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. 1985 Nov;4(11):2955–2959. doi: 10.1002/j.1460-2075.1985.tb04029.x

Efficient expression of an Epstein-Barr nuclear antigen in Drosophila cells transfected with Epstein-Barr virus DNA.

M J Allday, J H Sinclair, A J MacGillivray, J H Sang
PMCID: PMC554604  PMID: 2998769

Abstract

In a search for exogenous promoters which function in cultured Drosophila cells, we have co-transfected a D. melanogaster cell line with an Epstein-Barr virus (EBV) cosmid clone which encodes the Epstein-Barr nuclear antigen (EBNA-1). Here we report that Drosophila cells containing stably integrated copies of EBNA-1 encoding DNA synthesise a polypeptide of mol. wt. identical to that of authentic EBNA-1, which is detectable with EBNA-positive but not EBNA-negative human serum. As in EBV-transformed lymphoblastoid cells, this neo-antigen is associated with the nucleus of transfected cells suggesting that cellular localisation signals which operate in mammalian cells are also recognised in insect cells.

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Selected References

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

  1. Allday M. J., MacGillivray A. J. Epstein-Barr virus nuclear antigen (EBNA): size polymorphism of EBNA 1. J Gen Virol. 1985 Jul;66(Pt 7):1595–1600. doi: 10.1099/0022-1317-66-7-1595. [DOI] [PubMed] [Google Scholar]
  2. Baer R., Bankier A. T., Biggin M. D., Deininger P. L., Farrell P. J., Gibson T. J., Hatfull G., Hudson G. S., Satchwell S. C., Séguin C. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 1984 Jul 19;310(5974):207–211. doi: 10.1038/310207a0. [DOI] [PubMed] [Google Scholar]
  3. Bourouis M., Jarry B. Vectors containing a prokaryotic dihydrofolate reductase gene transform Drosophila cells to methotrexate-resistance. EMBO J. 1983;2(7):1099–1104. doi: 10.1002/j.1460-2075.1983.tb01552.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burke J. F., Pinchin S. M., Ish-Horowicz D., Sinclair J. H., Sang J. H. Integration of Drosophila heat-shock genes transfected into cultured Drosophila melanogaster cells. Somat Cell Mol Genet. 1984 Nov;10(6):579–588. doi: 10.1007/BF01535223. [DOI] [PubMed] [Google Scholar]
  5. Burke J. F., Sinclair J. H., Sang J. H., Ish-Horowicz D. An assay for transient gene expression in transfected Drosophila cells, using [3H]guanine incorporation. EMBO J. 1984 Nov;3(11):2549–2554. doi: 10.1002/j.1460-2075.1984.tb02172.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  7. Corces V., Pellicer A., Axel R., Meselson M. Integration, transcription, and control of a Drosophila heat shock gene in mouse cells. Proc Natl Acad Sci U S A. 1981 Nov;78(11):7038–7042. doi: 10.1073/pnas.78.11.7038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Di Nocera P. P., Dawid I. B. Transient expression of genes introduced into cultured cells of Drosophila. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7095–7098. doi: 10.1073/pnas.80.23.7095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dillner J., Sternås L., Kallin B., Alexander H., Ehlin-Henriksson B., Jörnvall H., Klein G., Lerner R. Antibodies against a synthetic peptide identify the Epstein-Barr virus-determined nuclear antigen. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4652–4656. doi: 10.1073/pnas.81.15.4652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Flavell A. J., Ish-Horowicz D. Extrachromosomal circular copies of the eukaryotic transposable element copia in cultured Drosophila cells. Nature. 1981 Aug 13;292(5824):591–595. doi: 10.1038/292591a0. [DOI] [PubMed] [Google Scholar]
  11. Griffin B. E., Karran L. Immortalization of monkey epithelial cells by specific fragments of Epstein-Barr virus DNA. Nature. 1984 May 3;309(5963):78–82. doi: 10.1038/309078a0. [DOI] [PubMed] [Google Scholar]
  12. Hennessy K., Kieff E. One of two Epstein-Barr virus nuclear antigens contains a glycine-alanine copolymer domain. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5665–5669. doi: 10.1073/pnas.80.18.5665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Luka J., Kreofsky T., Pearson G. R., Hennessy K., Kieff E. Identification and characterization of a cellular protein that cross-reacts with the Epstein-Barr virus nuclear antigen. J Virol. 1984 Dec;52(3):833–838. doi: 10.1128/jvi.52.3.833-838.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Miller G., Grogan E., Fischer D. K., Niederman J. C., Schooley R. T., Henle W., Lenoir G., Liu C. R. Antibody responses to two Epstein-Barr virus nuclear antigens defined by gene transfer. N Engl J Med. 1985 Mar 21;312(12):750–755. doi: 10.1056/NEJM198503213121204. [DOI] [PubMed] [Google Scholar]
