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. 1984 Jun;3(6):1235–1241. doi: 10.1002/j.1460-2075.1984.tb01958.x

Localization of T-antigen on simian virus 40 minichromosomes by immunoelectron microscopy.

F Harper, Y Florentin, E Puvion
PMCID: PMC557504  PMID: 6086310

Abstract

SV40 chromatin extracted from 42 h post-infected cells by a modification of the standard Triton X-100-EDTA procedure and purified on neutral sucrose gradients was partially immunoprecipitable by a specific SV40 T-antigen (T-Ag) antiserum. Electron microscopic observations of spread minichromosomes were made after labelling by the indirect colloidal gold immunological method using monoclonal antibodies specific for the SV40 T-Ag. In 1-2% of morphologically mature minichromosomes the labelling corresponding to tightly bound T-Ag was localized within the nucleosome-free region near one of its borders. Mapping with three single-cut restriction endonucleases: BamHI, EcoRI and BglI localized the labelling near to, or at the origin of, replication. In addition, we observed that the T-Ag specific antibodies were linked to a DNA-bound particle when the region was not masked by a large clump of antibodies. The variable size of this particle led us to suggest that it might be a complex of T-Ag with other proteins.

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Selected References

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

  1. Absolom D., Van Regenmortel M. H. Nucleosome structure studied with purified antibodies to histones H2B, H3 and H4. FEBS Lett. 1977 Dec 20;85(1):61–64. doi: 10.1016/0014-5793(78)81248-4. [DOI] [PubMed] [Google Scholar]
  2. Alwine J. C., Reed S. I., Stark G. R. Characterization of the autoregulation of simian virus 40 gene A. J Virol. 1977 Oct;24(1):22–27. doi: 10.1128/jvi.24.1.22-27.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beard P., Kaneko M., Cerutti P. N-Acetoxy-acetylaminofluorene reacts preferentially with a control region of intracellular SV40 chromosome. Nature. 1981 May 7;291(5810):84–85. doi: 10.1038/291084a0. [DOI] [PubMed] [Google Scholar]
  4. Bellard M., Oudet P., Germond J. E., Chambon P. Subunit structure of simian-virus-40 minichromosome. Eur J Biochem. 1976 Nov 15;70(2):543–553. doi: 10.1111/j.1432-1033.1976.tb11046.x. [DOI] [PubMed] [Google Scholar]
  5. Benoist C., Chambon P. In vivo sequence requirements of the SV40 early promotor region. Nature. 1981 Mar 26;290(5804):304–310. doi: 10.1038/290304a0. [DOI] [PubMed] [Google Scholar]
  6. Chou J. Y., Avila J., Martin R. G. Viral DNA synthesis in cells infected by temperature-sensitive mutants of simian virus 40. J Virol. 1974 Jul;14(1):116–124. doi: 10.1128/jvi.14.1.116-124.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cowan K., Tegtmeyer P., Anthony D. D. Relationship of replication and transcription of Simian Virus 40 DNA. Proc Natl Acad Sci U S A. 1973 Jul;70(7):1927–1930. doi: 10.1073/pnas.70.7.1927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cremisi C., Pignatti P. F., Croissant O., Yaniv M. Chromatin-like structures in polyoma virus and simian virus 10 lytic cycle. J Virol. 1975 Jan;17(1):204–211. doi: 10.1128/jvi.17.1.204-211.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dubochet J., Ducommun M., Zollinger M., Kellenberger E. A new preparation method for dark-field electron microscopy of biomacromolecules. J Ultrastruct Res. 1971 Apr;35(1):147–167. doi: 10.1016/s0022-5320(71)80148-x. [DOI] [PubMed] [Google Scholar]
  10. Garber E. A., Seidman M. M., Levine A. J. Intracellular SV40 nucleoprotein complexes: synthesis to encapsidation. Virology. 1980 Dec;107(2):389–401. doi: 10.1016/0042-6822(80)90306-2. [DOI] [PubMed] [Google Scholar]
  11. Germond J. E., Hirt B., Oudet P., Gross-Bellark M., Chambon P. Folding of the DNA double helix in chromatin-like structures from simian virus 40. Proc Natl Acad Sci U S A. 1975 May;72(5):1843–1847. doi: 10.1073/pnas.72.5.1843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ghose D., Weiss E., Homo J. C., Van Regenmortel M. H., Oudet P. Visualization of anti-histone antibodies on SV40 minichromosomes by scanning transmission electron microscopy (STEM). Exp Cell Res. 1983 Aug;147(1):135–142. doi: 10.1016/0014-4827(83)90278-1. [DOI] [PubMed] [Google Scholar]
  13. Ghosh P. K., Lebowitz P. Simian virus 40 early mRNA's contain multiple 5' termini upstream and downstream from a Hogness-Goldberg sequence; a shift in 5' termini during the lytic cycle is mediated by large T antigen. J Virol. 1981 Oct;40(1):224–240. doi: 10.1128/jvi.40.1.224-240.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goldblatt D., Bustin M., Sperling R. Heterogeneity in the interaction of chromatin subunits with anti-histone sera visulatized by immuno-electron microscopy. Exp Cell Res. 1978 Mar 1;112(1):1–14. doi: 10.1016/0014-4827(78)90519-0. [DOI] [PubMed] [Google Scholar]
