UMP SYNTHASE AND THE MOLECULAR BASIS OF OROTIC ACIDURIA

人UMP合成酶是一种双功能蛋白，约有55,000个 道尔顿由两种酶活性组成的Orotate 磷酸核糖基转移酶和核苷酸脱羧酶 多肽链。这些活动催化了从头开始的最后两个步骤 尿苷-5‘-单磷酸(UMP)的生物合成、花生酸的转化 奥替替丁-5‘-单磷酸(OMP)和OMP对UMP。在人类常染色体中 隐性疾病，遗传性口腔性酸尿，有严重的虚证 两种UMP合酶活性(I型)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 3例口腔性酸中毒患者的培养。使用UMP 合酶特异的cDNAs探查口腔畸形潜在的分子缺陷 酸尿症的特征将是。UMP合成酶DNA、mRNA和蛋白质的研究 将分析正常和缺陷细胞的数量变化。 或每个组件的结构。使用M13测序方法， 克隆的UMP合成酶cDNA的核苷酸序列将直接 确定了相应的mRNA和蛋白质的序列 推断出来的。UMP合成酶基因的结构将通过以下方式概述 人基因组伽玛文库中DNA片段的分离与分析 用限制性图谱和Southern方法对编码区和非编码区的研究 杂交技术。这些对突变性质的研究 口腔性酸尿症与正常和缺陷UMP的结构 合酶基因将加深我们对人类遗传缺陷的理解。这个 技术将提高我们诊断并最终治疗的能力 人类遗传病。 UMP合酶缺陷细胞也为研究 基因表达的调控。当某些药物被添加到生长中时 在缺陷细胞的培养液中，UMP合成酶的活性增加到 接近正常水平。这种增加表达的机制将是 正常人VMP合成酶基因表达水平和结构的研究 以及在药物存在下生长的缺陷细胞。脱氧核糖核酸 也将分析可能的二次UMP 合酶编码序列。

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.