GENETIC STUDY OF PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE

磷酸核糖焦磷酸合成酶的基因研究

• 批准号: 3426155
• 负责人: WILLIAM N KELLEY
• 金额: $ 2.37万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-01 至 1988-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3426155
• 关键词: complementary DNA; endonuclease; enzyme structure; gel electrophoresis; gene expression; genetic library; human tissue; messenger RNA; molecular cloning; molecular genetics; natural gene amplification; nucleic acid sequence; radiotracer; structural genes

Synthesis of 5-phosphoribosyl 1-pyrophosphate (PRPP), a regulatory determinant in the pathways of purine nucleotide synthesis, is catalyzed by PRPP synthetase (PS). Inherited superactivity of PS, an X chromosome-linked trait, results from an array of kinetic alterations in PS and is accompanied by gout with purine nucleotide overproduction. Much is known about the structure and kinetic properties of human PS; knowledge of the organization and control of PS gene expression in man is, however, rudimentary. The proposed Visiting Researcher has recently utilized labeled synthetic oligodeoxynucleotide probes, prepared from knowledge of partial amino acid sequence of human PS, to screen 2 human cDNA libraries for recombinant clones. Clones hybridizing with the probes have been isolated and purified. Some of these recombinants show preferential hybridization to genomic DNA enriched for or restricted to human X chromosomal DNA and appear likely to bear DNA sequences complementary to PS mRNA. The first specific aim of the proposed research is characterization of cDNAs isolated to date in order to determine their usefulness as probes for the PS gene and PS mRNA. Methods to be employed will include Southern and Northern blot analyses, DNA sequencing, and in situ hybridization. The sequences of several overlapping cDNAs will be utilized to derive the full-length sequence of PS cDNA and to deduce the amino acid sequence of PS. This amino acid sequence will be compared with primary structure data obtained by amino acid sequencing of PS peptides. These studies should expedite characterization of the human PS gene. The second specific aim is preparation of cDNA libraries derived from the mRNA of fibroblasts cultured from affected males with superactive forms of PS. Recombinant libraries will be screened for clones containing DNAs complementary to mRNA for superactive forms of PS. Sequencing of cDNA clones should permit identification of precise genetic defects associated with PS superactivity. Additional techniques required for these investigations will include mRNA isolation and cDNA library preparation. Dr. Kelley and his laboratory have extensive experience in the study of the molecular genetics of enzymes of purine metabolism. This experience encompasses the methods relevant to the research proposed. The Host's laboratory will be a unique environment for the Visiting Researcher to extend his long-term investigation of the structure and regulation of normal and aberrant forms of PS to the molecular genetic level.

5-磷酸核糖基1-焦磷酸（PRPP）的合成 嘌呤核苷酸途径中的调节决定簇 合成，由PRPP合成酶（PS）催化。 继承 PS的超活性是一种X染色体连锁性状， 一系列的动力学改变，并伴有痛风 嘌呤核苷酸过量产生。 很多人都知道 人PS的结构和动力学特性; PS基因表达的组织和控制， 然而，这是基本的。 拟议的访问研究员已 最近利用标记的合成寡脱氧核苷酸探针， 根据已知的人的部分氨基酸序列， PS，筛选2个人cDNA文库中的重组克隆。 与探针杂交的克隆已被分离， 提纯 这些重组体中的一些表现出优先性， 与富集或限制于人的基因组DNA杂交 X染色体DNA，并且似乎可能带有DNA序列 与PS mRNA互补。 拟议研究的第一个具体目标是表征 为了确定它们的有用性， 作为PS基因和PS mRNA的探针。 拟采用的方法 将包括Southern和北方印迹分析，DNA 测序和原位杂交。 几个序列 将利用重叠cDNA来获得全长cDNA， 序列，并推导出PS的氨基酸序列。 PS. 该氨基酸序列将与初级氨基酸序列进行比较。 通过PS肽的氨基酸测序获得的结构数据。 这些研究将加快人类PS的表征 基因 第二个具体目标是制备cDNA文库 来源于受影响的成纤维细胞培养的mRNA 患有超活跃型PS的男性 重组文库将 筛选含有与mRNA互补的DNA的克隆 超活性的PS cDNA克隆测序应 允许鉴定与以下疾病相关的精确遗传缺陷： PS超活性。 这些所需的其他技术 研究将包括mRNA分离和cDNA文库 准备. 博士凯利和他的实验室在这方面有着丰富的经验， 嘌呤代谢酶的分子遗传学研究。 这一经验包括与下列方面有关的方法： 研究提出。 宿主的实验室将是独一无二的 为访问研究员提供长期的环境， 研究正常和 PS的异常形式到分子遗传水平。