  15. Griffith J. D. Chromatin structure: deduced from a minichromosome. Science. 1975 Mar 28;187(4182):1202–1203. doi: 10.1126/science.187.4182.1202. [DOI] [PubMed] [Google Scholar]
  16. Griffith J., Dieckmann M., Berg P. Electron microscope localization of a protein bound near the origin of simian virus 40 DNA replication. J Virol. 1975 Jan;15(1):167–172. doi: 10.1128/jvi.15.1.167-172.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hansen U., Tenen D. G., Livingston D. M., Sharp P. A. T antigen repression of SV40 early transcription from two promoters. Cell. 1981 Dec;27(3 Pt 2):603–613. doi: 10.1016/0092-8674(81)90402-5. [DOI] [PubMed] [Google Scholar]
  18. Harlow E., Crawford L. V., Pim D. C., Williamson N. M. Monoclonal antibodies specific for simian virus 40 tumor antigens. J Virol. 1981 Sep;39(3):861–869. doi: 10.1128/jvi.39.3.861-869.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jakobovits E. B., Bratosin S., Aloni Y. A nucleosome-free region in SV40 minichromosomes. Nature. 1980 May 22;285(5762):263–265. doi: 10.1038/285263a0. [DOI] [PubMed] [Google Scholar]
  20. Jessel D., Landau T., Hudson J., Lalor T., Tenen D., Livingston D. M. Identification of regions of the SV40 genome which contain preferred SV40 T antigen-binding sites. Cell. 1976 Aug;8(4):535–545. doi: 10.1016/0092-8674(76)90222-1. [DOI] [PubMed] [Google Scholar]
  21. Kasamatsu H., Wu M. Protein-SV40 DNA complex stable in high salt and sodium dodecyl sulfate. Biochem Biophys Res Commun. 1976 Feb 9;68(3):927–936. doi: 10.1016/0006-291x(76)91234-1. [DOI] [PubMed] [Google Scholar]
  22. Kress M., May E., Cassingena R., May P. Simian virus 40-transformed cells express new species of proteins precipitable by anti-simian virus 40 tumor serum. J Virol. 1979 Aug;31(2):472–483. doi: 10.1128/jvi.31.2.472-483.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mann K., Hunter T. Association of simian virus 40 T antigen with simian virus 40 nucleoprotein complexes. J Virol. 1979 Jan;29(1):232–241. doi: 10.1128/jvi.29.1.232-241.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mathis D. J., Chambon P. The SV40 early region TATA box is required for accurate in vitro initiation of transcription. Nature. 1981 Mar 26;290(5804):310–315. doi: 10.1038/290310a0. [DOI] [PubMed] [Google Scholar]
  25. McKay R., DiMaio D. Binding of an SV40 T antigen-related protein to the DNA of SV40 regulatory mutants. Nature. 1981 Feb 26;289(5800):810–813. doi: 10.1038/289810a0. [DOI] [PubMed] [Google Scholar]
  26. Moyne G., Freeman R., Saragosti S., Yaniv M. A high-resolution electron microscopy study of nucleosomes from simian virus 40 chromatin. J Mol Biol. 1981 Jul 15;149(4):735–744. doi: 10.1016/0022-2836(81)90355-7. [DOI] [PubMed] [Google Scholar]
  27. Myers R. M., Rio D. C., Robbins A. K., Tjian R. SV40 gene expression is modulated by the cooperative binding of T antigen to DNA. Cell. 1981 Aug;25(2):373–384. doi: 10.1016/0092-8674(81)90056-8. [DOI] [PubMed] [Google Scholar]
  28. Myers R. M., Williams R. C., Tjian R. Oligomeric structure of a simian virus 40 T antigen in free form and bound to DNA. J Mol Biol. 1981 Jun 5;148(4):347–353. doi: 10.1016/0022-2836(81)90180-7. [DOI] [PubMed] [Google Scholar]
  29. Müller U., Zentgraf H., Eicken I., Keller W. Higher order structure of simian virus 40 chromatin. Science. 1978 Aug 4;201(4354):406–415. doi: 10.1126/science.208155. [DOI] [PubMed] [Google Scholar]
  30. Persico-DiLauro M., Martin R. G., Livingston D. M. Interaction of Simian Virus 40 chromatin with Simian Virus 40 T-antigen. J Virol. 1977 Nov;24(2):451–460. doi: 10.1128/jvi.24.2.451-460.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Reed S. I., Stark G. R., Alwine J. C. Autoregulation of simian virus 40 gene A by T antigen. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3083–3087. doi: 10.1073/pnas.73.9.3083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Reiser J., Renart J., Crawford L. V., Stark G. R. Specific association of simian virus 40 tumor antigen with simian virus 40 chromatin. J Virol. 1980 Jan;33(1):78–87. doi: 10.1128/jvi.33.1.78-87.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rio D. C., Tjian R. SV40 T antigen binding site mutations that affect autoregulation. Cell. 1983 Apr;32(4):1227–1240. doi: 10.1016/0092-8674(83)90305-7. [DOI] [PubMed] [Google Scholar]
