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. 1983 Jun;80(12):3608-12.
doi: 10.1073/pnas.80.12.3608.

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

D C Lee, D F Carmichael, E G Krebs, G S McKnight

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

D C Lee et al. Proc Natl Acad Sci U S A. 1983 Jun.

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.

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