Abstract
The nucleotide sequence of rat carbamyl phosphate synthetase I mRNA has been determined from the complementary DNA. The mRNA comprises minimally 5,645 nucleotides and codes for a polypeptide of 164,564 Da corresponding to the precursor form of the rat liver enzyme. The primary sequence of mature rat carbamyl phosphate synthetase I indicates that the precursor is cleaved at one of two leucines at residues 38 or 39. The derived amino acid sequence of carbamyl phosphate synthetase I is homologous to the sequences of carbamyl phosphate synthetase of Escherichia coli and yeast. The sequence homology extends along the entire length of the rat polypeptide and encompasses the entire sequences of both the small and large subunits of the E. coli and yeast enzymes. The protein sequence data provide strong evidence that the carbamyl phosphate synthetase I gene of rat, the carAB gene of E. coli, and the CPA1 and CPA2 genes of yeast were derived from common ancestral genes. Part of the rat carbamyl phosphate synthetase I gene has been characterized with two nonoverlapping phage clones spanning 28.7 kilobases of rat chromosomal DNA. This region contains 13 exons ranging in size from 68 to 195 base pairs and encodes the 453 carboxyl-terminal amino acids of the rat protein. Southern hybridization analysis of rat genomic DNA indicates the carbamyl phosphate synthetase I gene to be present in single copy.
Highlights
The nucleotide sequence of rat carbamyl phosphate encoding the NR-dependent carbamyl phosphate synthetase synthetase I mRNA has been determinedfrom the com- I evolved from the genes coding for the glutamine-dependent plementary DNA
MRNA were isolated from two different cDNA libraries ( 5 )
The cDNA inserts of representative plasmids isolated from ing region of the mRNA as well as 139 nucleotides of 5'
Summary
The amino acid composition, NH,-terminal sequence, and carboxylterminal amino acidsof mature carbamyl phosphate synthetasewere determined on the rat liver enzyme judged by SDS-polyacrylamide gel electrophoresis to be 98% pure. The carboxyl-terminal sequence of the enzymewas determined by digestion of the protein with phenylmethylsulfonyl fluoride-treated carboxypeptidase A according to the procedures described by Ambler (17). Genomic DNA (20 pg) was digested to completion with appropriate restriction endonucleases, separated on 0.75% agarose gels, and transferred to nitrocellulose as described by Southern (23). Hybridization with radioactive probes was carried out in 5 X SSC, 5 X Denhardt's, 100 pg/ml salmon sperm DNA, 50 mM sodium phosphate, pH 8, 10 mM EDTA, 0.1% SDS, 50% formamide, and 10% dextran sulfate overnight a t 42 "C. Blots were exposed to Kodak XAR-5 film with an ing 4215 nucleotides of the coding sequence This clone, intensifying screen for 24-48 h
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