REGULATORY AND STRUCTURAL ANALYSIS OF UMP SYNTHASE

UMP合成酶是一种多功能蛋白质，含有两种口香酸 磷酸核糖转移酶(OPRTase)和核苷-5‘-单磷酸 脱羧酶(ODCase)。这两种酶是一系列 UMP的前体UMP从头合成所需的六种酶 其他的嘧啶核苷酸。UMP合成酶在快速生长中升高 组织和细胞，如那些起源于肿瘤的组织和细胞，被认为是 用于化疗的靶酶。与肿瘤组织相比，基因 口腔性酸尿是一种疾病，已被描述，其直接原因是 缺乏OPRTase和ODCase(I型)或缺乏ODCase(II型)。 慢性尿苷疗法已被证明可以克服 这种疾病。最近，一个含有Ehrlich编码序列的cDNA 腹水UMP合成酶已在本实验室分离得到。DNA序列 对cdna的分析将允许氨基酸序列或蛋白质 要下定决心。通过使用适当的表达载体E.Coli 或者酵母将被编程从cDNA合成UMP合成酶。这个 C DNA的编码区会因移码突变或 顺序核裂解消化。这些DNA将用于编程 OPRTase缺陷型和ODCase缺陷型大肠杆菌生产截短型 只补充一种缺陷的蛋白质。这些DNA将被分析 通过限制性内切酶图谱和DNA测序来确定 整个氨基酸序列构成了单独的OPRTase和ODCase 催化域。目前UMP合成酶产量的纯化方案 由于胞内来源低，酶的变性程度很高。自.以来 在大肠杆菌中的表达可能导致更大的细胞内积累 超过15%的表达蛋白，这可能使一种 变性程度较低的蛋白质。这种蛋白质将作为结构蛋白的来源 实验室里已经计划好的研究。最后，cDNA3 将被用作探针来测量培养细胞中的mRNA水平 确定(A)细胞周期特异性和(B) 基因表达对UMP合成酶的转录调控。

UMP synthase is a multifunctional protein containing both orotate phosphoribosyltransferase (OPRTase) and orotidine-5'-monophosphate decarboxylase (ODCase). These two enzymes are the last of a sequence of six enzymes required for de novo biosynthesis of UMP, the precursor of all other pyrimidine nucleotides. UMP synthase is elevated in rapidly growing tissues and cells such as those of tumor origin and has been considered a target enzyme for chemotherapy. In contrast to tumor tissue, the genetic disease, orotic aciduria, has been described and directly results from a lack of both OPRTase and ODCase (Type I) or a lack of ODCase (Type II). Chronic uridine therapy has been shown to overcome the manifestations of the disease. Recently, a cDNA containing the coding sequence of Ehrlich ascites UMP synthase has been isolated in this laboratory. DNA sequence analysis of the cDNA will allow the amino acid sequence or the protein to be determined. By the use of the appropriate expression vectors, E. coli or yeast will be programmed to synthesize UMP synthase from the cDNA. The coding region of the cDNA will be altered by frameshift mutation or by sequential nucleolytic digestion. These DNAs will be used to program OPRTase-deficient and ODCase-deficient E. coli to produce truncated proteins which complement only one deficiency. These DNAs will be analyzed by restriction mapping and DNA sequencing to determine which amino acids of the entire amino acid sequence constitute the individual OPRTase and ODCase catalytic domains. The present purification scheme of UMP synthase yields extensively denatured enzyme due to the low intracellular source. Since expression in E. coli may lead to intracellular accumulation of greater than 15% of the expressed protein, this may enable the purification of a less-denatured protein. This protein will serve as a source for structural studies which are already planned in the laboratory. Finally, the cDNA will be used as a probe to measure the mRNA levels in cultured cells to determine (a) the cell-cycle specificity and (b) the degree of transcriptional regulation of UMP synthase by gene expression.