INBORN ERRORS OF A PURINE SALVAGE PATHWAY

嘌呤挽救途径的先天性错误

• 批准号: 3237451
• 负责人: JAY Arnold TISCHFIELD
• 金额: $ 18.04万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 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 1991-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3237451
• 关键词: adenine; adenine phosphoribosyltransferase; antiserum; biological polymorphism; cell transformation; clone cells; epidemiology; gel electrophoresis; gene expression; genetic promoter element; human tissue; inborn metabolism disorder; laboratory rabbit; messenger RNA; molecular cloning; molecular genetics; nucleic acid hybridization; nucleic acid sequence; pancreatic ribonuclease; purine /pyrimidine metabolism disorder

Adenine phosphoribosyltransferase (APRT), an enzyme of purine salvage, utilizes adenine and 5-phosphoribosyl-1-pyrophosphate to produce adenosine monophosphate and pyrophosphate. In man, complete APRT deficiency is a supposedly rare inborn error of purine metabolism that is inherited in an autosomal recessive manner. Adenine, which is primarily a byproduct of polyamine biosynthesis, accumulates to high levels in APRT deficient individuals and is ultimately oxidized to 2,8-dihydrixyadenine (DHA). Although the defect can be relatively benign, DHA is nephrotoxic and can lead to life-threatening DHA urolithiasis. A relatively high frequency of apparent heterozygosity for this disorder suggests that homozygosity could be more frequent than is currently recognized, presumably due to highly variable clinical expression coupled with problems of diagnosis. We have cloned a functional human APRT gene and have determined the nucleotide sequence of its coding regions and introns. We also have cell cultures from APRT deficient patients and APRT deficient human cell clones obtained by selection from normal somatic cells in vitro. Using our cloned gene to probe these cells, we will determine the molecular bases for expression of human APRT deficiency. By comparing mutant APRT genes in the human population to those obtained from cultured, human somatic cells we will ascertain whether or not somatic cell mutation in vitro, which forms the basis of most mutagenesis assays, is qualitatively different from mutation in germ cells in situ. Mutant cell nucleic acids will be analyzed by Southern and northern blots, RNase A digestion of DNA/RNA molecular hybrids and DNA sequence analysis. Mutant APRT proteins will also be analyzed using antisera directed against the N or C-termini of the normal enzyme.

腺嘌呤磷酸核糖转移酶（APRT）是一种嘌呤补救酶， 利用腺嘌呤和5-磷酸核糖-1-焦磷酸产生腺苷， 单磷酸盐和焦磷酸盐。 在人类中，完全APRT缺乏是一种 一种被认为是罕见的先天性嘌呤代谢缺陷， 常染色体隐性遗传方式。 腺嘌呤主要是 多胺生物合成，积累到高水平的APRT缺陷 个体，并最终氧化为2，8-二甲氧基腺嘌呤（DHA）。 虽然这种缺陷可能是相对良性的，但DHA具有肾毒性， 导致危及生命DHA尿石症。 相对较高的频率 这种疾病的明显杂合性表明纯合性可能 比目前认识到的更频繁，可能是由于高度 多变的临床表现加上诊断问题。 我们已经克隆了一个有功能的人APRT基因，并确定了其表达。 其编码区和内含子的核苷酸序列。 我们也有细胞 来自APRT缺陷患者和APRT缺陷人细胞克隆的培养物 通过从体外正常体细胞中选择获得。 利用我们克隆的 基因来探测这些细胞，我们将确定 人APRT缺陷的表达。 通过比较突变的APRT基因， 从人类群体中获得的那些， 将确定体细胞是否在体外发生突变， 大多数诱变试验的基础，在性质上不同于 生殖细胞的原位突变。 将分析突变细胞核酸 通过Southern和北方印迹，RNA酶A消化DNA/RNA分子 杂交和DNA序列分析。 突变的APRT蛋白也将是 使用针对正常人的N或C末端的抗血清进行分析， 酵素