Open AccessMolecular cloning of the cDNA coding for rat ornithine transcarbamoylase.
Author(s) -
Arthur L. Horwich,
Jan P. Kraus,
Kenneth R. Williams,
František Kalousek,
William H. Konigsberg,
León E. Rosenberg
Publication year - 1983
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.80.14.4258
Subject(s) - complementary dna , microbiology and biotechnology , biology , ornithine carbamoyltransferase , biochemistry , ornithine transcarbamylase , peptide sequence , protein subunit , polysome , messenger rna , amino acid , rna , gene , ornithine , ribosome , urea cycle , arginine
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.