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. 1982 Jul;79(14):4466–4470. doi: 10.1073/pnas.79.14.4466

Molecular cloning of cDNA coding for the gamma subunit of Torpedo acetylcholine receptor.

M Ballivet, J Patrick, J Lee, S Heinemann
PMCID: PMC346693  PMID: 6956875

Abstract

From the electric organ of Torpedo californica, we purified mRNA that, when translated in vitro, produces polypeptides immunoprecipitable by antibodies against purified acetylcholine receptor. A novel cloning system [Okayama, H. & Berg, P. (1982) Mol. Cell. Biol. 2, 161-170] was used to produce a cDNA library from this mRNA. This library contained clones with receptor sequences identified by differential hybridization and hybridization-selection. We describe a clone of 2,030 base pairs with sequences appropriate for the amino-terminal amino acids of the gamma subunit of acetylcholine receptor. This clone contains 82 bases 5' of the codon for the amino-terminal amino acid of the mature protein. A portion of this sequence codes for a methionine followed by a 16-amino acid polypeptide that is contiguous to the amino-terminal amino acid of the mature protein and that has the characteristics of a leader peptide. The cDNA insert hybridizes to a 2,100-base RNA present in electric organ but not in the brain of T. californica.

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Selected References

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

  1. Anderson D. J., Blobel G. In vitro synthesis, glycosylation, and membrane insertion of the four subunits of Torpedo acetylcholine receptor. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5598–5602. doi: 10.1073/pnas.78.9.5598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berg D. K., Hall Z. W. Loss of alpha-bungarotoxin from junctional and extrajunctional acetylcholine receptors in rat diaphragm muscle in vivo and in organ culture. J Physiol. 1975 Nov;252(3):771–789. doi: 10.1113/jphysiol.1975.sp011169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Glisin V., Crkvenjakov R., Byus C. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry. 1974 Jun 4;13(12):2633–2637. doi: 10.1021/bi00709a025. [DOI] [PubMed] [Google Scholar]
  6. Hallick R. B., Chelm B. K., Gray P. W., Orozco E. M., Jr Use of aurintricarboxylic acid as an inhibitor of nucleases during nucleic acid isolation. Nucleic Acids Res. 1977 Sep;4(9):3055–3064. doi: 10.1093/nar/4.9.3055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Inouye M., Halegoua S. Secretion and membrane localization of proteins in Escherichia coli. CRC Crit Rev Biochem. 1980;7(4):339–371. doi: 10.3109/10409238009105465. [DOI] [PubMed] [Google Scholar]
  8. Konkel D. A., Maizel J. V., Jr, Leder P. The evolution and sequence comparison of two recently diverged mouse chromosomal beta--globin genes. Cell. 1979 Nov;18(3):865–873. doi: 10.1016/0092-8674(79)90138-7. [DOI] [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  11. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. McCarty K. S., Jr, Vollmer R. T., McCarty K. S. Improved computer program data for the resolution and fractionation of macromolecules by isokinetic sucrose density gradient sedimentation. Anal Biochem. 1974 Sep;61(1):165–183. doi: 10.1016/0003-2697(74)90343-1. [DOI] [PubMed] [Google Scholar]
  13. Mendez B., Valenzuela P., Martial J. A., Baxter J. D. Cell-free synthesis of acetylcholine receptor polypeptides. Science. 1980 Aug 8;209(4457):695–697. doi: 10.1126/science.7394526. [DOI] [PubMed] [Google Scholar]
  14. Neher E., Sakmann B. Noise analysis of drug induced voltage clamp currents in denervated frog muscle fibres. J Physiol. 1976 Jul;258(3):705–729. doi: 10.1113/jphysiol.1976.sp011442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Okayama H., Berg P. High-efficiency cloning of full-length cDNA. Mol Cell Biol. 1982 Feb;2(2):161–170. doi: 10.1128/mcb.2.2.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Parnes J. R., Velan B., Felsenfeld A., Ramanathan L., Ferrini U., Appella E., Seidman J. G. Mouse beta 2-microglobulin cDNA clones: a screening procedure for cDNA clones corresponding to rare mRNAs. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2253–2257. doi: 10.1073/pnas.78.4.2253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Potter E., Nicolaisen A. K., Ong E. S., Evans R. M., Rosenfeld M. G. Thyrotropin-releasing hormone exerts rapid nuclear effects to increase production of the primary prolactin mRNA transcript. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6662–6666. doi: 10.1073/pnas.78.11.6662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Raftery M. A., Hunkapiller M. W., Strader C. D., Hood L. E. Acetylcholine receptor: complex of homologous subunits. Science. 1980 Jun 27;208(4451):1454–1456. doi: 10.1126/science.7384786. [DOI] [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. Rosen J. M., Woo S. L., Holder J. W., Means A. R., O'Malley B. W. Preparation and preliminary characterization of purified ovalbumin messenger RNA from the hen oviduct. Biochemistry. 1975 Jan 14;14(1):69–78. doi: 10.1021/bi00672a012. [DOI] [PubMed] [Google Scholar]
  22. Smith D. R., Calvo J. M. Nucleotide sequence of the E coli gene coding for dihydrofolate reductase. Nucleic Acids Res. 1980 May 24;8(10):2255–2274. doi: 10.1093/nar/8.10.2255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sumikawa K., Houghton M., Emtage J. S., Richards B. M., Barnard E. A. Active multi-subunit ACh receptor assembled by translation of heterologous mRNA in Xenopus oocytes. Nature. 1981 Aug 27;292(5826):862–864. doi: 10.1038/292862a0. [DOI] [PubMed] [Google Scholar]
  24. Summers J. Physical map of polyoma viral DNA fragments produced by cleavage with a restriction enzyme from Haemophilus aegyptius, endonuclease R-HaeIII. J Virol. 1975 Apr;15(4):946–953. doi: 10.1128/jvi.15.4.946-953.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Weinberg C. B., Hall Z. W. Antibodies from patients with myasthenia gravis recognize determinants unique to extrajunctional acetylcholine receptors. Proc Natl Acad Sci U S A. 1979 Jan;76(1):504–508. doi: 10.1073/pnas.76.1.504. [DOI] [PMC free article] [PubMed] [Google Scholar]

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