SOMATIC CELL GENETIC ANALYSIS OF PURINE SYNTHESIS

嘌呤合成的体细胞遗传学分析

• 批准号: 2764004
• 负责人: DAVID PATTERSON
• 金额: $ 22.96万
• 依托单位: ELEANOR ROOSEVELT INST FOR CANCER RES
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2764004
• 关键词: CHO cells; amidophosphoribosyltransferase; complementary DNA; cytogenetics; enzyme activity; enzyme complex; gene environment interaction; gene mutation; genetic regulation; genetic transcription; human genetic material tag; human tissue; hydroxymethyltransferases; hypoxanthine phosphoribosyltransferase; immunocytochemistry; immunoprecipitation; laboratory rabbit; northern blottings; nucleic acid sequence; polymerase chain reaction; posttranslational modifications; protein protein interaction; protein structure function; purine /pyrimidine metabolism; tissue /cell culture; western blottings

Purines are fundamental biological molecules. They are components of DNA and RNA, the genetic information of all living organisms. Purines are critical as intracellular signaling molecules in the form of cAMP and cGMP, and are important intercellular signaling molecules functioning as neurotransmitters and as signaling molecules important for vasodilation and other physiological processes. ATP serves as a key molecule in energy metabolism. Many coenzymes are purine based. Genetic and biochemical regulation of purine synthesis in mammals and humans must be complex, involving several enzymatic steps and proteins. The pathway is almost certainly developmentally regulated in mammals and humans as well. These conclusions are based almost entirely on studies of the biochemistry of the pathway in whole cells or in cell free extracts. That such regulation must exist seems obvious. However, the molecular mechanisms of regulation remain to be defined. In no case has a detailed molecular analysis of the regulation of purine synthesis in an animal or in animal cells in culture been carried out. This is largely because until now the molecular reagents necessary have not existed. Without this information, it will be difficult to understand the role of the pathway in human disease and to intervene appropriately. The first seven steps of the pathway and genes encoding these are critically involved in the environmental, developmental, and genetic regulation of the pathway, and propose to use a novel set of reagents and capabilities, including human and Chinese hamster cDNA and genomic clones, antibodies recognizing functional domains of the proteins carrying out the fist seven steps and which are suitable for Western blotting, immuneprecipitation, and immunocytochemistry, and Chinese hamster ovary (CHO) cell mutants to understand these regulatory mechanisms. The roles of multi-enzyme complexes, multi functional proteins, transcriptional, translational, and posttranslational regulation of these five enzymatic steps in the regulation of purine synthesis will be evaluated. The following specific aims are proposed: (1) Determination of the nature and effects of mutations in CHO cells in the first seven steps of the pathway on the regulation of purine synthesis and purine levels; (2) Determination of transcriptional translational, and posttranslational regulation of these enzymes in response to alterations in environmental conditions using CHO cell cultures; (3) Determination of transcriptional, translational or posttranslational regulation of the first seven steps of the pathway during human and mouse development, (4) Assessment of the existence of multi-enzyme complexes involving the first seven steps of the de novo pathway and assessment of whether these complexes have regulatory significance.

嘌呤是基本的生物分子。 它们是 DNA和RNA，所有生物体的遗传信息。 嘌呤 作为cAMP形式的细胞内信号分子， 和cGMP，是重要的细胞间信号分子 作为神经递质和信号分子， 用于血管舒张和其他生理过程。 ATP是关键 分子在能量代谢中的作用 许多辅酶是嘌呤基的。 哺乳动物嘌呤合成的遗传和生化调节 人体一定是复杂的，涉及几个酶步骤和蛋白质。 几乎可以肯定，在哺乳动物中， 人类也是。 这些结论几乎完全基于研究 在整个细胞或无细胞中， 萃取物 这种监管必须存在，这似乎是显而易见的。 但 调控的分子机制仍有待确定。 在任何情况下， 嘌呤合成调控的详细分子分析， 在动物或动物细胞培养中进行。这是 很大程度上是因为到目前为止所需的分子试剂还没有 存在过。 如果没有这些信息， 该途径在人类疾病中的作用，并进行适当的干预。 该途径的前七个步骤和编码这些步骤的基因是 在环境、发育和遗传方面有着重要的参与， 调节途径，并建议使用一套新的试剂 和能力，包括人类和中国仓鼠cDNA和基因组 克隆、识别蛋白质功能域的抗体 实施前七步，哪些适合西方 免疫印迹、免疫沉淀和免疫细胞化学， 仓鼠卵巢（CHO）细胞突变体，以了解这些调控 机制等 多酶复合物，多功能蛋白质， 转录、翻译和翻译后调节 嘌呤合成调节中的这五个酶促步骤将 被评价。 建议的具体目标如下：（1） 测定CHO细胞中突变的性质和影响， 嘌呤合成调节途径的前七步 和嘌呤水平;（2）确定转录翻译， 以及这些酶的翻译后调节， 使用CHO细胞培养物改变环境条件;（3） 确定转录、翻译或翻译后 在人类和哺乳动物期间， 小鼠发育，（4）多酶存在的评估 涉及从头途径的前七个步骤的复合物， 评估这些复合物是否具有调节意义。