INBORN ERRORS OF A PURINE SALVAGE PATHWAY

腺嘌呤磷酸核糖基转移酶(APRT)是一种嘌呤回收酶， 利用腺嘌呤和5-磷酸核糖基-1-焦磷酸生产腺苷 单磷酸盐和焦磷酸盐。在人类中，完全的aprt缺乏是一种 据推测，一种罕见的先天性嘌呤代谢错误，遗传于 常染色体隐性遗传方式。腺嘌呤，主要是一种副产品 多胺的生物合成，在APRT缺乏时积累到高水平 最终被氧化成2，8-二羟基腺嘌呤(DHA)。 虽然这种缺陷可能是相对良性的，但DHA是肾毒性的，可以 导致危及生命的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 比目前公认的更频繁，想必是由于高度 临床表现多变，加上诊断上的问题。 我们已经克隆了一个有功能的人类aprt基因，并确定了 其编码区和内含子的核苷酸序列。我们也有手机 APRT缺陷症患者和APRT缺陷型人细胞克隆的培养 从体外正常体细胞中选择获得。使用我们的克隆人 为了探测这些细胞，我们将确定 人类APRT缺乏症的表达。通过比较突变的aprt基因在 从培养的人类体细胞中获得的人的数量 将确定体细胞在体外是否发生突变，从而形成 大多数诱变试验的基础与 生殖细胞的原位突变。突变的细胞核酸将被分析 经Southern和Northern杂交，RNase A对DNA/RNA分子的消化 杂交种和DNA序列分析。突变的aprt蛋白也将是 用针对正常人N或C末端的抗血清进行分析 酵素。

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.