权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

SYNTHESIS/ASSEMBLY BRANCHED CHAIN KETOACID DEHYDROGENASE

合成/组装支链酮酸脱氢酶

基本信息

批准号：
2140465
负责人：
DEAN J DANNER
金额：
$ 21.73万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-08-15 至 1998-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2140465
关键词：
DNA footprinting aldehyde /ketone oxidoreductase embryo /fetus enzyme activity enzyme biosynthesis enzyme complex gel mobility shift assay gene expression genetic transcription human genetic material tag human tissue ketoacid laboratory mouse maple syrup urine disease mitochondria molecular cloning protein transport tissue /cell culture

项目摘要

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研究线粒体多酶的合成和组装配合物这种复合物的蛋白质在细胞核中编码，分离的基因，必须组装在一个特定的化学计量内，线粒体已知人类的遗传突变会影响这个复杂的功能。这些突变继续表达在从病人身上培养的细胞，因此可以用来研究突变体和野生型基因表达和蛋白质功能。计划这个项目是分离cDNA和基因组克隆的各种蛋白质成分这些DNA分子的核苷酸序列将并将野生型结构与突变型结构进行比较结构. 这可以很容易地做到使用聚合酶链反应来扩增基因的特定区域。细胞培养从一些表达支链脱氢酶缺陷的个体复合物活性已显示缺乏一种或另一种蛋白质组分复杂的。通过缺失成分的克隆，细胞可以用缺失成分的克隆DNA转染，功能可以确定。这样我们就可以开始定义导致枫糖浆尿病的基因水平突变。