title: Isolation of a cDNA clone for the type I regulatory subunit of bovine cAMP-dependent protein kinase.
abstract: 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 RI was enriched 50- to 100-fold by polysome immunoadsorption chromatography with affinity-purified rabbit anti-RI and 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 to 32P-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 RI mRNA; partial nucleotide sequence analysis confirmed its identity and indicated that it may contain the entire RI coding 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-RI antibodies. The function of this latter protein is unknown.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification; D002417:Cattle; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000235:genetics*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics; D013737:Testis / Q000201:enzymology*
chemical_list: D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; C015482:protein kinase modulator; D011061:Poly A; D012313:RNA; D004247:DNA; D011494:Protein Kinases; D004262:DNA Restriction Enzymes