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Chromatin Protocols pp 261–290Cite as

Transcriptional and Structural Analyses of Isolated SV40 Chromatin

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 119))

Abstract

An ideal biochemical source of a defined chromatin template assembled in vivo is the SV40 minichromosomes (see Note 1). At all stages in the viral lytic cycle, SV40 DNA is complexed with cellular histone and nonhistone proteins to form the episomal chromatin structure called a minichromosome (MC). MCs are therefore the viral template for both the host replication and host transcription machinery. The chromatin properties of MCs reflect transcriptionally competent host chromatin in every respect that has been examined. They contain more highly acetylated histones, high-mobility group proteins, and DNase I hypersensitive sites (1–4). For these reasons, SV40 MCs have long been used as a model system for transcriptionally active chromatin. Because of the transcriptionally competent nature of these templates, they provide chromatin with characteristics distinct from, but complementary to, most in vitro reconstituted chromatin. SV40 MCs are especially useful in approaching questions regarding stages in transcriptional activation from a potentially competent to a fully active state. Furthermore, because of the utility of SV40 as a viral vector for exogenous promoters (5,6), fully functional cellular promoters in a native chromatin context can also be isolated and studied in this manner.

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References

  1. Coca-Prados, M., Vidali, G., and Hsu, M.-T. (1980) Intracellular forms of Simian Virus 40 nucleoprotein complexes. III. Study of histone modifications. J. Virol. 36, 353–360.

    PubMed  CAS  Google Scholar 

  2. La Bella, F. and Vesco, C. (1980) Late modifications of Simian Virus 40 chromatin during the lytic cycle occur in an immature form of virion. J. Virol. 33, 1138–1150.

    PubMed  Google Scholar 

  3. La Bella, F., Romani, M., Vesco, C., and Vidali, G. (1981) High mobility group proteins 1 and 2 are present in simian virus 40 provirions, but not in virions. Nucleic Acids Res. 9, 121–131.

    Article  PubMed  Google Scholar 

  4. Ding, H.-F., Rimsky, S., Batson, S. C., Bustin, M., and Hansen, U. (1994) Stimulation of RNA polymerase II elongation by chromosomal protein HMG-14. Science 265, 796–799.

    Article  PubMed  CAS  Google Scholar 

  5. Lassar, A. B., Hamer, D. H., and Roeder, R. G. (1985) Stable transcription complex on a class III gene in a minichromosome. Mol. Cell. Biol. 5, 40–45.

    PubMed  CAS  Google Scholar 

  6. Hamer, D. H. (1980) DNA cloning in mammalian cells with SV40 vectors, in Genetic Engineering, Volume 2 (Setlow, J. K. and Hollaender, A., eds.), Plenum Press, New York, pp. 83–101.

    Google Scholar 

  7. Ding, H.-F., Bustin, M., and Hansen, U. (1997) Alleviation of histone H1-mediated transcriptional repression and chromatin compaction by the acidic activation region in chromosomal protein HMG-14. Mol. Cell. Biol. 17, 5843–5855.

    PubMed  CAS  Google Scholar 

  8. Scott, W. A. (1988) SV40 chromatin structure, in Molecular Aspects of Papovaviruses (Aloni, Y., ed. ) Martinus Nijhoff Publishing, Boston, pp. 199–217.

    Google Scholar 

  9. Ambrose, C., Blasquez, V., and Bina, M. (1986) A block in initiation of simian virus 40 assembly results in the accumulation of minichromosomes containing an exposed regulatory region. Proc. Natl. Acad. Sci. USA 83, 3287–3291.

    Article  PubMed  CAS  Google Scholar 

  10. Boyce, F. M., Sundin, O., Barsoum, J., and Varshavsky, A. (1982) New way to isolate Simian Virus 40 nucleoprotein complexes from infected cells: use of a thiol-specific reagent. J. Virol. 42, 292–296.

    PubMed  CAS  Google Scholar 

  11. Fernandez-Munoz, R., Coca-Prados, M., and Hsu, M.-T. (1979) Intracellular forms of Simian Virus 40 nucleoprotein complexes. I. Methods of isolation and characterization in CV-1 cells. J. Virol. 29, 612–623.

    PubMed  CAS  Google Scholar 

  12. Piette, J., Cereghini, S., Kryszke, M.-H., and Yaniv, M. (1986) Identification of cellular proteins that interact with polyomavirus or Simian Virus 40 enhancers, in Cancer Cells, 4th ed. (Botchan, M., Grodzicker, T., and Sharp, P. A., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 103–113.

