SYNTHESIS/ASSEMBLY BRANCHED CHAIN KETOACID DEHYDROGENASE

大多数线粒体是由核上编码的蛋白质提供的 基因。其中许多蛋白质相互作用，形成多酶复合体。 内膜的基质侧。这项提议旨在 阐明其中一个线粒体基因表达的具体步骤 人支链α-酮酸脱氢酶[BCKD]。这 复合体是有意义的，因为遗传突变发生在人类 降低BCKD的功能，导致一种被称为枫叶的表型 糖浆尿病[MSUD]三种基因产品是独一无二的 BCKD的催化成分，并提供这些研究的重点。五 与这三个基因相关的具体目标也被提及。1.使用克隆 直接上游的600个碱基对的DNA片段 各基因转录起始点、顺式元件和反式作用 DNA结合蛋白将使用足迹和凝胶进行鉴定 迟滞分析。三个推动者之间的相似之处将是 被追寻。2.已知培养的人类细胞的培养条件会改变 单个亚基的信使核糖核糖核酸的量。研究的目的是 区分是转录改变还是信使核糖核酸稳定性引起 变化以及这些变化如何影响BCKD活动。3.由于蛋白质 按照已知的化学计量比组装成复合体，研究要解决的问题 这些前蛋白的输入是否相互影响并发挥作用 在发展化学计量学方面。4.BCKD在胎儿中的表达从未 已经研究过了。小鼠胚胎将被用来定义基因模式 在整个胎儿发育过程中都有表达。重要的是要了解 这一模式提供了更好的怀孕管理 杂合子和新出现的纯合子MSUD母亲。5. 对这些基因的额外突变进行表征将导致 更好地了解基因型/表型关系和患者 管理层。了解这些突变将指导未来对 BCKD的组装和功能。最终目标是提供基因疗法。 用于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.