Skip to main content
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
. 1985 May;82(10):3154–3158. doi: 10.1073/pnas.82.10.3154

Cloning and sequence analysis of cDNA for the luminescent protein aequorin.

S Inouye, M Noguchi, Y Sakaki, Y Takagi, T Miyata, S Iwanaga, T Miyata, F I Tsuji
PMCID: PMC397733  PMID: 3858813

Abstract

The luminescent jellyfish Aequorea contains a photoprotein, aequorin, which emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. A cDNA library of Aequorea was constructed and clones carrying the cDNA for the Ca2+-dependent photoprotein were isolated by the method of colony hybridization using synthetic oligonucleotide probes. The primary structure of the protein deduced from the nucleotide sequence showed that the protein is composed of 189 amino acid residues and has three E-F hand structures that are characteristic for Ca2+-binding sites. The sequence also suggested that the protein has hydrophobic regions at which the protein may interact with its functional chromophore, coelenterazine.

Full text

PDF
3157

Images in this article

Selected References

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

  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blinks J. R., Prendergast F. G., Allen D. G. Photoproteins as biological calcium indicators. Pharmacol Rev. 1976 Mar;28(1):1–93. [PubMed] [Google Scholar]
  3. Collins J. H., Potter J. D., Horn M. J., Wilshire G., Jackman N. The amino acid sequence of rabbit skeletal muscle troponin C: gene replication and homology with calcium-binding proteins from carp and hake muscle. FEBS Lett. 1973 Nov 1;36(3):268–272. doi: 10.1016/0014-5793(73)80388-6. [DOI] [PubMed] [Google Scholar]
  4. Hastings J. W., Mitchell G., Mattingly P. H., Blinks J. R., Van Leeuwen M. Response of aequorin bioluminescence to rapid changes in calcium concentration. Nature. 1969 Jun 14;222(5198):1047–1050. doi: 10.1038/2221047a0. [DOI] [PubMed] [Google Scholar]
  5. Hunkapiller M. W., Hewick R. M., Dreyer W. J., Hood L. E. High-sensitivity sequencing with a gas-phase sequenator. Methods Enzymol. 1983;91:399–413. doi: 10.1016/s0076-6879(83)91038-8. [DOI] [PubMed] [Google Scholar]
  6. Johnsn F. H., Shimomura O. Preparation and use of aequorin for rapid microdetermination of Ca 2+ in biological systems. Nat New Biol. 1972 Jun 28;237(78):287–288. doi: 10.1038/newbio237287a0. [DOI] [PubMed] [Google Scholar]
  7. Kohama Y., Shimomura O., Johnson F. H. Molecular weight of the photoprotein aequorin. Biochemistry. 1971 Oct 26;10(22):4149–4152. doi: 10.1021/bi00798a022. [DOI] [PubMed] [Google Scholar]
  8. Kretsinger R. H., Nockolds C. E. Carp muscle calcium-binding protein. II. Structure determination and general description. J Biol Chem. 1973 May 10;248(9):3313–3326. [PubMed] [Google Scholar]
  9. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Miyata T., Hiranaga M., Umezu M., Iwanaga S. Amino acid sequence of the coagulogen from Limulus polyphemus hemocytes. J Biol Chem. 1984 Jul 25;259(14):8924–8933. [PubMed] [Google Scholar]
  12. Miyata T., Iwanaga S., Sakata Y., Aoki N. Plasminogen Tochigi: inactive plasmin resulting from replacement of alanine-600 by threonine in the active site. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6132–6136. doi: 10.1073/pnas.79.20.6132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Miyata T., Iwanaga S., Sakata Y., Aoki N., Takamatsu J., Kamiya T. Plasminogens Tochigi II and Nagoya: two additional molecular defects with Ala-600----Thr replacement found in plasmin light chain variants. J Biochem. 1984 Aug;96(2):277–287. doi: 10.1093/oxfordjournals.jbchem.a134836. [DOI] [PubMed] [Google Scholar]
  14. Miyata T., Usui K., Iwanaga S. The amino acid sequence of coagulogen isolated from southeast Asian horseshoe crab, Tachypleus gigas. J Biochem. 1984 Jun;95(6):1793–1801. doi: 10.1093/oxfordjournals.jbchem.a134792. [DOI] [PubMed] [Google Scholar]
