Molecular cloning of the cDNA coding for rat ornithine transcarbamoylase.
Molecular cloning of the cDNA coding for rat ornithine transcarbamoylase.
复制标题
编码大鼠鸟氨酸转氨甲酰酶的 cDNA 的分子克隆。
DOI：
10.1073/pnas.80.14.4258
复制
发表时间：
1983
影响因子：
11.1
通讯作者：
Rosenberg,LE
中科院分区：
文献类型：
--
作者：
Horwich,AL;Kraus,JP;Williams,K;Kalousek,F;Konigsberg,W;Rosenberg,LE
鸟氨酸转氨甲酰酶是一种线粒体基质酶，由X染色体上编码的三个相同亚基组成。该亚基作为前体在细胞质多聚核糖体上合成，在转运到线粒体期间被裂解。我们在这里报告的分离和表征的cDNA克隆含有相应的mRNA编码的鸟氨酸氨甲酰转移酶亚基的序列。cDNA合成使用大鼠肝脏mRNA富集的多核糖体免疫吸附的低丰度信使物种编码的酶亚基。将cDNA插入质粒pBR 322并在大肠杆菌中克隆后，通过（i）使用从各种组织的mRNA合成的cDNA探针的差异菌落杂交;（ii）使用从富含或耗尽鸟氨酸转氨甲酰酶信息的mRNA合成的cDNA探针的差异印迹杂交;（iii）杂交选择翻译测定;和（iv）最确定的是，结构分析，其匹配25个连续氨基酸残基，所述25个连续氨基酸残基通过纯化的酶亚基的羧基末端部分的连续Edman分析与存在于质粒之一的插入物中的编码序列相匹配。
Ornithine transcarbamoylase is a mitochondrial matrix enzyme composed of three identical subunits encoded on the X chromosome. The subunit is synthesized on cytoplasmic polysomes as a precursor that is cleaved during transport into mitochondria. We report here the isolation and characterization of cDNA clones containing sequences corresponding to the mRNA encoding the ornithine transcarbamoylase subunit. cDNA was synthesized using rat liver mRNA enriched by polysome immunoadsorption for the low-abundance messenger species encoding the enzyme subunit. After insertion of cDNA into plasmid pBR322 and cloning in Escherichia coli, identification of the desired plasmids was accomplished by (i) differential colony hybridization using cDNA probes synthesized from mRNA of various tissues; (ii) differential blot hybridization using cDNA probes synthesized from mRNA enriched for or depleted of the ornithine transcarbamoylase message; (iii) hybrid-selected translation assays; and (iv) most definitively, structural analysis, which matched 25 consecutive amino acid residues determined by sequential Edman analysis of the carboxyl-terminal portion of the purified enzyme subunit with coding sequence present in the insert of one of the plasmids.