  16. Mulligan R. C., Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2072–2076. doi: 10.1073/pnas.78.4.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Reedman B. M., Klein G. Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. Int J Cancer. 1973 May;11(3):499–520. doi: 10.1002/ijc.2910110302. [DOI] [PubMed] [Google Scholar]
  18. Reeves R., Gorman C. M., Howard B. In vivo incorporation of Drosophila H2a histone into mammalian chromatin. Nucleic Acids Res. 1983 May 11;11(9):2681–2700. doi: 10.1093/nar/11.9.2681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Robins D. M., Ripley S., Henderson A. S., Axel R. Transforming DNA integrates into the host chromosome. Cell. 1981 Jan;23(1):29–39. doi: 10.1016/0092-8674(81)90267-1. [DOI] [PubMed] [Google Scholar]
  20. Robinson J., Smith D. Infection of human B lymphocytes with high multiplicities of Epstein-Barr virus: kinetics of EBNA expression, cellular DNA synthesis, and mitosis. Virology. 1981 Mar;109(2):336–343. doi: 10.1016/0042-6822(81)90504-3. [DOI] [PubMed] [Google Scholar]
  21. Rubin G. M., Spradling A. C. Genetic transformation of Drosophila with transposable element vectors. Science. 1982 Oct 22;218(4570):348–353. doi: 10.1126/science.6289436. [DOI] [PubMed] [Google Scholar]
  22. Scott R. W., Vogt T. F., Croke M. E., Tilghman S. M. Tissue-specific activation of a cloned alpha-fetoprotein gene during differentiation of a transfected embryonal carcinoma cell line. Nature. 1984 Aug 16;310(5978):562–567. doi: 10.1038/310562a0. [DOI] [PubMed] [Google Scholar]
  23. Sculley T. B., Kreofsky T., Pearson G. R., Spelsberg T. C. Partial purification of the Epstein-Barr virus nuclear antigen(s). J Biol Chem. 1983 Mar 25;258(6):3974–3982. [PubMed] [Google Scholar]
  24. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  25. Summers W. C., Klein G. Inhibition of Epstein-Barr virus DNA synthesis and late gene expression by phosphonoacetic acid. J Virol. 1976 Apr;18(1):151–155. doi: 10.1128/jvi.18.1.151-155.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Summers W. P., Grogan E. A., Shedd D., Robert M., Liu C. R., Miller G. Stable expression in mouse cells of nuclear neoantigen after transfer of a 3.4-megadalton cloned fragment of Epstein-Barr virus DNA. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5688–5692. doi: 10.1073/pnas.79.18.5688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tanguay R. M., Vincent M. Intracellular translocation of cellular and heat shock induced proteins upon heat shock in Drosophila Kc cells. Can J Biochem. 1982 Mar;60(3):306–315. doi: 10.1139/o82-037. [DOI] [PubMed] [Google Scholar]
  28. Weigel R., Fischer D. K., Heston L., Miller G. Constitutive expression of Epstein-Barr virus-encoded RNAs and nuclear antigen during latency and after induction of Epstein-Barr virus replication. J Virol. 1985 Jan;53(1):254–259. doi: 10.1128/jvi.53.1.254-259.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Weigel R., Miller G. Latent and viral replicative transcription in vivo from the BamHI K fragment of Epstein-Barr virus DNA. J Virol. 1985 May;54(2):501–508. doi: 10.1128/jvi.54.2.501-508.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yates J. L., Warren N., Sugden B. Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. 1985 Feb 28-Mar 6Nature. 313(6005):812–815. doi: 10.1038/313812a0. [DOI] [PubMed] [Google Scholar]
  31. Yates J., Warren N., Reisman D., Sugden B. A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3806–3810. doi: 10.1073/pnas.81.12.3806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. van Santen V., Cheung A., Kieff E. Epstein-Barr virus RNA VII: size and direction of transcription of virus-specified cytoplasmic RNAs in a transformed cell line. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1930–1934. doi: 10.1073/pnas.78.3.1930. [DOI] [PMC free article] [PubMed] [Google Scholar]

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