  15. Norgard M. V., Tocci M. J., Monahan J. J. On the cloning of eukaryotic total poly(A)-RNA populations in Escherichia coli. J Biol Chem. 1980 Aug 25;255(16):7665–7672. [PubMed] [Google Scholar]
  16. 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]
  17. Pless D. D., Lennarz W. J. Enzymatic conversion of proteins to glycoproteins. Proc Natl Acad Sci U S A. 1977 Jan;74(1):134–138. doi: 10.1073/pnas.74.1.134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Prasher D., McCann R. O., Cormier M. J. Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein. Biochem Biophys Res Commun. 1985 Feb 15;126(3):1259–1268. doi: 10.1016/0006-291x(85)90321-3. [DOI] [PubMed] [Google Scholar]
  19. Proudfoot N. J., Brownlee G. G. 3' non-coding region sequences in eukaryotic messenger RNA. Nature. 1976 Sep 16;263(5574):211–214. doi: 10.1038/263211a0. [DOI] [PubMed] [Google Scholar]
  20. SHIMOMURA O., JOHNSON F. H., SAIGA Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol. 1962 Jun;59:223–239. doi: 10.1002/jcp.1030590302. [DOI] [PubMed] [Google Scholar]
  21. Sherman L., Dafni N., Lieman-Hurwitz J., Groner Y. Nucleotide sequence and expression of human chromosome 21-encoded superoxide dismutase mRNA. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5465–5469. doi: 10.1073/pnas.80.18.5465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shimomura O., Johnson F. H. Calcium binding, quantum yield, and emitting molecule in aequorin bioluminescence. Nature. 1970 Sep 26;227(5265):1356–1357. doi: 10.1038/2271356a0. [DOI] [PubMed] [Google Scholar]
  23. Shimomura O., Johnson F. H., Morise H. Mechanism of the luminescent intramolecular reaction of aequorin. Biochemistry. 1974 Jul 30;13(16):3278–3286. doi: 10.1021/bi00713a016. [DOI] [PubMed] [Google Scholar]
  24. Shimomura O., Johnson F. H. Peroxidized coelenterazine, the active group in the photoprotein aequorin. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2611–2615. doi: 10.1073/pnas.75.6.2611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shimomura O., Johnson F. H. Properties of the bioluminescent protein aequorin. Biochemistry. 1969 Oct;8(10):3991–3997. doi: 10.1021/bi00838a015. [DOI] [PubMed] [Google Scholar]
  26. Shimomura O., Johnson F. H. Regeneration of the photoprotein aequorin. Nature. 1975 Jul 17;256(5514):236–238. doi: 10.1038/256236a0. [DOI] [PubMed] [Google Scholar]
  27. Shimomura O., Johnson F. H., Saiga Y. Microdetermination of Calcium by Aequorin Luminescence. Science. 1963 Jun 21;140(3573):1339–1340. doi: 10.1126/science.140.3573.1339. [DOI] [PubMed] [Google Scholar]
  28. Shimomura O., Johnson F. H. Structure of the light-emitting moiety of aequorin. Biochemistry. 1972 Apr 25;11(9):1602–1608. doi: 10.1021/bi00759a009. [DOI] [PubMed] [Google Scholar]
  29. Shimomura O., Shimomura A. EDTA-binding and acylation of the Ca2+-sensitive photoprotein aequorin. FEBS Lett. 1982 Feb 22;138(2):201–204. doi: 10.1016/0014-5793(82)80441-9. [DOI] [PubMed] [Google Scholar]
  30. Shimomura O., Shimomura A. Effect of calcium chelators on the Ca2+-dependent luminescence of aequorin. Biochem J. 1984 Aug 1;221(3):907–910. doi: 10.1042/bj2210907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wallace R. B., Johnson M. J., Hirose T., Miyake T., Kawashima E. H., Itakura K. The use of synthetic oligonucleotides as hybridization probes. II. Hybridization of oligonucleotides of mixed sequence to rabbit beta-globin DNA. Nucleic Acids Res. 1981 Feb 25;9(4):879–894. doi: 10.1093/nar/9.4.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Watterson D. M., Sharief F., Vanaman T. C. The complete amino acid sequence of the Ca2+-dependent modulator protein (calmodulin) of bovine brain. J Biol Chem. 1980 Feb 10;255(3):962–975. [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