Isolation of a cDNA clone for the type I regulatory subunit of bovine cAMP-dependent protein kinase

• Published in: Proceedings of the National Academy of Sciences - PNAS 1983-06, Vol.80 (12), p.3608-3612
• Main Authors: Lee, David C., Carmichael, David F., Krebs, Edwin G., McKnight, G. Stanley
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Animals; Base Sequence; Biochemistry; Carrier Proteins - genetics; Carrier Proteins - isolation & purification; Cattle; Chromatography, Affinity; Cloning, Molecular; Complementary DNA; DNA; DNA - isolation & purification; DNA Restriction Enzymes; Intracellular Signaling Peptides and Proteins; Male; Messenger RNA; Plasmids; Poly A - genetics; Poly A - isolation & purification; Polyribosomes; Protein Biosynthesis; Protein Kinases - genetics; RNA; RNA - genetics; RNA - isolation & purification; RNA, Messenger - genetics; Sodium; Testis - enzymology; Ungulates
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.80.12.3608

A cDNA clone for the type I regulatory subunit (RI) of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) was isolated from bovine testis by a differential screening method. mRNA coding for RIwas enriched 50- to 100-fold by polysome immunoadsorption chromatography with affinity-purified rabbit anti-RIand protein A-Sepharose. Poly(A)+RNA from these polysomes was utilized to construct a cDNA library in pBR322, and this library was screened for hybridization to32P-labeled cDNAs synthesized from either total or RI-enriched poly(A)+RNA. Plasmids isolated from colonies showing preferential hybridization to the latter probe were further characterized by hybrid selection and DNA sequence analysis. One of these plasmids (designated 62C12) contains a 1,350-nucleotide insert that hybridized to RImRNA; partial nucleotide sequence analysis confirmed its identify and indicated that it may contain the entire RIcoding region. We also have identified a recombinant plasmid with a 1,550-nucleotide insert that selected through hybridization a mRNA coding for a 55,000-dalton protein that crossreacts with anti-RIantibodies. The function of this latter protein is unknown.