UMP SYNTHASE AND THE MOLECULAR BASIS OF OROTIC ACIDURIA

UMP 合酶和乳清酸尿的分子基础

• 批准号: 3154568
• 负责人: DALE P SUTTLE
• 金额: $ 9.19万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-05-01 至 1987-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3154568
• 关键词: autosomal recessive trait; fibroblasts; gene expression; genetic disorder diagnosis; genetic library; human tissue; isozymes; messenger RNA; molecular pathology; nucleic acid sequence; orotate phosphoribosyltransferase; orotic aciduria; orphan disease /drug; tissue /cell culture

Human UMP synthase is a bifunctional protein of approximately 55,000 daltons comprising the two enzymic activities orotate phosphoribosyltransferase and orotidylate decarboxylase in a single polypeptide chain. The activities catalyze the last two steps of de novo uridine-5' monophosphate (UMP) biosynthesis, the conversion of orotic acid to orotidine-5'-monophosphate (OMP) and OMP to UMP. In the human autosomal recessive disease, hereditary orotic aciduria, there is a severe deficiency of both activities of UMP synthase (Type I) or a deficiency of only the decarboxylase (Type II). This disease is the only known human disorder of pyrimidine nucleotide biosynthesis. Fibroblast cell lines are available in culture from three patients with orotic acidura. Using UMP synthase-specific cDNA probes the underlying molecular defects of orotic aciduria will be characterized. UMP synthase DNA, mRNA and protein from normal and deficient cells will be analyzed for alterations in the amount or structure of each component. Using the M13 sequencing methods, the nucleotide sequence of cloned UMP synthase cDNAs will be directly determined and the sequences of the corresponding mRNA and protein deduced. The structure of the UMP synthase gene will be outlined by isolation of DNA fragments from human genomic Gamma libraries and analysis of coding and non-coding regions by restriction mapping and Southern hybridization techniques. These studies into the nature of the mutations in orotic aciduria and the structure of the normal and deficient UMP synthase gene will enhance our understanding of human genetic defects. The techniques will improve our ability to diagnosis and eventually to treat human genetic diseases. The UMP synthase-deficient cells also provide an opportunity to study the regulation of gene expression. When certain drugs are added to the growth media of the deficient cells, the activity for UMP synthase is increased to near normal levels. The mechanism for this increased expression will be studied by determining levels and structures of VMP synthase mRNA in normal and deficient cells following growth in the presence of the drugs. DNA from the cell lines will also be analyzed for possible secondary UMP synthase coding sequences.

人UMP合酶是一种双功能蛋白质，约55，000 包含两种酶活性乳清酸盐的道尔顿 磷酸核糖基转移酶和乳清酸脱羧酶 多肽链 这些活动催化了de novo的最后两步 尿苷-5 '-磷酸（UMP）生物合成，乳清酸的转化 乳清酸核苷-5 '-单磷酸（OMP）和OMP至UMP。 在人类常染色体 隐性疾病，遗传性乳清酸尿症，有一个严重的不足 UMP合酶（I型）的两种活性或仅缺乏 脱羧酶（II型）。 这种疾病是唯一已知的人类疾病， 嘧啶核苷酸生物合成。 成纤维细胞系可在 三名乳清酸尿患者的培养物。 使用UMP 乳清蛋白酶特异性cDNA探针检测乳清蛋白酶的潜在分子缺陷 酸尿将被表征。 UMP合成酶DNA、mRNA和蛋白质来自 将分析正常和缺陷细胞的量的改变 或每个组件的结构。 使用M13测序方法， 克隆的UMP合酶cDNA的核苷酸序列将直接 确定相应的mRNA和蛋白质的序列 推导 UMP合酶基因的结构将由以下概述 从人类基因组γ文库中分离DNA片段并分析 通过限制性酶切作图和Southern杂交， 杂交技术 这些对突变本质的研究 乳清酸尿症和正常和缺乏UMP的结构 合成酶基因将增强我们对人类遗传缺陷的理解。 的 技术将提高我们的诊断能力， 人类遗传疾病。 UMP脱氢酶缺陷细胞也提供了一个研究 基因表达的调控。 当某些药物被添加到生长的 在缺陷细胞的培养基中，UMP合酶的活性增加至 接近正常水平。 这种表达增加的机制将是 通过测定正常人VMP合成酶mRNA的水平和结构， 以及在药物存在下生长后的缺陷细胞。 DNA 还将分析可能的继发性UMP 合成酶编码序列。