Dennis J A, Healy P J, Beaudet A L, O'Brien W E
Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030.
Proc Natl Acad Sci U S A. 1989 Oct;86(20):7947-51. doi: 10.1073/pnas.86.20.7947.
Citrullinemia is an inborn error of metabolism due to deficiency of the urea cycle enzyme, argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5]. The disease was first described in humans but was recently reported in dairy cattle in Australia. Here we report the nucleotide sequence of the normal bovine cDNA for argininosuccinate synthetase and the mutation present in animals with citrullinemia. Analysis of DNA from affected animals by Southern blotting did not readily identify the mutation in the bovine gene. RNA (Northern) blotting revealed a major reduction in the steady-state amount of mRNA in the liver of affected animals to less than 5% of controls. The bovine cDNA was cloned and sequenced and revealed 96% identity with the deduced human sequence at the amino acid level. Starting with mutant bovine liver, the mRNA was reverse-transcribed; the cDNA product was amplified with the polymerase chain reaction, cloned, and sequenced. The sequence revealed a C----T transition converting arginine-86 (CGA) to a nonsense codon (TGA). A second C----T transition represented a polymorphism in proline-175 (CCC----CCT). The mutation and the polymorphism were confirmed by amplification of genomic DNA and demonstration with restriction endonuclease enzymes of both the loss of an Ava II site in DNA from mutant animals at codon 86 and the presence or absence of a Dde I site at codon 175. The loss of the Ava II site can be used for rapid, economical, nonradioactive detection of heterozygotes for bovine citrullinemia.
瓜氨酸血症是一种由于尿素循环酶精氨琥珀酸合成酶[L-瓜氨酸:L-天冬氨酸连接酶(AMP形成),EC 6.3.4.5]缺乏导致的先天性代谢紊乱。该疾病最初在人类中被描述，但最近在澳大利亚的奶牛中也有报道。在此，我们报告了精氨琥珀酸合成酶正常牛cDNA的核苷酸序列以及患有瓜氨酸血症动物中存在的突变。通过Southern印迹法对患病动物的DNA进行分析，未能轻易鉴定出牛基因中的突变。RNA(Northern)印迹显示，患病动物肝脏中mRNA的稳态量大幅减少，降至对照组的5%以下。克隆并测序了牛cDNA，结果显示其在氨基酸水平上与推导的人类序列有96%的同一性。从突变的牛肝脏开始，将mRNA进行逆转录；用聚合酶链反应扩增cDNA产物，克隆并测序。序列显示一个C→T转换，将精氨酸-86(CGA)转变为一个无义密码子(TGA)。另一个C→T转换代表脯氨酸-175(CCC→CCT)处的多态性。通过扩增基因组DNA并用限制性内切酶证明突变动物DNA中86密码子处Ava II位点的缺失以及175密码子处Dde I位点的有无，证实了该突变和多态性。Ava II位点的缺失可用于快速、经济、非放射性检测牛瓜氨酸血症的杂合子。
