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. 1990 May 25;18(10):2909–2915. doi: 10.1093/nar/18.10.2909

Activation in vitro of RNA polymerase II and III directed transcription by baculovirus produced E1A protein.

G Patel 1, N C Jones 1
PMCID: PMC330818  PMID: 2140886

Abstract

The baculovirus expression system has been successfully used to overproduce a number of different protein products. In this report we describe the construction of a recombinant baculovirus containing the adenovirus E1A 13s cDNA sequence. Infection of insect cells with this virus results in the production of phosphorylated E1A protein. The phosphorylation pattern appears to be similar to the complex pattern associated with E1A protein synthesis in mammalian cells. Purified baculovirus generated E1A protein activated transcription of specific poIIII promoters both in microinjected Xenopus laevis oocytes and in HeLa cell in vitro transcription extracts. The protein also stimulates in vitro transcription of the poIIII transcribed VA1 gene.

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Selected References

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

  1. Berger S. L., Folk W. R. Differential activation of RNA polymerase III-transcribed genes by the polyomavirus enhancer and the adenovirus E1A gene products. Nucleic Acids Res. 1985 Feb 25;13(4):1413–1428. doi: 10.1093/nar/13.4.1413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berk A. J. Adenovirus promoters and E1A transactivation. Annu Rev Genet. 1986;20:45–79. doi: 10.1146/annurev.ge.20.120186.000401. [DOI] [PubMed] [Google Scholar]
  3. Borrelli E., Hen R., Chambon P. Adenovirus-2 E1A products repress enhancer-induced stimulation of transcription. Nature. 1984 Dec 13;312(5995):608–612. doi: 10.1038/312608a0. [DOI] [PubMed] [Google Scholar]
  4. Branton P. E., Rowe D. T. Stabilities and interrelations of multiple species of human adenovirus type 5 early region 1 proteins in infected and transformed cells. J Virol. 1985 Nov;56(2):633–638. doi: 10.1128/jvi.56.2.633-638.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Estes M. K., Crawford S. E., Penaranda M. E., Petrie B. L., Burns J. W., Chan W. K., Ericson B., Smith G. E., Summers M. D. Synthesis and immunogenicity of the rotavirus major capsid antigen using a baculovirus expression system. J Virol. 1987 May;61(5):1488–1494. doi: 10.1128/jvi.61.5.1488-1494.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ferguson B., Jones N., Richter J., Rosenberg M. Adenovirus E1a gene product expressed at high levels in Escherichia coli is functional. Science. 1984 Jun 22;224(4655):1343–1346. doi: 10.1126/science.6374895. [DOI] [PubMed] [Google Scholar]
  7. Fowlkes D. M., Shenk T. Transcriptional control regions of the adenovirus VAI RNA gene. Cell. 1980 Nov;22(2 Pt 2):405–413. doi: 10.1016/0092-8674(80)90351-7. [DOI] [PubMed] [Google Scholar]
  8. Gaynor R. B., Feldman L. T., Berk A. J. Transcription of class III genes activated by viral immediate early proteins. Science. 1985 Oct 25;230(4724):447–450. doi: 10.1126/science.2996135. [DOI] [PubMed] [Google Scholar]
  9. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  10. Green M., Loewenstein P. M., Pusztai R., Symington J. S. An adenovirus E1A protein domain activates transcription in vivo and in vitro in the absence of protein synthesis. Cell. 1988 Jun 17;53(6):921–926. doi: 10.1016/s0092-8674(88)90429-1. [DOI] [PubMed] [Google Scholar]
  11. Greenfield C., Patel G., Clark S., Jones N., Waterfield M. D. Expression of the human EGF receptor with ligand-stimulatable kinase activity in insect cells using a baculovirus vector. EMBO J. 1988 Jan;7(1):139–146. doi: 10.1002/j.1460-2075.1988.tb02793.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haley K. P., Overhauser J., Babiss L. E., Ginsberg H. S., Jones N. C. Transformation properties of type 5 adenovirus mutants that differentially express the E1A gene products. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5734–5738. doi: 10.1073/pnas.81.18.5734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harlow E., Franza B. R., Jr, Schley C. Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products. J Virol. 1985 Sep;55(3):533–546. doi: 10.1128/jvi.55.3.533-546.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hen R., Borrelli E., Chambon P. Repression of the immunoglobulin heavy chain enhancer by the adenovirus-2 E1A products. Science. 1985 Dec 20;230(4732):1391–1394. doi: 10.1126/science.2999984. [DOI] [PubMed] [Google Scholar]
  15. Hoeffler W. K., Kovelman R., Roeder R. G. Activation of transcription factor IIIC by the adenovirus E1A protein. Cell. 1988 Jun 17;53(6):907–920. doi: 10.1016/s0092-8674(88)90409-6. [DOI] [PubMed] [Google Scholar]
  16. Hoeffler W. K., Roeder R. G. Enhancement of RNA polymerase III transcription by the E1A gene product of adenovirus. Cell. 1985 Jul;41(3):955–963. doi: 10.1016/s0092-8674(85)80076-3. [DOI] [PubMed] [Google Scholar]
  17. Jeang K. T., Giam C. Z., Nerenberg M., Khoury G. Abundant synthesis of functional human T-cell leukemia virus type I p40x protein in eucaryotic cells by using a baculovirus expression vector. J Virol. 1987 Mar;61(3):708–713. doi: 10.1128/jvi.61.3.708-713.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jones N. C., Rigby P. W., Ziff E. B. Trans-acting protein factors and the regulation of eukaryotic transcription: lessons from studies on DNA tumor viruses. Genes Dev. 1988 Mar;2(3):267–281. doi: 10.1101/gad.2.3.267. [DOI] [PubMed] [Google Scholar]
  19. Kao H. T., Nevins J. R. Transcriptional activation and subsequent control of the human heat shock gene during adenovirus infection. Mol Cell Biol. 1983 Nov;3(11):2058–2065. doi: 10.1128/mcb.3.11.2058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Krippl B., Ferguson B., Rosenberg M., Westphal H. Functions of purified E1A protein microinjected into mammalian cells. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6988–6992. doi: 10.1073/pnas.81.22.6988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kuroda K., Hauser C., Rott R., Klenk H. D., Doerfler W. Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector. EMBO J. 1986 Jun;5(6):1359–1365. doi: 10.1002/j.1460-2075.1986.tb04367.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Luckow V. A., Summers M. D. Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors. Virology. 1988 Nov;167(1):56–71. doi: 10.1016/0042-6822(88)90054-2. [DOI] [PubMed] [Google Scholar]
  23. Manley J. L., Fire A., Cano A., Sharp P. A., Gefter M. L. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855–3859. doi: 10.1073/pnas.77.7.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Miller L. K. Baculoviruses as gene expression vectors. Annu Rev Microbiol. 1988;42:177–199. doi: 10.1146/annurev.mi.42.100188.001141. [DOI] [PubMed] [Google Scholar]
  25. Miyamoto C., Smith G. E., Farrell-Towt J., Chizzonite R., Summers M. D., Ju G. Production of human c-myc protein in insect cells infected with a baculovirus expression vector. Mol Cell Biol. 1985 Oct;5(10):2860–2865. doi: 10.1128/mcb.5.10.2860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Moran E., Grodzicker T., Roberts R. J., Mathews M. B., Zerler B. Lytic and transforming functions of individual products of the adenovirus E1A gene. J Virol. 1986 Mar;57(3):765–775. doi: 10.1128/jvi.57.3.765-775.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nevins J. R. Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus E1A gene product. Cell. 1982 Jul;29(3):913–919. doi: 10.1016/0092-8674(82)90453-6. [DOI] [PubMed] [Google Scholar]
  28. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  29. Ollo R., Maniatis T. Drosophila Krüppel gene product produced in a baculovirus expression system is a nuclear phosphoprotein that binds to DNA. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5700–5704. doi: 10.1073/pnas.84.16.5700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Patel G., Stabel S. Expression of a functional protein kinase C-gamma using a baculovirus vector: purification and characterisation of a single protein kinase C iso-enzyme. Cell Signal. 1989;1(3):227–240. doi: 10.1016/0898-6568(89)90040-5. [DOI] [PubMed] [Google Scholar]
  31. Richter J. D., Slavicek J. M., Schneider J. F., Jones N. C. Heterogeneity of adenovirus type 5 E1A proteins: multiple serine phosphorylations induce slow-migrating electrophoretic variants but do not affect E1A-induced transcriptional activation or transformation. J Virol. 1988 Jun;62(6):1948–1955. doi: 10.1128/jvi.62.6.1948-1955.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Richter J. D., Young P., Jones N. C., Krippl B., Rosenberg M., Ferguson B. A first exon-encoded domain of E1A sufficient for posttranslational modification, nuclear-localization, and induction of adenovirus E3 promoter expression in Xenopus oocytes. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8434–8438. doi: 10.1073/pnas.82.24.8434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schneider J. F., Fisher F., Goding C. R., Jones N. C. Mutational analysis of the adenovirus E1a gene: the role of transcriptional regulation in transformation. EMBO J. 1987 Jul;6(7):2053–2060. doi: 10.1002/j.1460-2075.1987.tb02470.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Simanis V., Lane D. P. An immunoaffinity purification procedure for SV40 large T antigen. Virology. 1985 Jul 15;144(1):88–100. doi: 10.1016/0042-6822(85)90308-3. [DOI] [PubMed] [Google Scholar]