    Google Scholar 

  13. Batson, S. C., Rimsky, S., Sundseth, R., and Hansen, U. (1993) Association of nucleosome-free regions and basal transcription factors with in vivo-assembled chromatin templates active in vitro. Nucleic Acids Res. 21, 3459–3468.

    Article  PubMed  CAS  Google Scholar 

  14. DePamphilis, M. L. and Bradley, M. K. (1986) Replication of SV40 and polyoma virus chromosomes, in The Papovaviridae, Vol. 1: The Polyomaviruses (Salzman, N. P., ed.) Plenum, New York, NY, pp. 99–246.

    Google Scholar 

  15. Saragosti, S., Moyne, G., and Yaniv, M. (1980) Absence of nucleosomes in a fraction of SV40 chromatin between the origin of replication and the region coding for the late leader RNA. Cell 20, 65–73.

    Article  PubMed  CAS  Google Scholar 

  16. Powers, J. H. and Bina, M. (1991) In vitro assembly of a positioned nucleosome near the hypersensitive region in simian virus 40 chromatin. J. Mol. Biol. 221, 795–803.

    Article  PubMed  CAS  Google Scholar 

  17. Ambrose, C., Lowman, H., Rajadhyaksha, A., Blasquez, V., and Bina, M. (1990) Location of nucleosomes in Simian Virus 40 chromatin. J. Mol. Biol. 214, 875–884.

    Article  PubMed  CAS  Google Scholar 

  18. Jakobovits, E. B., Bratosin, S., and Aloni, Y. (1982) Formation of a nucleosome-free region in SV40 minichromosomes is dependent upon a restricted segment of DNA. Virology 120, 340–348.

    Article  PubMed  CAS  Google Scholar 

  19. Scott, W. A. and Wigmore, D. J. (1978) Sites in Simian Virus 40 chromatin which are preferentially cleaved by endonucleases. Cell 15, 1511–1518.

    Article  PubMed  CAS  Google Scholar 

  20. Varshavsky, A. J., Sundin, O., and Bohn, M. (1979) A stretch of “late” SV40 viral DNA about 400 bp long which includes the origin of replication is specifically exposed in SV40 minichromosomes. Cell 16, 453–466.

    Article  PubMed  CAS  Google Scholar 

  21. Waldeck, W., Föhring, B., Chowdhury, K., Gruss, P., and Sauer, G. (1978) Origin of DNA replication in papovavirus chromatin is recognized by endogeonous endonuclease. Proc. Natl. Acad. Sci. USA 75, 5964–5968.

    Article  PubMed  CAS  Google Scholar 

  22. Choder, M., Bratosin, S., and Aloni, Y. (1984) A direct analysis of transcribed minichromosomes: all transcribed SV40 minichromosomes have a nuclease-hypersensitive region within a nucleosome-free domain. EMBO J. 3, 2929–2936.

    PubMed  CAS  Google Scholar 

  23. Weiss, E., Ruhlmann, C., and Oudet, P. (1986) Transcriptionally active SV40 minichromosomes are restriction enzyme sensitive and contain a nucleosome-free origin region. Nucleic Acids Res. 14, 2045–2058.

    Article  PubMed  CAS  Google Scholar 

  24. Weiss, E., Regnier, E., and Oudet, P. (1987) Restriction enzyme accessibility and RNA polymerase localization on transcriptionally active SV40 minichromosomoes isolated late in infection. Virology 159, 84–93.

    Article  PubMed  CAS  Google Scholar 

  25. Blasquez, V., Stein, A., Ambrose, C., and Bina, M. (1986) Simian virus 40 protein VP1 is involved in spacing nucleosomes in minichromosomes. J. Mol. Biol. 191, 97–106.

    Article  PubMed  CAS  Google Scholar 

  26. Hansen, U. and Sharp, P. A. (1983) Sequences controlling in vitro transcription of SV40 promoters. EMBO J. 2, 2293–2303.

    PubMed  CAS  Google Scholar 

  27. Batson, S. C., Sundseth, R., Heath, C. V., Samuels, M., and Hansen, U. (1992) In vitro initiation of transcription by RNA polymerase II on in vivo-assembled chromatin templates. Mol. Cell. Biol. 12, 1639–1651.

    PubMed  CAS  Google Scholar 

  28. Gruss, C., Gutierrez, C., Burhans, W. C., DePamphilis, M. L., Koller, T., and Sogo, J. M. (1990) Nucleosome assembly in mammalian cell extracts before and after DNA replication. EMBO J. 9, 2911–2922.

    PubMed  CAS  Google Scholar 

  29. Stillman, B. (1986) Chromatin assembly during SV40 DNA replication in vitro. Cell 45, 555–565.

    Article  PubMed  CAS  Google Scholar 

  30. Sewack, G. F. and Hansen, U. (1997) Nucleosome positioning and transcription-associated chromatin alterations on the human estrogen-responsive pS2 promoter. J. Biol. Chem. 272, 31,118–31,129.

    Article  PubMed  CAS  Google Scholar 

  31. Brady, J. N., Winston, V. D., and Consigli, R. A. (1978) Characterization of a DNA-protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis-N,N’-tetraacetic acid and dithiothreitol. J. Virol. 27, 193–204.

    PubMed  CAS  Google Scholar 

  32. Hirt, B. (1967) Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365–369.

    Article  PubMed  CAS  Google Scholar 

  33. Gerard, R. D. and Gluzman, Y. (1985) New host cell system for regulated Simian Virus 40 DNA replication. Mol. Cell. Biol. 5, 3231–3240.

    PubMed  CAS  Google Scholar 

  34. Griffith, O. M. (1979) Techniques of Preparative, Zonal, and Continuous Flow Ultracentrifugation. Beckman Instruments, Palo Alto, CA.

    Google Scholar 

  35. Manley, J. L., Fire, A., Cano, A., Sharp, P. A., and Gefter, M. L. (1980) DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc. Natl. Acad. Sci. USA 77, 3855–3859.

    Article  PubMed  CAS  Google Scholar 

  36. Croston, G. E., Kerrigan, L. A., Lira, L. M., Marshak, D. R., and Kadonaga, J. T. (1991) Sequence-specific antirepression of histone H1-mediated inhibition of basal RNA polymerase II transcription. Science 251, 643–649.

    Article  PubMed  CAS  Google Scholar 

  37. Chang, X.-B. and Wilson, J. H. (1986) Formation of deletions after initiation of simian virus 40 replication: Influence of packaging limit of the capsid. J. Virol. 58, 393–401.

    PubMed  CAS  Google Scholar 

  38. O’Reilly, D. R., Miller, L. K., and Luckow, V. A. (1992) Baculovirus Expression Vectors: A Laboratory Manual. W. H. Freeman, New York, NY.

    Google Scholar 

  39. Cartwright, I. L. and Elgin, S. C. R. (1989) Nonenzymatic cleavage of chromatin. Methods Enzymol. 170, 359–369.

    Article  PubMed  CAS  Google Scholar 

  40. Wu, C. (1989) Analysis of hypersensitive sites in chromatin. Methods Enzymol. 170, 269–289.

    Article  PubMed  CAS  Google Scholar 

  41. Bellard, M., Dretzen, G., Giangrande, A., and Ramain, P. (1989) Nuclease digestion of transcriptionally active chromatin. Methods Enzymol. 170, 317–346.

    Article  PubMed  CAS  Google Scholar 

  42. Mueller, P. R. and Wold, B. (1989) In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246, 780–786.

    Article  PubMed  CAS  Google Scholar 

  43. Garrity, P. A. and Wold, B. J. (1992) Effects of different DNA polymerases in ligation-mediated PCR: Enhanced genomic sequencing and in vivo footprinting. Proc. Natl. Acad. Sci. USA 89, 1021–1025.

    Article  PubMed  CAS  Google Scholar 

  44. McPherson, C. E., Shim, E.-Y., Friedman, D. S., and Zaret, K. S. (1993) An active tissue-specific enhancer and bound transcription factors existing in a precisely positioned nucleosomal array. Cell 75, 387–398.

    Article  PubMed  CAS  Google Scholar 

  45. Pfeifer, G. P. and Riggs, A. D. (1991) Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation-mediated PCR. Genes Dev. 5, 1102–1113.

    Article  PubMed  CAS  Google Scholar 

  46. Grodzicker, T. and Hopkins, N. (1980) Origins of contemporary DNA tumor virus research, in DNA Tumor Viruses, Part 2, 2nd edition (Tooze, J., ed.) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 1–59.

    Google Scholar 

  47. Cereghini, S. and Yaniv, M. (1984) Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication. EMBO J. 3, 1243–1253.

    PubMed  CAS  Google Scholar 

  48. Innis, J. W. and Scott, W. A. (1983) Chromatin structure of simian virus 40-pBR322 recombinant plasmids in COS-1 cells. Mol. Cell. Biol. 3, 2203–2210.