  34. Robinson G. W., Hallick L. M. Mapping the in vivo arrangement of nucleosomes on simian virus 40 chromatin by the photoaddition of radioactive hydroxymethyltrimethylpsoralen. J Virol. 1982 Jan;41(1):78–87. doi: 10.1128/jvi.41.1.78-87.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rudolph K., Mann K. Salt-resistant association of simian virus 40 T antigen with simian virus 40 DNA in nucleoprotein complexes. J Virol. 1983 Aug;47(2):276–286. doi: 10.1128/jvi.47.2.276-286.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Saragosti S., Moyne G., Yaniv M. Absence of nucleosomes in a fraction of SV40 chromatin between the origin of replication and the region coding for the late leader RNA. Cell. 1980 May;20(1):65–73. doi: 10.1016/0092-8674(80)90235-4. [DOI] [PubMed] [Google Scholar]
  37. Scott W. A., Wigmore D. J. Sites in simian virus 40 chromatin which are preferentially cleaved by endonucleases. Cell. 1978 Dec;15(4):1511–1518. doi: 10.1016/0092-8674(78)90073-9. [DOI] [PubMed] [Google Scholar]
  38. Segawa M., Sugano S., Yamaguchi N. Association of simian virus 40 T antigen with replicating nucleoprotein complexes of simian virus 40. J Virol. 1980 Aug;35(2):320–330. doi: 10.1128/jvi.35.2.320-330.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shalloway D., Kleinberger T., Livingston D. M. Mapping of SV40 DNA replication origin region binding sites for the SV40 T antigen by protection against exonuclease III digestion. Cell. 1980 Jun;20(2):411–422. doi: 10.1016/0092-8674(80)90627-3. [DOI] [PubMed] [Google Scholar]
  40. Shortle D. R., Margolskee R. F., Nathans D. Mutational analysis of the simian virus 40 replicon: pseudorevertants of mutants with a defective replication origin. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6128–6131. doi: 10.1073/pnas.76.12.6128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stahl H., Knippers R. Simian virus 40 large tumor antigen on replicating viral chromatin: tight binding and localization on the viral genome. J Virol. 1983 Jul;47(1):65–76. doi: 10.1128/jvi.47.1.65-76.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Switzer R. C., 3rd, Merril C. R., Shifrin S. A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem. 1979 Sep 15;98(1):231–237. doi: 10.1016/0003-2697(79)90732-2. [DOI] [PubMed] [Google Scholar]
  43. Tack L. C., DePamphilis M. L. Analysis of simian virus 40 chromosome-T-antigen complexes: T-antigen is preferentially associated with early replicating DNA intermediates. J Virol. 1983 Oct;48(1):281–295. doi: 10.1128/jvi.48.1.281-295.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tegtmeyer P., Andersen B., Shaw S. B., Wilson V. G. Alternative interactions of the SV40 A protein with DNA. Virology. 1981 Nov;115(1):75–87. doi: 10.1016/0042-6822(81)90090-8. [DOI] [PubMed] [Google Scholar]
  45. Tegtmeyer P., Lewton B. A., DeLucia A. L., Wilson V. G., Ryder K. Topography of simian virus 40 A protein-DNA complexes: arrangement of protein bound to the origin of replication. J Virol. 1983 Apr;46(1):151–161. doi: 10.1128/jvi.46.1.151-161.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tegtmeyer P. Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon. J Virol. 1972 Oct;10(4):591–598. doi: 10.1128/jvi.10.4.591-598.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tjian R. Protein-DNA interactions at the origin of simian virus 40 DNA replication. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):655–661. doi: 10.1101/sqb.1979.043.01.073. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Varshavsky A. J., Nedospasov S. A., Schmatchenko V. V., Bakayev V. V., Chumackov P. M., Georgiev G. P. Compact form of SV40 viral minichromosome is resistant to nuclease: possible implications for chromatin structure. Nucleic Acids Res. 1977 Oct;4(10):3303–3325. doi: 10.1093/nar/4.10.3303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Varshavsky A. J., Sundin O. H., Bohn M. J. SV40 viral minichromosome: preferential exposure of the origin of replication as probed by restriction endonucleases. Nucleic Acids Res. 1978 Oct;5(10):3469–3477. doi: 10.1093/nar/5.10.3469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Varshavsky A. J., Sundin O., Bohn M. A stretch of "late" SV40 viral DNA about 400 bp long which includes the origin of replication is specifically exposed in SV40 minichromosomes. Cell. 1979 Feb;16(2):453–466. doi: 10.1016/0092-8674(79)90021-7. [DOI] [PubMed] [Google Scholar]
  52. Waldeck W., Föhring B., Chowdhury K., Gruss P., Sauer G. Origin of DNA replication in papovavirus chromatin is recognized by endogenous endonuclease. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5964–5968. doi: 10.1073/pnas.75.12.5964. [DOI] [PMC free article] [PubMed] [Google Scholar]

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