1887

Abstract

SUMMARY

The major enhancer, extending from nucleotides –530 to –120 upstream of the transcription initiation site of immediate early (IE) genes 1 and 2 in human cytomegalovirus (HCMV), contains four groups of repeated sequence motifs that consist of 17, 18, 19 or 21 bp, respectively. One of these elements, the 19 bp repeat, is a symmetrical palindrome that is also part of IE regulatory sequences of other cytomegalovirus-type herpesviruses, but not of unrelated members of the herpesvirus group. Synthetic oligonucleotides representing the 19 bp repeat unit strongly reduced the activity of the IE1/2 enhancer/promoter in cotransfection assays after transient expression. The HCMV enhancer can substitute for the 72 bp repeats of simian virus 40 (SV40). Replication-competent deletion mutants of SV40/HCMV enhancer recombinants were constructed that contained a single palindromic 19 bp repeat with a central cleavage site for II. If deletions were introduced into the single remaining 19 bp repeat most of the mutant viruses were still replication-competent in CV-1 monkey kidney cells. Insertion of two nucleotides into the single II site did not significantly alter transient SV40 T antigen expression. Deletion of four nucleotides or more from the single 19 bp palindrome reduced the stimulation of T antigen synthesis by the HCMV enhancer/SV40 promoter unit down to about 50%. More extended deletions (28 to 80 bp) did not further reduce T antigen expression. All mutants without an intact 19 bp repeat contained the 18 bp and/or the 21 bp sequence motif. DNase I footprinting and gel retardation assays indicated sequence-specific protein binding by the 19 bp palindrome. Altered palindromes, correlating with reduced enhancer activity, lost most of their protein-binding properties. Thus, the 19 bp repeat element is one of several protein-binding sites that contribute to enhancer strength. However, the 19 bp sequence motif can be deleted entirely to leave reduced activity. The HCMV IE1/2 upstream sequence appears to be the perfect model of an enhancer as a complex of multiple binding sites for trans-activating proteins in a modular fashion.

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1989-01-01
2024-12-09
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References

  1. Akrigg A., Wilkinson G. W. G., Oram J. D. 1985; The structure of the major immediate early gene of human cytomegalovirus strain AD 169. Virus Research 2:107–121
    [Google Scholar]
  2. Angel P., Imagawa M., Chiu R., Stein B., Imbra R. J., Rahmsdorf H. J., Jonat C., Hbrrlich P., Karin M. 1987; Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated transacting factor. Cell 49:729–739
    [Google Scholar]
  3. Baer R., Bankier A. T., Biggin M. D., Deininger P. L., Farrell P. J., Gibson T. J., Hatfull G., Hudson G. S., Satchwell S. C., Séguin C., Tuffnell P. s., Barrell B. G. 1984; DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature London: 310207–211
    [Google Scholar]
  4. Banerji J. S., Rusconi S., Schaffner W. 1981; Expression of a β-globin gene is enhanced by remote SV40 DNA sequences. Cell 27:299–308
    [Google Scholar]
  5. Benoist C., Chambon P. 1981; In vivo sequence requirements of the SV40 early promoter region. Nature London: 290304–310
    [Google Scholar]
  6. Bilofsky H. S., Burks C., Fickett J. W., Goad W. B., Lewitter F. I., Rindone W. P., Swindell C. D., Tung C.-S. 1986; The GenBankR genetic sequence databank. Nucleic Acids Research 14:1–4
    [Google Scholar]
  7. Boshart M., Weber F., Jahn G., Dorsch-Häsler K., Fleckenstein B., Schaffner W. 1985; A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell 41:521–530
    [Google Scholar]
  8. Comb M., Birnberg N. C., Seasholtz A., Nerbert E., Goodman H. M. 1987; A cyclic AMP- and phorbol ester-inducible DNA element. Nature London: 323353–356
    [Google Scholar]
  9. Davidson I., Fromental C., Augereau P., Wildeman A., Zenke M., Chambon P. 1986; Cell-type specific protein binding to the enhancer of simian virus 40 in nuclear extracts. Nature London: 323544–548
    [Google Scholar]
  10. Davison A. J., Scott J. E. 1986; The complete DNA sequence of varicella zoster-virus. Journal of General Virology 67:1759–1816
    [Google Scholar]
  11. Delegeane A. M., Ferland L. H., Mellon P. L. 1987; Tissue-specific enhancer of the human glycoprotein hormone alpha-subunit gene: dependence on cyclic AMP-inducible elements. Molecular and Cellular Biology 7:3994–4002
    [Google Scholar]
  12. Devereux J., Haeberli P., Smithies O. 1984; A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12:387–395
    [Google Scholar]
  13. De Villiers J., Schaffner W. 1981; A small segment of polyoma virus DNA enhances the expression of a cloned β-globin gene over a distance of 1400 base pairs. Nucleic Acids Research 9:6251–6264
    [Google Scholar]
  14. de Villiers J., Olson L., Tyndall C., Schaffner W. 1982; Transcriptional ‘enhancers’ from SV40 and polyoma virus show a cell type preference. Nucleic Acids Research 10:7965–7976
    [Google Scholar]
  15. Dignam J., Lebovitz R. M., Roeder G. 1983; Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Research 11:1475–1489
    [Google Scholar]
  16. Dorsch-Häsler K., Keil G. M., Weber F., Jasin M., Schaffner W., Koszinowski U. H. 1985; A long and complex enhancer activates transcription of the gene coding for the highly abundant immediate early mRNA in murine cytomegalovirus. Proceedings of the National Academy of SciencesU.S.A. 82:8325–8329
    [Google Scholar]
  17. Ellis J. G., Llewellyn D. J., Walker J. C., Dennis E. S., Peacock W. J. 1987; The ocs element: a 16 base pair palindrome element for activity of the octopine synthase enhancer. EMBO Journal 6:3203–3208
    [Google Scholar]
  18. Foecking M. K., Hofstetter H. 1986; Powerful and versatile enhancer-promoter unit for mammalian expression vectors. Gene 45:101–105
    [Google Scholar]
  19. Ghazal P., Lubon H., Fleckenstein B., Hennighausen L. 1987; Binding of transcription factors and the creation of a large nucleoprotein complex on the human cytomegalovirus enhancer. Proceedings of the National Academy of SciencesU.S.A. 84:3658–3662
    [Google Scholar]
  20. Ghazal P., Lubon H., Hennighausen L. 1988a; Specific interactions between transcription factors and the promoter-regulatory region of the human cytomegalovirus major immediate-early gene. Journal of Virology 62:1076–1079
    [Google Scholar]
  21. Ghazal P., Lubon H., Hennighausen L. 1988b; Multiple sequence-specific transcription factors modulate cytomegalovirus enhancer activity in vitro. Molecular and Cellular Biology 8:1809–1811
    [Google Scholar]
  22. Gielen J., Debeuckeleer M., Seurinck J., Deboeck F., Degreve H., Lemmers M., Van Montagu M., Schell J. 1984; The complete nucleotide sequence of the TL-DNA of the Agrobacterium tumefaciens plasmid pTiAch5. EMBO Journal 3:835–846
    [Google Scholar]
  23. Gorman C. M., Moffat L. F., Howard B. H. 1982; Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Molecular and Cellular Biology 2:1044–1051
    [Google Scholar]
  24. Graham F. L., Van Der Eb A. J. 1973; A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456–467
    [Google Scholar]
  25. Gruss P., Dhar R., Khoury G. 1981; Simian virus 40 tandem repeated sequences as an element of the early promoter. Proceedings of the National Academy of SciencesU.S.A 78:943–947
    [Google Scholar]
  26. Hardy S., Shenk T. 1988; Adenoviral control regions activated by E1 A and the cAMP response element bind to the same factor. Proceedings of the National Academy of SciencesU.S.A. 85:4171–4175
    [Google Scholar]
  27. Haugen T. H., Cripe T. P., Ginder G. D., Karin M., Turek L. P. 1987; Trans-activation of an upstream early gene promoter of bovine papilloma virus-1 by a product of the viral E2 gene. EMBO Journal 6:145–152
    [Google Scholar]
  28. Hearing P., Shenk T. 1983; The adenovirus type 5 E1A transcriptional control region contains a duplicated enhancer element. Cell 33:695–703
    [Google Scholar]
