症的基因水平上的突变。