    PubMed  CAS  Google Scholar 

  49. Gilmour, R. S., Gow, J. W., and Spandidos, D. A. (1982) In vivo assembly of regularly spaced nucleosomes on mouse βmaj-globin DNA cloned in an SV40 recombinant. Bioscience Reports 2, 1031–1040.

    Article  PubMed  CAS  Google Scholar 

  50. Reeves, R., Gorman, C. M., and Howard, B. (1985) Minichromosome assembly of non-integrated plasmid DNA transfected into mammalian cells. Nucleic Acids Res. 13, 3599–3615.

    Article  PubMed  CAS  Google Scholar 

  51. Shapiro, D. J., Sharp, P. A., Wahli, W. W., and Keller, M. J. (1988) A high-efficiency HeLa cell nuclear transcription extract. DNA 7, 47–55.

    Article  PubMed  CAS  Google Scholar 

  52. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489.

    Article  PubMed  CAS  Google Scholar 

  53. Conaway, R. C. and Conaway, J. W. (1993) General initiation factors for RNA polymerase II. Annu. Rev. Bioch. 62, 161–190.

    Article  CAS  Google Scholar 

  54. Zawel, L. and Reinberg, D. (1993) Initiation of transcription by RNA polymerase II: A multi-step process. Prog. Nucleic Acid Res. Mol. Biol. 44, 67–108.

    Article  PubMed  CAS  Google Scholar 

  55. Hansen, U., Tenen, D. G., Livingston, D. M., and Sharp, P. A. (1981) T antigen repression of SV40 early transcription from two promoters. Cell 27, 603–612.

    Article  PubMed  CAS  Google Scholar 

  56. Fromm, M. and Berg, P. (1982) Deletion mapping of DNA regions required for SV40 early region promoter function in vivo. J. Mol. Appl. Gen. 1, 457–481.

    CAS  Google Scholar 

  57. Fromm, M. and Berg, P. (1983) 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. 3, 991–999.

    PubMed  CAS  Google Scholar 

  58. Oudet, P., Weiss, E., and Regnier, E. (1989) Preparation of Simian Virus 40 minichromosomes. Methods Enzymol. 170, 14–25.

    Article  PubMed  CAS  Google Scholar 

  59. Varshavsky, A. J., Bakayev, V. V., Chumackov, P. M., and Georgiev, G. P. (1976) Minichromosome of simian virus 40: presence of histone H1. Nucleic Acids Res. 3, 2101–2113.

    PubMed  CAS  Google Scholar 

  60. Huang, H.-C. and Cole, R. D. (1984) The distribution of H1 histone is nonuniform in chromatin and correlates with different degrees of condensation. J. Biol. Chem. 259, 14,237–14,242.

    PubMed  CAS  Google Scholar 

  61. Yoshida, M., Horinouchi, S., and Beppu, T. (1995) Trichostatin A and trapoxin: novel chemical probes for the role of histone acetylation in chromatin structure and function. BioEssays 17, 423–430.

    Article  PubMed  CAS  Google Scholar 

  62. Sundseth, R. and Hansen, U. M. (1990) A systematic approach to the study of RNA polymerase II mediated transcription in vitro. DNA Prot. Eng. Tech. 2, 57–65.

    Google Scholar 

  63. Chodosh, L. A., Fire, A., Samuels, M., and Sharp, P. A. (1989) 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole inhibits transcription elongation by RNA polymerase II in vitro. J. Biol. Chem. 264, 2250–2257.

    PubMed  CAS  Google Scholar 

  64. Messing, J. (1983) New M13 vectors for cloning. Methods Enzymol. 101, 20–78.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Division of Molecular Genetics, Dana Faber Cancer Institute, Boston, MA

    Ulla Hansen

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  1. Ulla Hansen
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  1. Adolf-Butenandt-Institut-Molekularbiologie, Ludwig-Maximilians-Universität, München, Germany

    Peter B. Becker

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© 1999 Humana Press Inc.

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Hansen, U. (1999). Transcriptional and Structural Analyses of Isolated SV40 Chromatin. In: Becker, P.B. (eds) Chromatin Protocols. Methods in Molecular Biology™, vol 119. Humana Press. https://doi.org/10.1385/1-59259-681-9:261

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  • DOI: https://doi.org/10.1385/1-59259-681-9:261

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-665-9

  • Online ISBN: 978-1-59259-681-2

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