Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1983 Jun;80(12):3608–3612. 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
PMCID: PMC394099  PMID: 6190178

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.

Full text

PDF
3608

Images in this article

3609Image
on p.3609
3609Image
on p.3609
3610Image
on p.3610
3610Image
on p.3610
3610Image
on p.3610
3611Image
on p.3611
3611Image
on p.3611
3611Image
on p.3611

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Builder S. E., Beavo J. A., Krebs E. G. Stoichiometry of cAMP and 1,N6-etheno-cAMP binding to protein kinase. J Biol Chem. 1980 Mar 25;255(6):2350–2354. [PubMed] [Google Scholar]
  2. Cochet M., Perrin F., Gannon F., Krust A., Chambon P., McKnight G. S., Lee D. C., Mayo K. E., Palmiter R. Cloning of an almost full-length chicken conalbumin double-stranded cDNA. Nucleic Acids Res. 1979 Jun 11;6(7):2435–2452. doi: 10.1093/nar/6.7.2435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Corbin J. D., Sugden P. H., West L., Flockhart D. A., Lincoln T. M., McCarthy D. Studies on the properties and mode of action of the purified regulatory subunit of bovine heart adenosine 3':5'-monophosphate-dependent protein kinase. J Biol Chem. 1978 Jun 10;253(11):3997–4003. [PubMed] [Google Scholar]
  4. Cuatrecasas P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J Biol Chem. 1970 Jun;245(12):3059–3065. [PubMed] [Google Scholar]
  5. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  6. Gergen J. P., Stern R. H., Wensink P. C. Filter replicas and permanent collections of recombinant DNA plasmids. Nucleic Acids Res. 1979 Dec 20;7(8):2115–2136. doi: 10.1093/nar/7.8.2115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gharrett A. J., Malkinson A. M., Sheppard J. R. Cyclic AMP-dependent protein kinases from normal and SV40-transformed 3T3 cells. Nature. 1976 Dec 16;264(5587):673–675. doi: 10.1038/264673a0. [DOI] [PubMed] [Google Scholar]
  8. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hixson C. S., Krebs E. G. Characterization of a cyclic AMP-binding protein from bakers' yeast. Identification as a regulatory subunit of cyclic AMP-dependent protein kinase. J Biol Chem. 1980 Mar 10;255(5):2137–2145. [PubMed] [Google Scholar]
  10. Krebs E. G., Beavo J. A. Phosphorylation-dephosphorylation of enzymes. Annu Rev Biochem. 1979;48:923–959. doi: 10.1146/annurev.bi.48.070179.004423. [DOI] [PubMed] [Google Scholar]
  11. Land H., Grez M., Hauser H., Lindenmaier W., Schütz G. 5'-Terminal sequences of eucaryotic mRNA can be cloned with high efficiency. Nucleic Acids Res. 1981 May 25;9(10):2251–2266. doi: 10.1093/nar/9.10.2251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lee D. C., McKnight G. S., Palmiter R. D. The action of estrogen and progesterone on the expression of the transferrin gene. A comparison of the response in chick liver and oviduct. J Biol Chem. 1978 May 25;253(10):3494–3503. [PubMed] [Google Scholar]
  13. Lee P. C., Radloff D., Schweppe J. S., Jungmann R. A. Testicular protein kinases. Characterization of multiple forms and ontogeny. J Biol Chem. 1976 Feb 25;251(4):914–921. [PubMed] [Google Scholar]
  14. Liu A. Y. Differentiation-specific increase of cAMP-dependent protein kinase in the 3T3-L1 cells. J Biol Chem. 1982 Jan 10;257(1):298–306. [PubMed] [Google Scholar]
  15. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  16. Nimmo H. G., Cohen P. Hormonal control of protein phosphorylation. Adv Cyclic Nucleotide Res. 1977;8:145–266. [PubMed] [Google Scholar]
  17. Palmiter R. D. Magnesium precipitation of ribonucleoprotein complexes. Expedient techniques for the isolation of undergraded polysomes and messenger ribonucleic acid. Biochemistry. 1974 Aug 13;13(17):3606–3615. doi: 10.1021/bi00714a032. [DOI] [PubMed] [Google Scholar]
  18. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  19. Richards J. S., Rolfes A. I. Hormonal regulation of cyclic AMP binding to specific receptor proteins in rat ovarian follicles. Characterization by photoaffinity labeling. J Biol Chem. 1980 Jun 10;255(11):5481–5489. [PubMed] [Google Scholar]
  20. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  21. Takai Y., Yamamura H., Nishizuka Y. Adenosine 3':5'-monophosphate-dependent protein kinase from yeast. J Biol Chem. 1974 Jan 25;249(2):530–535. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES