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 Jun;82(11):3567–3571. doi: 10.1073/pnas.82.11.3567

Overproduction of the protein product of a nonselected foreign gene carried by an adenovirus vector.

M Yamada, J A Lewis, T Grodzicker
PMCID: PMC397826  PMID: 2987934

Abstract

We have constructed a recombinant adenovirus that carries the herpes simplex virus type I gene for thymidine kinase (EC 2.7.1.21) and expresses thymidine kinase under control of adenovirus major late promoter. A DNA fragment carrying thymidine kinase coding sequences but lacking the thymidine kinase promoter was sandwiched between a piece of adenoviral DNA and simian virus 40 early DNA on a plasmid. The aligned fragment was then inserted into the adenoviral genome, replacing internal adenoviral DNA. Hybrid viruses carrying the thymidine kinase gene were obtained by selecting for viruses that express simian virus 40 tumor antigen (T antigen) in monkey cells. The thymidine kinase gene was positioned in the third segment of the adenovirus tripartite leader downstream from the major late promoter by in vivo DNA recombination between the duplicated adenoviral sequences present in the plasmid insert and the viral vector. Levels of thymidine kinase activity in human or monkey cells infected with this hybrid virus were several times higher than in cells infected with herpes simplex virus. Infected cells produced thymidine kinase protein at very high levels, similar to those found for adenovirus late major capsid proteins. The thymidine kinase protein represented 10% of the newly synthesized protein in late infected cells and accumulated to represent 1% of total cell protein under optimal conditions. This vector system offers a procedure by which a variety of gene products that are biologically active and properly modified can be produced at high levels in mammalian cells.

Full text

PDF
3567

Images in this article

Selected References

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

  1. 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]
  2. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  3. Elder J. T., Spritz R. A., Weissman S. M. Simian virus 40 as a eukaryotic cloning vehicle. Annu Rev Genet. 1981;15:295–340. doi: 10.1146/annurev.ge.15.120181.001455. [DOI] [PubMed] [Google Scholar]
  4. Friedman M. P., Lyons M. J., Ginsberg H. S. Biochemical consequences of type 2 adenovirus and Simian virus 40 double infections of African green monkey kidney cells. J Virol. 1970 May;5(5):586–597. doi: 10.1128/jvi.5.5.586-597.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gingeras T. R., Sciaky D., Gelinas R. E., Bing-Dong J., Yen C. E., Kelly M. M., Bullock P. A., Parsons B. L., O'Neill K. E., Roberts R. J. Nucleotide sequences from the adenovirus-2 genome. J Biol Chem. 1982 Nov 25;257(22):13475–13491. [PubMed] [Google Scholar]
  6. Goldberg D. A. Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5794–5798. doi: 10.1073/pnas.77.10.5794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  8. Harlow E., Pim D. C., Crawford L. V. Complex of simian virus 40 large-T antigen and host 53,000-molecular-weight protein in monkey cells. J Virol. 1981 Feb;37(2):564–573. doi: 10.1128/jvi.37.2.564-573.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  10. Jamieson A. T., Subak-Sharpe J. H. Biochemical studies on the herpes simplex virus-specified deoxypyrimidine kinase activity. J Gen Virol. 1974 Sep;24(3):481–492. doi: 10.1099/0022-1317-24-3-481. [DOI] [PubMed] [Google Scholar]
  11. Klemperer H. G., Haynes G. R., Shedden W. I., Watson D. H. A virus-specific thymidine kinase in BHK-21 cells infected with herpes simplex virus. Virology. 1967 Jan;31(1):120–128. doi: 10.1016/0042-6822(67)90015-3. [DOI] [PubMed] [Google Scholar]
  12. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984 Jan 25;12(2):857–872. doi: 10.1093/nar/12.2.857. [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. Logan J., Shenk T. Adenovirus tripartite leader sequence enhances translation of mRNAs late after infection. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3655–3659. doi: 10.1073/pnas.81.12.3655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maniatis T., Jeffrey A., Kleid D. G. Nucleotide sequence of the rightward operator of phage lambda. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1184–1188. doi: 10.1073/pnas.72.3.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mansour S. L., Grodzicker T., Tjian R. An adenovirus vector system used to express polyoma virus tumor antigens. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1359–1363. doi: 10.1073/pnas.82.5.1359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marsden H. S., Haarr L., Preston C. M. Processing of herpes simplex virus proteins and evidence that translation of thymidine kinase mRNA is initiated at three separate AUG codons. J Virol. 1983 May;46(2):434–445. doi: 10.1128/jvi.46.2.434-445.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McKnight S. L. Functional relationships between transcriptional control signals of the thymidine kinase gene of herpes simplex virus. Cell. 1982 Dec;31(2 Pt 1):355–365. doi: 10.1016/0092-8674(82)90129-5. [DOI] [PubMed] [Google Scholar]
  19. Nakajima K., Oda K. The alteration of ribosomes for mRNA selection concerned with adenovirus growth in SV40-infected simian cells. Virology. 1975 Sep;67(1):85–93. doi: 10.1016/0042-6822(75)90406-7. [DOI] [PubMed] [Google Scholar]
  20. Preston C. M., McGeoch D. J. Identification and mapping of two polypeptides encoded within the herpes simplex virus type 1 thymidine kinase gene sequences. J Virol. 1981 May;38(2):593–605. doi: 10.1128/jvi.38.2.593-605.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. RABSON A. S., O'CONOR G. T., BEREZESKY I. K., PAUL F. J. ENHANCEMENT OF ADENOVIRUS GROWTH IN AFRICAN GREEN MONKEY KIDNEY CELL CULTURES BY SV40. Proc Soc Exp Biol Med. 1964 May;116:187–190. doi: 10.3181/00379727-116-29197. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Tegtmeyer P., Schwartz M., Collins J. K., Rundell K. Regulation of tumor antigen synthesis by simain virus 40 gene A. J Virol. 1975 Jul;16(1):168–178. doi: 10.1128/jvi.16.1.168-178.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Thummel C., Tjian R., Grodzicker T. Construction of adenovirus expression vectors by site-directed in vivo recombination. J Mol Appl Genet. 1982;1(5):435–446. [PubMed] [Google Scholar]
  27. Thummel C., Tjian R., Grodzicker T. Expression of SV40 T antigen under control of adenovirus promoters. Cell. 1981 Mar;23(3):825–836. doi: 10.1016/0092-8674(81)90447-5. [DOI] [PubMed] [Google Scholar]
  28. Thummel C., Tjian R., Hu S. L., Grodzicker T. Translational control of SV40 T antigen expressed from the adenovirus late promoter. Cell. 1983 Jun;33(2):455–464. doi: 10.1016/0092-8674(83)90427-0. [DOI] [PubMed] [Google Scholar]
  29. Tjian R. The binding site on SV40 DNA for a T antigen-related protein. Cell. 1978 Jan;13(1):165–179. doi: 10.1016/0092-8674(78)90147-2. [DOI] [PubMed] [Google Scholar]
  30. Wagner M. J., Sharp J. A., Summers W. C. Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1441–1445. doi: 10.1073/pnas.78.3.1441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Weinstock G. M., ap Rhys C., Berman M. L., Hampar B., Jackson D., Silhavy T. J., Weisemann J., Zweig M. Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4432–4436. doi: 10.1073/pnas.80.14.4432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yamada M., Takeda Y., Okamoto K., Hirota Y. Physical map of the nrdA-nrdB-ftsB-glpT region of the chromosomal DNA of Escherichia coli. Gene. 1982 Jun;18(3):309–318. doi: 10.1016/0378-1119(82)90169-x. [DOI] [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