Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1988 Jan;8(1):353–360. doi: 10.1128/mcb.8.1.353

A specific domain of the adenovirus EIV promoter is necessary to maintain susceptibility of the integrated promoter to EIA transactivation.

T Nishigaki 1, S Hanaka 1, R E Kingston 1, H Handa 1
PMCID: PMC363129  PMID: 2961981

Abstract

We constructed a series of mutations that delete sequences in the promoter region of the early-region IV (EIV) promoter of adenovirus type 5. We fused these promoter mutations to the coding sequences of either the chloramphenicol acetyltransferase or the dihydrofolate reductase (DHFR) gene and tested the ability of a cotransfected EIa gene to stimulate EIV expression. All of the mutations tested were stimulated in these assays, implying that no specific sequence is required for stimulation. Two mutant promoters, deleted for either the TATA box or the region residing between -39 and -177 upstream from the cap site of EIV mRNA, did show a reduced level of stimulation by the EIa products. To assess the effects of the EIA gene products on expression from an EIV promoter integrated into the chromosome, we isolated CHO cell lines containing EIV-DHFR chimeric genes. After introduction of the EIa gene with a second selectable marker, expression from all mutant EIV-DHFR genes was increased. Surprisingly, one mutant promoter, deleted for sequences between -39 and -177, lost the ability to respond to the EIa region on passage of cells, although deletions in any part of the region still retained this ability. These results demonstrate that multiple elements residing between -39 and -177 in the EIV promoter are necessary to maintain susceptibility of the integrated promoter to regulation.

Full text

PDF
353

Images in this article

Selected References

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

  1. Berk A. J., Lee F., Harrison T., Williams J., Sharp P. A. Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs. Cell. 1979 Aug;17(4):935–944. doi: 10.1016/0092-8674(79)90333-7. [DOI] [PubMed] [Google Scholar]
  2. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  3. Bienz M., Pelham H. R. Heat shock regulatory elements function as an inducible enhancer in the Xenopus hsp70 gene and when linked to a heterologous promoter. Cell. 1986 Jun 6;45(5):753–760. doi: 10.1016/0092-8674(86)90789-0. [DOI] [PubMed] [Google Scholar]
  4. Bos J. L., ten Wolde-Kraamwinkel H. C. The E1b promoter of Ad12 in mouse L tk- cells is activated by adenovirus region E1a. EMBO J. 1983;2(1):73–76. doi: 10.1002/j.1460-2075.1983.tb01383.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Elkaim R., Goding C., Kédinger C. The adenovirus-2 EIIa early gene promoter: sequences required for efficient in vitro and in vivo transcription. Nucleic Acids Res. 1983 Oct 25;11(20):7105–7117. doi: 10.1093/nar/11.20.7105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gaynor R. B., Hillman D., Berk A. J. Adenovirus early region 1A protein activates transcription of a nonviral gene introduced into mammalian cells by infection or transfection. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1193–1197. doi: 10.1073/pnas.81.4.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gilardi P., Perricaudet M. The E4 promoter of adenovirus type 2 contains an E1A dependent cis-acting element. Nucleic Acids Res. 1986 Nov 25;14(22):9035–9049. doi: 10.1093/nar/14.22.9035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gilardi P., Perricaudet M. The E4 transcriptional unit of Ad2: far upstream sequences are required for its transactivation by E1A. Nucleic Acids Res. 1984 Oct 25;12(20):7877–7888. doi: 10.1093/nar/12.20.7877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Green M. R., Treisman R., Maniatis T. Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products. Cell. 1983 Nov;35(1):137–148. doi: 10.1016/0092-8674(83)90216-7. [DOI] [PubMed] [Google Scholar]
  13. Hanaka S., Nishigaki T., Sharp P. A., Handa H. Regulation of in vitro and in vivo transcription of early-region IV of adenovirus type 5 by multiple cis-acting elements. Mol Cell Biol. 1987 Jul;7(7):2578–2587. doi: 10.1128/mcb.7.7.2578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Handa H., Mizumoto K., Oda K., Okamoto T., Fukasawa T. Transcription of the human adenovirus E1a gene in Saccharomyces cerevisiae. Gene. 1985;33(2):159–168. doi: 10.1016/0378-1119(85)90090-3. [DOI] [PubMed] [Google Scholar]
  15. Handa H., Sharp P. A. Requirement for distal upstream sequences for maximal transcription in vitro of early region IV of adenovirus. Mol Cell Biol. 1984 Apr;4(4):791–798. doi: 10.1128/mcb.4.4.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Imperiale M. J., Kao H. T., Feldman L. T., Nevins J. R., Strickland S. Common control of the heat shock gene and early adenovirus genes: evidence for a cellular E1A-like activity. Mol Cell Biol. 1984 May;4(5):867–874. doi: 10.1128/mcb.4.5.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Imperiale M. J., Nevins J. R. Adenovirus 5 E2 transcription unit: an E1A-inducible promoter with an essential element that functions independently of position or orientation. Mol Cell Biol. 1984 May;4(5):875–882. doi: 10.1128/mcb.4.5.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jones N., Shenk T. An adenovirus type 5 early gene function regulates expression of other early viral genes. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3665–3669. doi: 10.1073/pnas.76.8.3665. [DOI] [PMC free article] [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. Kaufman R. J., Sharp P. A. Construction of a modular dihydrofolate reductase cDNA gene: analysis of signals utilized for efficient expression. Mol Cell Biol. 1982 Nov;2(11):1304–1319. doi: 10.1128/mcb.2.11.1304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kingston R. E., Baldwin A. S., Jr, Sharp P. A. Regulation of heat shock protein 70 gene expression by c-myc. Nature. 1984 Nov 15;312(5991):280–282. doi: 10.1038/312280a0. [DOI] [PubMed] [Google Scholar]
  22. Kingston R. E., Kaufman R. J., Sharp P. A. Regulation of transcription of the adenovirus EII promoter by EIa gene products: absence of sequence specificity. Mol Cell Biol. 1984 Oct;4(10):1970–1977. doi: 10.1128/mcb.4.10.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Leff T., Corden J., Elkaim R., Sassone-Corsi P. Transcriptional analysis of the adenovirus-5 EIII promoter: absence of sequence specificity for stimulation by EIa gene products. Nucleic Acids Res. 1985 Feb 25;13(4):1209–1221. doi: 10.1093/nar/13.4.1209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McKnight S., Tjian R. Transcriptional selectivity of viral genes in mammalian cells. Cell. 1986 Sep 12;46(6):795–805. doi: 10.1016/0092-8674(86)90061-9. [DOI] [PubMed] [Google Scholar]
  25. McMaster G. K., Carmichael G. G. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. doi: 10.1073/pnas.74.11.4835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mulligan R. C., Berg P. Expression of a bacterial gene in mammalian cells. Science. 1980 Sep 19;209(4463):1422–1427. doi: 10.1126/science.6251549. [DOI] [PubMed] [Google Scholar]
  27. Murthy S. C., Bhat G. P., Thimmappaya B. Adenovirus EIIA early promoter: transcriptional control elements and induction by the viral pre-early EIA gene, which appears to be sequence independent. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2230–2234. doi: 10.1073/pnas.82.8.2230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nagata K., Guggenheimer R. A., Hurwitz J. Specific binding of a cellular DNA replication protein to the origin of replication of adenovirus DNA. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6177–6181. doi: 10.1073/pnas.80.20.6177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Nevins J. R. Mechanism of activation of early viral transcription by the adenovirus E1A gene product. Cell. 1981 Oct;26(2 Pt 2):213–220. doi: 10.1016/0092-8674(81)90304-4. [DOI] [PubMed] [Google Scholar]
  31. Nielsen D. A., Chou J., MacKrell A. J., Casadaban M. J., Steiner D. F. Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5198–5202. doi: 10.1073/pnas.80.17.5198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. O'Farrell P. H., Kutter E., Nakanishi M. A restriction map of the bacteriophage T4 genome. Mol Gen Genet. 1980;179(2):421–435. doi: 10.1007/BF00425473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Steffen D., Bird S., Rowe W. P., Weinberg R. A. Identification of DNA fragments carrying ecotropic proviruses of AKR mice. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4554–4558. doi: 10.1073/pnas.76.9.4554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Wu L., Rosser D. S., Schmidt M. C., Berk A. A TATA box implicated in E1A transcriptional activation of a simple adenovirus 2 promoter. Nature. 1987 Apr 2;326(6112):512–515. doi: 10.1038/326512a0. [DOI] [PubMed] [Google Scholar]
  39. Zajchowski D. A., Boeuf H., Kédinger C. The adenovirus-2 early EIIa transcription unit possesses two overlapping promoters with different sequence requirements for EIa-dependent stimulation. EMBO J. 1985 May;4(5):1293–1300. doi: 10.1002/j.1460-2075.1985.tb03775.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES