Complete hamster CAD protein and the carbamylphosphate synthetase domain of CAD complement mammalian cell mutants defective in de novo pyrimidine biosynthesis.
The mammalian CAD gene codes for a 240-kDa multifunctional protein that catalyzes the first three steps of de novo pyrimidine biosynthesis. Previously, the longest cDNA construct available was missing approximately 500 bp of coding sequence at the 5' end, thereby lacking the sequence to encode the entire carbamylphosphate synthetase (CPSase) domain. Here, a complete CAD hamster cDNA is constructed, placed into a mammalian expression vector, and transfected into hamster cells deficient in CAD. Transfectants show coordinately restored levels of all three enzyme activities and the presence of full-length CAD protein. A derivative construct of the CAD cDNA was generated that should encode only the CPSase domain. When transfected into mammalian cells, a protein was synthesized that had significant CPSase activity both in vivo and in vitro. The two constructs generated in this study will facilitate the study of CAD structure, function, and allosteric regulation.
Department of Microbiology and Immunology, University of Kentucky, Lexington 40536-0084.
L A
LA
Musmanno
R S
RS
Jamison
R S
RS
Barnett
E
E
Buford
J N
JN
Davidson
D000595:Amino Acid Sequence; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000235:genetics*; D001483:Base Sequence; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004080:Dihydroorotase / Q000235:genetics*; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D011487:Protein Conformation; D011743:Pyrimidines / Q000096:biosynthesis*
C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D011743:Pyrimidines; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)