Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1989 Sep;63(9):3634–3642. doi: 10.1128/jvi.63.9.3634-3642.1989

Deletion analysis of the polyomavirus late promoter: evidence for both positive and negative elements in the absence of early proteins.

K B Cahill 1, G G Carmichael 1
PMCID: PMC250953  PMID: 2547989

Abstract

We have been interested in understanding more about the sequences that constitute the polyomavirus late promoter. Our approach has been to target specific deletions to the viral intergenic region by oligonucleotide-directed mutagenesis. Wild-type and mutant promoter cassettes with defined deletions were then inserted into a promoterless expression vector containing the bacterial chloramphenicol acetyltransferase (CAT) gene (cat). Plasmids were introduced into mouse NIH 3T3 cells by transfection, and promoter activities were assessed by quantitation of both CAT enzyme and cat mRNA levels. In this report, we present the results of experiments designed to map promoter elements which affect late transcription in the absence of early viral proteins and viral DNA replication. Using this approach, we mapped two major cis-acting elements (a positive and a negative one) which affect transcription in our transient expression system. The first, positive, element coincided with the enhancer A element, which is known to be important for early transcription and viral DNA replication. Removal of this element reduced late transcription by 50- to 100-fold. The second element was a negative one; removal of 89 base pairs that included two high-affinity large-T-antigen-binding sites just to the early side of the inverted repeat structure within the replication origin resulted in a 5- to 10-fold increase in late promoter activity. The implications of these findings for late promoter function and regulation are discussed.

Full text

PDF
3637

Images in this article

3639Image
on p.3639
3639Image
on p.3639

Selected References

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

  1. Acheson N. H. Polyoma virus giant RNAs contain tandem repeats of the nucleotide sequence of the entire viral genome. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4754–4758. doi: 10.1073/pnas.75.10.4754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alwine J. C., Picardi J. Activity of simian virus 40 late promoter elements in the absence of large T antigen: evidence for repression of late gene expression. J Virol. 1986 Nov;60(2):400–404. doi: 10.1128/jvi.60.2.400-404.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Banerji J., Olson L., Schaffner W. A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell. 1983 Jul;33(3):729–740. doi: 10.1016/0092-8674(83)90015-6. [DOI] [PubMed] [Google Scholar]
  4. Bendig M. M., Folk W. R. Deletion mutants of polyoma virus defining a nonessential region between the origin of replication and the initiation codon for early proteins. J Virol. 1979 Nov;32(2):530–535. doi: 10.1128/jvi.32.2.530-535.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brady J., Bolen J. B., Radonovich M., Salzman N., Khoury G. Stimulation of simian virus 40 late gene expression by simian virus 40 tumor antigen. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2040–2044. doi: 10.1073/pnas.81.7.2040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  8. Böhnlein E., Gruss P. Interaction of distinct nuclear proteins with sequences controlling the expression of polyomavirus early genes. Mol Cell Biol. 1986 May;6(5):1401–1411. doi: 10.1128/mcb.6.5.1401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  11. Cohen B., Carmichael G. G. A method for constructing multiple tandem repeats of specific DNA fragments. DNA. 1986 Aug;5(4):339–343. doi: 10.1089/dna.1986.5.339. [DOI] [PubMed] [Google Scholar]
  12. Contreras R., Gheysen D., Knowland J., van de Voorde A., Fiers W. Evidence for the direct involvement of DNA replication origin in synthesis of late SV40 RNA. Nature. 1982 Dec 9;300(5892):500–505. doi: 10.1038/300500a0. [DOI] [PubMed] [Google Scholar]
  13. Cowie A., Kamen R. Multiple binding sites for polyomavirus large T antigen within regulatory sequences of polyomavirus DNA. J Virol. 1984 Dec;52(3):750–760. doi: 10.1128/jvi.52.3.750-760.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cowie A., Tyndall C., Kamen R. Sequences at the capped 5'-ends of polyoma virus late region mRNAs: an example of extreme terminal heterogeneity. Nucleic Acids Res. 1981 Dec 11;9(23):6305–6322. doi: 10.1093/nar/9.23.6305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Deb S., DeLucia A. L., Koff A., Tsui S., Tegtmeyer P. The adenine-thymine domain of the simian virus 40 core origin directs DNA bending and coordinately regulates DNA replication. Mol Cell Biol. 1986 Dec;6(12):4578–4584. doi: 10.1128/mcb.6.12.4578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ernoult-Lange M., May P., Moreau P., May E. Simian virus 40 late promoter region able to initiate simian virus 40 early gene transcription in the absence of the simian virus 40 origin sequence. J Virol. 1984 Apr;50(1):163–173. doi: 10.1128/jvi.50.1.163-173.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Farmerie W. G., Folk W. R. Regulation of polyomavirus transcription by large tumor antigen. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6919–6923. doi: 10.1073/pnas.81.22.6919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Freund R., Mandel G., Carmichael G. G., Barncastle J. P., Dawe C. J., Benjamin T. L. Polyomavirus tumor induction in mice: influences of viral coding and noncoding sequences on tumor profiles. J Virol. 1987 Jul;61(7):2232–2239. doi: 10.1128/jvi.61.7.2232-2239.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fromm M., Berg P. Deletion mapping of DNA regions required for SV40 early region promoter function in vivo. J Mol Appl Genet. 1982;1(5):457–481. [PubMed] [Google Scholar]
  20. Fromm M., Berg P. Simian virus 40 early- and late-region promoter functions are enhanced by the 72-base-pair repeat inserted at distant locations and inverted orientations. Mol Cell Biol. 1983 Jun;3(6):991–999. doi: 10.1128/mcb.3.6.991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fujimura F. K. Nuclear activity from F9 embryonal carcinoma cells binding specifically to the enhancers of wild-type polyoma virus and PyEC mutant DNAs. Nucleic Acids Res. 1986 Apr 11;14(7):2845–2861. doi: 10.1093/nar/14.7.2845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gaudray P., Tyndall C., Kamen R., Cuzin F. The high affinity binding site on polyoma virus DNA for the viral large-T protein. Nucleic Acids Res. 1981 Nov 11;9(21):5697–5710. doi: 10.1093/nar/9.21.5697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Hart R. P., McDevitt M. A., Ali H., Nevins J. R. Definition of essential sequences and functional equivalence of elements downstream of the adenovirus E2A and the early simian virus 40 polyadenylation sites. Mol Cell Biol. 1985 Nov;5(11):2975–2983. doi: 10.1128/mcb.5.11.2975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hartzell S. W., Byrne B. J., Subramanian K. N. Mapping of the late promoter of simian virus 40. Proc Natl Acad Sci U S A. 1984 Jan;81(1):23–27. doi: 10.1073/pnas.81.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hartzell S. W., Byrne B. J., Subramanian K. N. The simian virus 40 minimal origin and the 72-base-pair repeat are required simultaneously for efficient induction of late gene expression with large tumor antigen. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6335–6339. doi: 10.1073/pnas.81.20.6335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hearing P., Shenk T. The adenovirus type 5 E1A transcriptional control region contains a duplicated enhancer element. Cell. 1983 Jul;33(3):695–703. doi: 10.1016/0092-8674(83)90012-0. [DOI] [PubMed] [Google Scholar]
  28. Herbomel P., Bourachot B., Yaniv M. Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell. 1984 Dec;39(3 Pt 2):653–662. doi: 10.1016/0092-8674(84)90472-0. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Hyde-DeRuyscher R., Carmichael G. G. Polyomavirus early-late switch is not regulated at the level of transcription initiation and is associated with changes in RNA processing. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8993–8997. doi: 10.1073/pnas.85.23.8993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Johnson P. F., Landschulz W. H., Graves B. J., McKnight S. L. Identification of a rat liver nuclear protein that binds to the enhancer core element of three animal viruses. Genes Dev. 1987 Apr;1(2):133–146. doi: 10.1101/gad.1.2.133. [DOI] [PubMed] [Google Scholar]
  32. Katinka M., Yaniv M. DNA replication origin of polyoma virus: early proximal boundary. J Virol. 1983 Jul;47(1):244–248. doi: 10.1128/jvi.47.1.244-248.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Keller J. M., Alwine J. C. Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication. Cell. 1984 Feb;36(2):381–389. doi: 10.1016/0092-8674(84)90231-9. [DOI] [PubMed] [Google Scholar]
  34. Keller J. M., Alwine J. C. Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation. Mol Cell Biol. 1985 Aug;5(8):1859–1869. doi: 10.1128/mcb.5.8.1859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kern F. G., Basilico C. Transcription from the polyoma late promoter in cells stably transformed by chimeric plasmids. Mol Cell Biol. 1985 Apr;5(4):797–807. doi: 10.1128/mcb.5.4.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kern F. G., Dailey L., Basilico C. Common regulatory elements control gene expression from polyoma early and late promoters in cells transformed by chimeric plasmids. Mol Cell Biol. 1985 Aug;5(8):2070–2079. doi: 10.1128/mcb.5.8.2070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kern F. G., Pellegrini S., Cowie A., Basilico C. Regulation of polyomavirus late promoter activity by viral early proteins. J Virol. 1986 Oct;60(1):275–285. doi: 10.1128/jvi.60.1.275-285.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kovesdi I., Satake M., Furukawa K., Reichel R., Ito Y., Nevins J. R. A factor discriminating between the wild-type and a mutant polyomavirus enhancer. Nature. 1987 Jul 2;328(6125):87–89. doi: 10.1038/328087a0. [DOI] [PubMed] [Google Scholar]
  39. Luthman H., Nilsson M. G., Magnusson G. Non-contiguous segments of the polyoma genome required in cis for DNA replication. J Mol Biol. 1982 Nov 15;161(4):533–550. doi: 10.1016/0022-2836(82)90406-5. [DOI] [PubMed] [Google Scholar]
  40. Martin M. E., Piette J., Yaniv M., Tang W. J., Folk W. R. Activation of the polyomavirus enhancer by a murine activator protein 1 (AP1) homolog and two contiguous proteins. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5839–5843. doi: 10.1073/pnas.85.16.5839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Mueller C. R., Mes-Masson A. M., Bouvier M., Hassell J. A. Location of sequences in polyomavirus DNA that are required for early gene expression in vivo and in vitro. Mol Cell Biol. 1984 Dec;4(12):2594–2609. doi: 10.1128/mcb.4.12.2594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Mueller C. R., Muller W. J., Hassell J. A. The polyomavirus enhancer comprises multiple functional elements. J Virol. 1988 May;62(5):1667–1678. doi: 10.1128/jvi.62.5.1667-1678.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Muller W. J., Mueller C. R., Mes A. M., Hassell J. A. Polyomavirus origin for DNA replication comprises multiple genetic elements. J Virol. 1983 Sep;47(3):586–599. doi: 10.1128/jvi.47.3.586-599.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ostapchuk P., Diffley J. F., Bruder J. T., Stillman B., Levine A. J., Hearing P. Interaction of a nuclear factor with the polyomavirus enhancer region. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8550–8554. doi: 10.1073/pnas.83.22.8550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Piette J., Kryszke M. H., Yaniv M. Specific interaction of cellular factors with the B enhancer of polyoma virus. EMBO J. 1985 Oct;4(10):2675–2685. doi: 10.1002/j.1460-2075.1985.tb03987.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Piette J., Yaniv M. Molecular analysis of the interaction between an enhancer binding factor and its DNA target. Nucleic Acids Res. 1986 Dec 22;14(24):9595–9611. doi: 10.1093/nar/14.24.9595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Piette J., Yaniv M. Two different factors bind to the alpha-domain of the polyoma virus enhancer, one of which also interacts with the SV40 and c-fos enhancers. EMBO J. 1987 May;6(5):1331–1337. doi: 10.1002/j.1460-2075.1987.tb02372.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Pomerantz B. J., Mueller C. R., Hassell J. A. Polyomavirus large T antigen binds independently to multiple, unique regions on the viral genome. J Virol. 1983 Sep;47(3):600–610. doi: 10.1128/jvi.47.3.600-610.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Ruley H. E., Fried M. Sequence repeats in a polyoma virus DNA region important for gene expression. J Virol. 1983 Jul;47(1):233–237. doi: 10.1128/jvi.47.1.233-237.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Salbaum J. M., Weidemann A., Lemaire H. G., Masters C. L., Beyreuther K. The promoter of Alzheimer's disease amyloid A4 precursor gene. EMBO J. 1988 Sep;7(9):2807–2813. doi: 10.1002/j.1460-2075.1988.tb03136.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Schöler H. R., Gruss P. Specific interaction between enhancer-containing molecules and cellular components. Cell. 1984 Feb;36(2):403–411. doi: 10.1016/0092-8674(84)90233-2. [DOI] [PubMed] [Google Scholar]
  54. Soeda E., Arrand J. R., Smolar N., Walsh J. E., Griffin B. E. Coding potential and regulatory signals of the polyoma virus genome. Nature. 1980 Jan 31;283(5746):445–453. doi: 10.1038/283445a0. [DOI] [PubMed] [Google Scholar]
  55. Tang W. J., Berger S. L., Triezenberg S. J., Folk W. R. Nucleotides in the polyomavirus enhancer that control viral transcription and DNA replication. Mol Cell Biol. 1987 May;7(5):1681–1690. doi: 10.1128/mcb.7.5.1681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Taylor J. W., Schmidt W., Cosstick R., Okruszek A., Eckstein F. The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8749–8764. doi: 10.1093/nar/13.24.8749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Treisman R. Characterisation of polyoma late mRNA leader sequences by molecular cloning and DNA sequence analysis. Nucleic Acids Res. 1980 Nov 11;8(21):4867–4888. doi: 10.1093/nar/8.21.4867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Triezenberg S. J., Folk W. R. Essential nucleotides in the polyomavirus origin region. J Virol. 1984 Aug;51(2):437–444. doi: 10.1128/jvi.51.2.437-444.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Tyndall C., La Mantia G., Thacker C. M., Favaloro J., Kamen R. A region of the polyoma virus genome between the replication origin and late protein coding sequences is required in cis for both early gene expression and viral DNA replication. Nucleic Acids Res. 1981 Dec 11;9(23):6231–6250. doi: 10.1093/nar/9.23.6231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Valerio D., Duyvesteyn M. G., Dekker B. M., Weeda G., Berkvens T. M., van der Voorn L., van Ormondt H., van der Eb A. J. Adenosine deaminase: characterization and expression of a gene with a remarkable promoter. EMBO J. 1985 Feb;4(2):437–443. doi: 10.1002/j.1460-2075.1985.tb03648.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Veldman G. M., Lupton S., Kamen R. Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression. Mol Cell Biol. 1985 Apr;5(4):649–658. doi: 10.1128/mcb.5.4.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Weiher H., Botchan M. R. An enhancer sequence from bovine papilloma virus DNA consists of two essential regions. Nucleic Acids Res. 1984 Mar 26;12(6):2901–2916. doi: 10.1093/nar/12.6.2901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Weiher H., König M., Gruss P. Multiple point mutations affecting the simian virus 40 enhancer. Science. 1983 Feb 11;219(4585):626–631. doi: 10.1126/science.6297005. [DOI] [PubMed] [Google Scholar]
  65. Wigler M., Pellicer A., Silverstein S., Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. doi: 10.1016/0092-8674(78)90254-4. [DOI] [PubMed] [Google Scholar]
  66. de Villiers J., Schaffner W. A small segment of polyoma virus DNA enhances the expression of a cloned beta-globin gene over a distance of 1400 base pairs. Nucleic Acids Res. 1981 Dec 11;9(23):6251–6264. doi: 10.1093/nar/9.23.6251. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES