SYNTHESIS/ASSEMBLY BRANCHED CHAIN KETOACID DEHYDROGENASE

合成/组装支链酮酸脱氢酶

• 批准号: 2140466
• 负责人: DEAN J DANNER
• 金额: $ 22.29万
• 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 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2140466
• 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

The majority of mitochondria are furnished by proteins encoded on nuclear genes. Many of these proteins interact to form multienzyme complexes on the matrix side of the inner membrane. This proposal is designed to elucidate specific steps in gene expression for one of these mitochondrial complexes, human branched chain alpha-ketoacid dehydrogenase [BCKD]. This complex is of interest since inherited mutations occur in humans which decrease the function of BCKD resulting in a phenotype known as maple syrup urine disease [MSUD]. Three gene products are unique for the catalytic components of BCKD and provide the focus of these studies. Five specific aims are addressed related to these three genes. 1. Using cloned DNA fragments for the immediate 600 bp upstream from each of the transcriptional start site of each gene, the cis elements and trans-acting DNA binding proteins will be identified using footprinting and gel retardation analysis. Similarities among the three promoters will be sought. 2. Media conditions for cultured human cells are known which alter the amount of mRNA for the individual subunits. Studies are designed to differentiate whether altered transcription or mRNA stability cause these changes and how the changes affect BCKD activity. 3. Since the proteins assemble into the complex with known stoichiometry, studies to address whether the import of these preproteins affect each other and play a role in developing the stoichiometry. 4. Fetal expression of BCKD has never been studied. Murine embryos will be used to define the pattern of gene expression throughout fetal development. It is important to understand this pattern to provide improved management of pregnancies of the heterozygote and newly emerging homozygous MSUD mothers. 5. Characterization of additional mutations in these genes will lead to a better understanding of genotype/phenotype relations and patient management. Understanding the mutations will direct future studies on the assembly and function of BCKD. An ultimate goal is to provide gene therapy for MSUD.

大多数线粒体由核上编码的蛋白质提供 基因。许多这些蛋白质相互作用形成多酶复合物 内膜的基质侧。该提案旨在 阐明这些线粒体之一的基因表达的具体步骤 复合物，人支链α-酮酸脱氢酶[BCKD]。这 复杂的现象引起了人们的兴趣，因为遗传突变发生在人类身上， 降低 BCKD 功能，导致称为枫树的表型 糖浆尿病[MSUD]。三种基因产物是独一无二的 BCKD 的催化成分并提供了这些研究的重点。五 解决与这三个基因相关的具体目标。 1.使用克隆 每个片段上游紧邻 600 bp 的 DNA 片段 每个基因的转录起始位点、顺式元件和反式元件 DNA 结合蛋白将使用足迹法和凝胶进行鉴定 延迟分析。三个发起人之间的相似之处是 寻求。 2. 已知培养人类细胞的培养基条件会改变 各个亚基的 mRNA 量。研究旨在 区分转录改变或 mRNA 稳定性是否导致这些 变化以及这些变化如何影响 BCKD 活动。 3.由于蛋白质 以已知的化学计量组装成复合物，研究解决 这些前蛋白的导入是否相互影响并发挥作用 发展化学计量。 4.BCKD的胎儿表达从未 被研究过。小鼠胚胎将用于定义基因模式 在整个胎儿发育过程中表达。了解这一点很重要 这种模式可以改善怀孕管理 杂合子和新出现的纯合子 MSUD 母亲。 5. 这些基因中额外突变的表征将导致 更好地了解基因型/表型关系和患者 管理。了解突变将指导未来的研究 BCKD的组装和功能。最终目标是提供基因治疗 对于 MSUD。