Molecular cloning of a human cDNA encoding a trifunctional enzyme of carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase in de Novo pyrimidine synthesis.
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.
Iwahana
H
H
Otsuka Department of Clinical and Molecular Nutrition, University of Tokushima, Japan.
Fujimura
M
M
Ii
S
S
Kondo
M
M
Moritani
M
M
Takahashi
Y
Y
Yamaoka
T
T
Yoshimoto
K
K
Itakura
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D004080:Dihydroorotase / Q000096:biosynthesis* / Q000235:genetics; D004284:Dogfish; D005347:Fibroblasts; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011743:Pyrimidines / Q000096:biosynthesis; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
C033626:CAD trifunctional enzyme; D017931:DNA Primers; D018076:DNA, Complementary; D009097:Multienzyme Complexes; D011743:Pyrimidines; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)