  29. Hen R., Borrelli E., Sassone-Corsi P., Chambon P. 1983; An enhancer element is located 340 base pairs upstream from the adenovirus-2 E1A capsite. Nucleic Acids Research 11:8748–8760
    [Google Scholar]
  30. Hennighausen L., Fleckenstein B. 1986; Nuclear factor 1 interacts with five DNA elements in the promoter region of the human cytomegalovirus major immediate early gene. EMBO Journal 5:1367–1371
    [Google Scholar]
  31. Hill D. E., Hope I. A., Macke J. P., Struhl K. 1986; Saturation mutagenesis of the yeast his3 regulatory site: requirements for transcriptional induction and for binding by GCN4 activator protein. Science 234:451–457
    [Google Scholar]
  32. Hirt B. 1967; Selective extraction of polyoma DNA from infected mouse cell cultures. Journal of Molecular Biology 26:365–369
    [Google Scholar]
  33. Holmes D. S., Quigley M. 1981; A rapid boiling method for the preparation of bacterial plasmids. Analytical Biochemistry 114:193–197
    [Google Scholar]
  34. Hope I. A., Struhl K. 1987; GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA. EMBO Journal 6:2781–2784
    [Google Scholar]
  35. Imagawa M., Chiu R., Karin M. 1987; Transcription factor AP-2 mediates induction by two different signal-transduction pathways: protein kinase C and cAMP. Cell 51:251–260
    [Google Scholar]
  36. Jahn G., Knust E., Schmolla H., Sarre T., Nelson J. A., McDll J. K., Fleckenstein B. 1984; Predominant immediate-early transcripts of human cytomegalovirus AD 169. Journal of Virology 49:363–370
    [Google Scholar]
  37. Jeang K-T., Cho M-S., Hayward G. S. 1984; Abundant constitutive expression of the immediate early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line. Molecular and Cellular Biology 4:2214–2223
    [Google Scholar]
  38. Jeang K-T., Rawlins D. R., Rosenfeld P., Shero J. H., Kelly T., Hayward G. S. 1987; Multiple tandemly repeated binding sites for cellular nuclear factor 1 that surround the major immediate-early promoters of simian and human cytomegalovirus. Journal of Virology 61:1559–1570
    [Google Scholar]
  39. Keil G. M., Ebeling Keil A., Koszinowski U. H. 1987; Sequence and structural organization of murine cytomegalovirus immediate-early gene 1. Journal of Virology 61:1901–1908
    [Google Scholar]
  40. Koszinowski U. H., Keil G. M., Volkmer H., Fibi M. R., Ebeling-Keil A., Münch K. 1986; The 89,000-Mr murine cytomegalovirus immediate-early protein activates gene transcription. Journal of Virology 58:59–66
    [Google Scholar]
  41. Laimins L. A., Gruss P., Pozzatti R., Khoury G. 1984; Characterization of enhancer elements in the long terminal repeat of Moloney murine sarcoma virus. Journal of Virology 49:183–189
    [Google Scholar]
  42. Lee W., Mitchell P., Than R. 1987; Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements. Cell 49:741–752
    [Google Scholar]
  43. Levinson B., Khoury G., Van De Woude G. V., Gruss P. 1982; Activation of SV40 genome by 72-base pair tandem repeats of Moloney sarcoma virus. Nature London: 295568–572
    [Google Scholar]
  44. Leza M. A., Hearing P. 1988; Cellular transcription factor binds to adenovirus early region promoters and to a cyclic AMP response element. Journal of Virology 62:3003–3013
    [Google Scholar]
  45. Lopata M. A., Cleveland D. W., Sollner-Webb B. 1984; High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Research 12:5707–5717
    [Google Scholar]
  46. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: A Laboratory Manual New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  47. Messing J. 1983; New M13 vectors for cloning. Methods in Enzymology 101:20–78
    [Google Scholar]
  48. Mitchell P. J., Wang C., Than R. 1987; Positive and negative regulation of transcription in vitro: enhancer-binding protein AP-2 is inhibited by SV40 T antigen. Cell 50:847–861
    [Google Scholar]
  49. Montminy M. R., Bilezikuian L. M. 1987; Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature London: 328175–178
    [Google Scholar]
  50. Montminy M. R., Sevarino K. A., Wagner J. A., Mandel G., Goodman R. H. 1986; Identification of a cyclic-AMP-responsive element within the rat somatostatin gene. Proceedings of the National Academy of SciencesU.S.A. 83:6682–6686
    [Google Scholar]
  51. Mosca J. D., Jeang K-T., Pitha P. M., Hayward G. S. 1987; Novel induction by herpes simplex virus of hybrid interferon gene transcripts driven by the strong cytomegalovirus IE 94 promoter. Journal of Virology 61:819–828
    [Google Scholar]
  52. Mosthaf L., Pawlita M., Gruss P. 1985; A viral enhancer element specifically active in human haematopoietic cells. Nature, London 315:597–600
    [Google Scholar]
  53. Nelson J. A., Groudine M. 1986; Transcriptional regulation of the human cytomegalovirus major immediate-early gene is associated with induction of DNase I-hypersensitive sites. Molecular and Cellular Biology 6:452–461
    [Google Scholar]
  54. Nelson J. A., Reynolds-Kohler C., Smith B. A. 1987; Negative and positive regulation by a short segment in the 5′-flanking region of the human cytomegalovirus major immediate-early gene. Molecular and Cellular Biology 7:4125–4129
    [Google Scholar]
  55. Ondek B., Shepard A., Herr W. 1987; Discrete elements within the SV40 enhancer region display different cell-specific enhancer activities. EMBO Journal 6:1017–1025
    [Google Scholar]
  56. Ondek B., Gloss L., Herr W. 1988; The SV40 enhancer contains two distinct levels of organization. Nature London 333:40–45
    [Google Scholar]
  57. Parker C. S., Topol J. 1984; A drosophila RNA polymerase II transcription factor binds to the regulatory site of an hsp 70 gene. Cell 37:273–283
    [Google Scholar]
  58. Ptashne M. 1986 A Genetic Switch Oxford: Cell Press/Blackwell Scientific Publications;
    [Google Scholar]
  59. Rosenthal N., Kress M., Gruss P., Khoury G. 1983; BK viral enhancer element and a human cellular homolog. Science 222:749–755
    [Google Scholar]
  60. Rüger R., Bornkamm G. W., Fleckenstein B. 1984; Human cytomegalovirus DNA sequences with homologies to the cellular genome. Journal of General Virology 65:1351–1364
    [Google Scholar]
  61. Schaffner G., Schirm S., Müller-Baden B., Weber F., Schaffner W. 1988; Redundancy of information in enhancers as a principle of mammalian transcription control. Journal of Molecular Biology 201:81–90
    [Google Scholar]
  62. Schaffner W. 1985; Introduction. In Eukaryotic Transcription. The Role of Cis- and Trans-acting Elements in Initiation1–18 Gluzman Y. New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  63. Schirm S., Weber F., Schaffner W., Fleckenstein B. 1985; A transcription enhancer in the Herpesvirus saimiri genome. EMBO Journal 4:2669–2674
    [Google Scholar]
  64. Schirm S., Jiricny J., Schaffner W. 1987; The SV40 enhancer can be dissected into multiple segments, each with a different cell type specificity. Genes and Development 1:65–74
    [Google Scholar]
  65. Schlokat U. 1987; Immunoglobulin Enhancer bindende Faktoren: Identifikation, Charakterisierung, Reinigung und Wechselwirkung mit anderen Enhancer Elementen. Ph.D. thesis University of Heidelberg:
    [Google Scholar]
  66. Seeberger R., Haugen T., Turek L., Pfister H. 1987; An enhancer of human papilloma virus 8 is trans-activated by the bovine papilloma virus 1 E2-function. Cancer Cells 5:33–38
    [Google Scholar]
  67. Serfling E., Jasin M., Schaffner W. 1985; Enhancers and eukaryotic gene transcription. Trends in Genetics 1:224–230
    [Google Scholar]
  68. Shaul Y., Rutter W. J., Laub O. 1985; A human hepatitis B viral enhancer element. EMBO Journal 4:427–430
    [Google Scholar]
  69. Silhavy T. J., Berman M. L., Enquist L. W. 1984; Experiments with Gene Fusions. New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  70. Silver B. J., Bokar J. A., Virgin J. B., Vallen E. A., Milstedt A., Nilson J. H. 1987; Cyclic AMP regulation of the human glycoprotein hormone alpha-subunit gene is mediated by an 18-base-pair element. Proceedings of the National Academy of SciencesU.S.A. 84:2198–2202
    [Google Scholar]
  71. Spalholz B. A., Yang Y-C., Howley P. M. 1985; Transactivation of a bovine papilloma virus transcriptional regulatory element by the E2 gene product. Cell 42:183–191
    [Google Scholar]
  72. Stenberg R. M., Thomsen D. R., Stinski M. F. 1984; Structural analysis of the major immediate gene of human cytomegalovirus. Journal of Virology 49:190–199
    [Google Scholar]
  73. Stenberg R. M., Witte P. R., Stinski M. F. 1985; Multiple spliced and unspliced transcripts from human cytomegalovirus immediate-early region 2 and evidence for a common initiation site within immediate-early region 1. Journal of Virology 56:665–675
    [Google Scholar]
  74. Stinski M. F., Thomsen D. R., Stenberg R. M., Goldstein L. C. 1983; Organization and expression of the immediate early genes of human cytomegalovirus. Journal of Virology 46:1–14
    [Google Scholar]
  75. Stinski M. F., Roehr T. J. 1985; Activation of the major immediate early gene of human cytomegalovirus by cis- acting elements in the promoter-regulatory sequence and by virus-specific trans-acting components. Journal of Virology 55:431–141
    [Google Scholar]
  76. Struhl K. 1987; The DNA-binding domains of the jun oncoprotein and the yeast GCN4 transcriptional activator protein are functionally homologous. Cell 50:841–846
    [Google Scholar]
  77. Swift F. V., Bhat K., Younghusband H. B., Hamada H. 1987; Characterization of a cell-type specific enhancer found in the human papilloma-virus type 18 genome. EMBO Journal 6:1339–1344
    [Google Scholar]
  78. Takeda Y., Ohlendorf D. H., Anderson W. F., Matthews B. W. 1983; DNA-binding proteins. Science 221:1020–1026
    [Google Scholar]
  79. Terao M., Watanabe Y., Mishina M., Numa S. 1983; Sequence requirement for transcription in vivo of the human preproenkephalin A gene. EMBO Journal 2:2223–2228
    [Google Scholar]
  80. Thomsen D. R., Stenberg R. M., Goins W. F., Stinski M. F. 1984; Promoter-regulatory region of the major immediate early gene of human cytomegalovirus. Proceedings of the National Academy of SciencesU.S.A. 81:659–663
    [Google Scholar]
  81. Tognoni A., Cattaneo R., Serfling E., Schaffner W. 1985; A novel expression selection approach allows precise mapping of the hepatitis B virus enhancer. Nucleic Acids Research 13:7457–7472
    [Google Scholar]
  82. Tsukada T., Horovitch S. J., Montminy M. R., Mandel G., Goodman R. H. 1985; Structure of the human vasoactive intestinal polypeptide gene. DNA 4:293–300
    [Google Scholar]
  83. Van Beveren C., Van Straaten F., Curran T., Müller R., Verma I. M. 1983; Analysis of FBJ-MuSV provirus and c-fos (mouse) gene reveals that viral and cellular fos gene products have different carboxy termini. Cell 32:1241–1255
    [Google Scholar]
  84. Weber F., De Villiers J., Schaffner W. 1984; An SV40 ‘enhancer trap’ incorporates exogenous enhancers or generates enhancers from its own sequences. Cell 36:983–992
    [Google Scholar]
  85. Weintraub H. 1988; Formation of stable transcription complexes as assayed by analysis of individual templates. Proceedings of the National Academy of SciencesU.S.A. 85:5819–5823
    [Google Scholar]
  86. Weston K. 1988; An enhancer in the short unique region of human cytomegalovirus regulates the production of a group of abundant immediate early transcripts. Virology 162:406–416
    [Google Scholar]
  87. Wilbur W. J., Lipman D. J. 1983; Rapid similarity searches of nucleic acids and protein data banks. Proceedings of the National Academy of SciencesU.S.A. 80:726–730
    [Google Scholar]
  88. Wilkinson G. W. G., Akrigg A., Greenaway P. J. 1984; Transcription of the immediate early genes of human cytomegalovirus strain AD 169. Virus Research 1:101–116
    [Google Scholar]
  89. Wynshaw-Boris A., Short J. M., Loose D. S., Hanson R. W. 1986; Characterization of the phosphoenolpyruvate carboxykinase (GTP) promoter-regulatory region. Journal of Biological Chemistry 261:9714–9720
    [Google Scholar]
  90. Zagursky R. J., Baumeister K., Lomax N., Berman M. L. 1985; Rapid and easy sequencing of large linear double-stranded DNA and supercoiled plasmid DNA. Gene Analysis Techniques 2:89–94
    [Google Scholar]
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