  35. Slavicek J. M., Jones N. C., Richter J. D. Rapid turnover of adenovirus E1A is determined through a co-translational mechanism that requires an aminoterminal domain. EMBO J. 1988 Oct;7(10):3171–3180. doi: 10.1002/j.1460-2075.1988.tb03184.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Smith C. L., Debouck C., Rosenberg M., Culp J. S. Phosphorylation of serine residue 89 of human adenovirus E1A proteins is responsible for their characteristic electrophoretic mobility shifts, and its mutation affects biological function. J Virol. 1989 Apr;63(4):1569–1577. doi: 10.1128/jvi.63.4.1569-1577.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Smith G. E., Ju G., Ericson B. L., Moschera J., Lahm H. W., Chizzonite R., Summers M. D. Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8404–8408. doi: 10.1073/pnas.82.24.8404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Spangler R., Bruner M., Dalie B., Harter M. L. Activation of adenovirus promoters by the adenovirus E1A protein in cell-free extracts. Science. 1987 Aug 28;237(4818):1044–1046. doi: 10.1126/science.2956686. [DOI] [PubMed] [Google Scholar]
  39. Spindler K. R., Berk A. J. Rapid intracellular turnover of adenovirus 5 early region 1A proteins. J Virol. 1984 Nov;52(2):706–710. doi: 10.1128/jvi.52.2.706-710.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stein R. W., Ziff E. B. Repression of insulin gene expression by adenovirus type 5 E1a proteins. Mol Cell Biol. 1987 Mar;7(3):1164–1170. doi: 10.1128/mcb.7.3.1164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stein R., Ziff E. B. HeLa cell beta-tubulin gene transcription is stimulated by adenovirus 5 in parallel with viral early genes by an E1a-dependent mechanism. Mol Cell Biol. 1984 Dec;4(12):2792–2801. doi: 10.1128/mcb.4.12.2792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stephens C., Harlow E. Differential splicing yields novel adenovirus 5 E1A mRNAs that encode 30 kd and 35 kd proteins. EMBO J. 1987 Jul;6(7):2027–2035. doi: 10.1002/j.1460-2075.1987.tb02467.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Timmers H. T., De Wit D., Bos J. L., Van der Eb A. J. E1A products of adenoviruses reduce the expression of cellular proliferation-associated genes. Oncogene Res. 1988;3(1):67–76. [PubMed] [Google Scholar]
  44. Timmers H. T., van Dam H., Pronk G. J., Bos J. L., Van der Eb A. J. Adenovirus E1A represses transcription of the cellular JE gene. J Virol. 1989 Mar;63(3):1470–1473. doi: 10.1128/jvi.63.3.1470-1473.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tremblay M. L., McGlade C. J., Gerber G. E., Branton P. E. Identification of the phosphorylation sites in early region 1A proteins of adenovirus type 5 by amino acid sequencing of peptide fragments. J Biol Chem. 1988 May 5;263(13):6375–6383. [PubMed] [Google Scholar]
  46. Tsukamoto A. S., Ponticelli A., Berk A. J., Gaynor R. B. Genetic mapping of a major site of phosphorylation in adenovirus type 2 E1A proteins. J Virol. 1986 Jul;59(1):14–22. doi: 10.1128/jvi.59.1.14-22.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ulfendahl P. J., Linder S., Kreivi J. P., Nordqvist K., Sevensson C., Hultberg H., Akusjärvi G. A novel adenovirus-2 E1A mRNA encoding a protein with transcription activation properties. EMBO J. 1987 Jul;6(7):2037–2044. doi: 10.1002/j.1460-2075.1987.tb02468.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Velcich A., Ziff E. Adenovirus E1a proteins repress transcription from the SV40 early promoter. Cell. 1985 Mar;40(3):705–716. doi: 10.1016/0092-8674(85)90219-3. [DOI] [PubMed] [Google Scholar]
  49. Weeks D. L., Jones N. C. Adenovirus E3-early promoter: sequences required for activation by E1A. Nucleic Acids Res. 1985 Jul 25;13(14):5389–5402. doi: 10.1093/nar/13.14.5389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wu B. J., Hurst H. C., Jones N. C., Morimoto R. I. The E1A 13S product of adenovirus 5 activates transcription of the cellular human HSP70 gene. Mol Cell Biol. 1986 Aug;6(8):2994–2999. doi: 10.1128/mcb.6.8.2994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yoshinaga S., Dean N., Han M., Berk A. J. Adenovirus stimulation of transcription by RNA polymerase III: evidence for an E1A-dependent increase in transcription factor IIIC concentration. EMBO J. 1986 Feb;5(2):343–354. doi: 10.1002/j.1460-2075.1986.tb04218.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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