SYNTHESIS/ASSEMBLY BRANCHED CHAIN KETOACID DEHYDROGENASE

大多数线粒体由核上编码的蛋白质提供 基因.这些蛋白质中的许多相互作用形成多酶复合物， 内膜的基质侧。这项建议旨在 阐明其中一种线粒体基因表达的具体步骤 复合物，人支链α-酮酸脱氢酶[BCKD]。这 复杂的是感兴趣的，因为遗传突变发生在人类， 降低BCKD的功能，导致称为枫树的表型 糖浆尿病。三种基因产物是独特的， BCKD的催化组分，并提供这些研究的重点。五 针对这三个基因提出了具体目标。1.使用克隆 从每一个DNA片段的上游600 bp的DNA片段， 每个基因的转录起始位点，顺式元件和反式作用元件 DNA结合蛋白将使用足迹法和凝胶电泳法进行鉴定。 迟滞分析三位发起人的相似之处将是 寻求。2.用于培养的人细胞的培养基条件是已知的， 单个亚基的mRNA量。研究旨在 区分是否改变转录或mRNA稳定性导致这些 这些变化以及这些变化如何影响BCKD活性。3.因为蛋白质 组装成具有已知化学计量的复合物， 这些前体蛋白的输入是否相互影响， 化学计量的发展。4. BCKD的胎儿表达从未 本文研究了小鼠胚胎将用于定义基因的模式 在整个胎儿发育过程中表达。重要的是要了解 这种模式提供了改善的怀孕管理， 杂合子和新出现的纯合子MSUD母亲。5. 这些基因中的额外突变的表征将导致 更好地理解基因型/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。

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.