REGULATORY AND STRUCTURAL ANALYSIS OF UMP SYNTHASE

UMP合成酶是一种多功能蛋白， 磷酸核糖基转移酶和乳清酸核苷-5'-单磷酸 脱羧酶（ODCase）。 这两种酶是最后一种 UMP的从头生物合成所需的六种酶，UMP是所有酶的前体。 其他嘧啶核苷酸。 UMP合成酶在快速生长的 组织和细胞，如肿瘤来源的那些，并且已经被认为是 用于化疗靶向酶。 与肿瘤组织相比， 疾病乳清酸尿症，已被描述，并直接导致从 缺乏OPRTase和ODCase（I型）或缺乏ODCase（II型）。 慢性尿苷治疗已被证明可以克服 这种疾病 最近，一个含有埃利希基因编码序列的cDNA 腹水UMP合酶已在本实验室分离。 dna序列 cDNA的分析将允许氨基酸序列或蛋白质 被确定。 通过使用合适的表达载体，E.杆菌 或者酵母将被编程以从cDNA合成UMP合酶。 的 cDNA的编码区将通过移码突变或 顺序溶核消化。 这些DNA将被用于编程 OPRTase缺陷型和ODCase缺陷型E.大肠杆菌，以产生截短的 只补充一种缺陷的蛋白质。 这些DNA将被分析 通过限制性酶切图谱和DNA测序来确定 整个氨基酸序列构成单独的OPRTase和ODCase 催化域 UMP合酶的本纯化方案产生 由于细胞内来源低，酶广泛变性。 以来 在大肠杆菌中的表达大肠杆菌中可能导致细胞内积累更多的 超过15%的表达蛋白，这可以使得能够纯化蛋白质。 低变性蛋白质 这种蛋白质将作为一种结构来源， 实验室里已经计划好的研究。 最后，cDNA 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）细胞周期特异性的程度， UMP合成酶通过基因表达的转录调节。

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.