Iwahana H, Fujimura M, Ii S, Kondo M, Moritani M, Takahashi Y, Yamaoka T, Yoshimoto K, Itakura M
Otsuka Department of Clinical and Molecular Nutrition, University of Tokushima, Japan.
Biochem Biophys Res Commun. 1996 Feb 6;219(1):249-55. doi: 10.1006/bbrc.1996.0213.
A human CAD cDNA encoding a trifunctional enzyme of carbamoylphosphate synthetase-aspartate transcarbamoylase-dihydroorotase, which catalyzes the first three steps of de novo pyrimidine nucleotide biosynthesis, was cloned from a human fibroblast cell line of TIG-1-20 by polymerase chain reaction (PCR). The predicted open reading frame encodes a protein of 2,225 amino acids with a deduced molecular weight (Mr) OF 242,913. The deduced amino acid sequence exhibits 95.3 and 76.1% identity with the CAD sequences of hamster and Squalus acanthias. The DNA fragment of 6,679 bp containing the full-length coding sequence was amplified by nested PCR using the first-strand cDNA of human cell lines of TIG-1-20 and COLO205 as a template. Southern blot analysis suggested that the CAD gene exists as a single copy in the human genome.
通过聚合酶链反应（PCR）从TIG-1-20人成纤维细胞系中克隆出一个编码氨甲酰磷酸合成酶-天冬氨酸转氨甲酰酶-二氢乳清酸酶三功能酶的人CAD cDNA，该酶催化从头嘧啶核苷酸生物合成的前三个步骤。预测的开放阅读框编码一个2225个氨基酸的蛋白质，推导分子量（Mr）为242913。推导的氨基酸序列与仓鼠和棘鲨的CAD序列分别具有95.3%和76.1%的同一性。以TIG-1-20和COLO205人细胞系的第一链cDNA为模板，通过巢式PCR扩增出包含全长编码序列的6679 bp DNA片段。Southern印迹分析表明CAD基因在人类基因组中以单拷贝形式存在。
