MOLECULAR CLONING OF HUMAN UMP SYNTHASE

为了明确遗传性羊角性酸尿的原因，从正常人红细胞中纯化了UMP合成酶（一种同时具有羊角酸磷酸基转移酶和OMP脱羧酶活性的多功能蛋白）。酶蛋白的分子量为51,000。利用合成的寡核苷酸探针从<lambda>gtll小鼠脾脏cDNA文库中分离出小鼠OMP脱羧酶cDNA片段。筛选<lambda>gtll人胎盘cDNA文库，获得两个阳性克隆，与小鼠cDNA片段杂交强烈。对其中一个克隆的全长cDNA插入片段（1.7 kb）的核苷酸序列分析表明，其包含一个开放阅读框（1,444 bp），编码一个分子量为52,000的蛋白。该插入物3′-一半的氨基酸序列与从埃利希腹水癌OMP脱羧酶cDNA中推断的氨基酸序列同源性为89%。Northern blot分析显示，该cDNA中存在约1.8 kb的单条带，表明克隆的cDNA包含了人类UMP合酶的全部信息。

In order to clarify the cause of hereditary orotic aciduria, UMP synthase (a multifunctional protein having both orotate phosporibosyltransferase and OMP decarboxylase activities) was purified from normal human erythrocytes. The enzyme protein had a molecular weight of 51,000. Then a cDNA fragment of mouse OMP decarboxylase was isolated from a <lambda>gtll mouse spleen cDNA library by the use of a synthesized oligonucleotide probe. Screening of a <lambda>gtll human placenta cDNA library yielded two positive clones which hybridized strongly to the mouse cDNA fragment. Analysis of the nucleotide sequence of the entire cDNA insert (1.7 kb) of one of the clones indicated that it contained an open reading frame (1,444 bp) encoding a protein with a molecular weight of 52,000. The deduced amino acid sequence of the 3'-half of the insert showed 89% homology with that deduced from Ehrlich ascites carcinoma OMP decarboxlase cDNA. Northern blot analysis revealed a presence of a single band of approximately 1.8 kb, which suggests that the cloned cDNA contains the whole message for human UMP synthase.