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. 1990 Sep;87(17):6743–6747. doi: 10.1073/pnas.87.17.6743

Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells.

O Elroy-Stein 1, B Moss 1
PMCID: PMC54613  PMID: 2204064

Abstract

A mouse cell line that constitutively synthesizes the bacteriophage T7 RNA polymerase was constructed. Fluorescence microscopy indicated that the T7 RNA polymerase was present in the cytoplasmic compartment. The system provided, therefore, a unique opportunity to study structural elements of mRNA that affect stability and translation. The in vivo activity of the bacteriophage polymerase was demonstrated by transfection of a plasmid containing the chloramphenicol acetyltransferase (CAT) gene flanked by T7 promoter and termination signals. Synthesis of CAT was dependent on the presence of a cDNA copy of the untranslated region of encephalomyocarditis virus (ECMV) RNA downstream of the T7 promoter, consistent with the absence of RNA-capping activity in the cytoplasm. CAT expression from a plasmid, pT7EMCAT, containing the T7 and EMCV regulatory elements was detected within 4 hr after transfection and increased during the next 20 hr, exceeding that obtained by transfection of a plasmid with the CAT gene attached to a retrovirus promoter and enhancer. Nevertheless, the presumably cap-independent transient expression of CAT from pT7EMCAT was increased more than 500-fold when the transfected cells also were infected with wild-type vaccinia virus. A protocol for high-level expression involved the infection of the T7 RNA polymerase cell line with a single recombinant vaccinia virus containing the target gene regulated by a T7 promoter and EMCV untranslated region.

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Selected References

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

  1. Barrett N., Mitterer A., Mundt W., Eibl J., Eibl M., Gallo R. C., Moss B., Dorner F. Large-scale production and purification of a vaccinia recombinant-derived HIV-1 gp160 and analysis of its immunogenicity. AIDS Res Hum Retroviruses. 1989 Apr;5(2):159–171. doi: 10.1089/aid.1989.5.159. [DOI] [PubMed] [Google Scholar]
  2. Benton B. M., Eng W. K., Dunn J. J., Studier F. W., Sternglanz R., Fisher P. A. Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae nucleus and specific transcription of target genes. Mol Cell Biol. 1990 Jan;10(1):353–360. doi: 10.1128/mcb.10.1.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Broyles S. S., Moss B. Sedimentation of an RNA polymerase complex from vaccinia virus that specifically initiates and terminates transcription. Mol Cell Biol. 1987 Jan;7(1):7–14. doi: 10.1128/mcb.7.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen W., Tabor S., Struhl K. Distinguishing between mechanisms of eukaryotic transcriptional activation with bacteriophage T7 RNA polymerase. Cell. 1987 Sep 25;50(7):1047–1055. doi: 10.1016/0092-8674(87)90171-1. [DOI] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Cochran M. A., Mackett M., Moss B. Eukaryotic transient expression system dependent on transcription factors and regulatory DNA sequences of vaccinia virus. Proc Natl Acad Sci U S A. 1985 Jan;82(1):19–23. doi: 10.1073/pnas.82.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davanloo P., Rosenberg A. H., Dunn J. J., Studier F. W. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2035–2039. doi: 10.1073/pnas.81.7.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DeLange A. M., McFadden G. Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells. Proc Natl Acad Sci U S A. 1986 Feb;83(3):614–618. doi: 10.1073/pnas.83.3.614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deuschle U., Pepperkok R., Wang F. B., Giordano T. J., McAllister W. T., Ansorge W., Bujard H. Regulated expression of foreign genes in mammalian cells under the control of coliphage T3 RNA polymerase and lac repressor. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5400–5404. doi: 10.1073/pnas.86.14.5400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dolph P. J., Racaniello V., Villamarin A., Palladino F., Schneider R. J. The adenovirus tripartite leader may eliminate the requirement for cap-binding protein complex during translation initiation. J Virol. 1988 Jun;62(6):2059–2066. doi: 10.1128/jvi.62.6.2059-2066.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dunn J. J., Krippl B., Bernstein K. E., Westphal H., Studier F. W. Targeting bacteriophage T7 RNA polymerase to the mammalian cell nucleus. Gene. 1988 Sep 7;68(2):259–266. doi: 10.1016/0378-1119(88)90028-5. [DOI] [PubMed] [Google Scholar]
  12. Elroy-Stein O., Bernstein Y., Groner Y. Overproduction of human Cu/Zn-superoxide dismutase in transfected cells: extenuation of paraquat-mediated cytotoxicity and enhancement of lipid peroxidation. EMBO J. 1986 Mar;5(3):615–622. doi: 10.1002/j.1460-2075.1986.tb04255.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Elroy-Stein O., Fuerst T. R., Moss B. Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6126–6130. doi: 10.1073/pnas.86.16.6126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fuerst T. R., Earl P. L., Moss B. Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes. Mol Cell Biol. 1987 Jul;7(7):2538–2544. doi: 10.1128/mcb.7.7.2538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fuerst T. R., Moss B. Structure and stability of mRNA synthesized by vaccinia virus-encoded bacteriophage T7 RNA polymerase in mammalian cells. Importance of the 5' untranslated leader. J Mol Biol. 1989 Mar 20;206(2):333–348. doi: 10.1016/0022-2836(89)90483-x. [DOI] [PubMed] [Google Scholar]
  16. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gorman C. M., Merlino G. T., Willingham M. C., Pastan I., Howard B. H. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6777–6781. doi: 10.1073/pnas.79.22.6777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Herman R. C. Internal initiation of translation on the vesicular stomatitis virus phosphoprotein mRNA yields a second protein. J Virol. 1986 Jun;58(3):797–804. doi: 10.1128/jvi.58.3.797-804.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jackson R. J. RNA translation. Picornaviruses break the rules. Nature. 1988 Jul 28;334(6180):292–293. doi: 10.1038/334292a0. [DOI] [PubMed] [Google Scholar]
  20. Jang S. K., Davies M. V., Kaufman R. J., Wimmer E. Initiation of protein synthesis by internal entry of ribosomes into the 5' nontranslated region of encephalomyocarditis virus RNA in vivo. J Virol. 1989 Apr;63(4):1651–1660. doi: 10.1128/jvi.63.4.1651-1660.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lehrach H., Diamond D., Wozney J. M., Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
  22. Lieber A., Kiessling U., Strauss M. High level gene expression in mammalian cells by a nuclear T7-phase RNA polymerase. Nucleic Acids Res. 1989 Nov 11;17(21):8485–8493. doi: 10.1093/nar/17.21.8485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pilipenko E. V., Blinov V. M., Romanova L. I., Sinyakov A. N., Maslova S. V., Agol V. I. Conserved structural domains in the 5'-untranslated region of picornaviral genomes: an analysis of the segment controlling translation and neurovirulence. Virology. 1989 Feb;168(2):201–209. doi: 10.1016/0042-6822(89)90259-6. [DOI] [PubMed] [Google Scholar]
  24. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  25. Sarnow P. Translation of glucose-regulated protein 78/immunoglobulin heavy-chain binding protein mRNA is increased in poliovirus-infected cells at a time when cap-dependent translation of cellular mRNAs is inhibited. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5795–5799. doi: 10.1073/pnas.86.15.5795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  27. Subramani S., Mulligan R., Berg P. Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors. Mol Cell Biol. 1981 Sep;1(9):854–864. doi: 10.1128/mcb.1.9.854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]

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