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. 1985 May 24;13(10):3599–3615. doi: 10.1093/nar/13.10.3599

Minichromosome assembly of non-integrated plasmid DNA transfected into mammalian cells.

R Reeves, C M Gorman, B Howard
PMCID: PMC341261  PMID: 3859838

Abstract

The nucleoprotein structures formed on various plasmid expression vectors transfected into mammalian cells by both the calcium phosphate and DEAE-dextran methods have been studied. We demonstrate by a variety of means that mammalian cells are capable of rapidly assembling non-integrated circular plasmids (both replicating and non-replicating) into typical "minichromosomes" containing nucleosomes with a 190 bp repetitive spacing. Treatment of recipient cells with sodium butyrate for a short period of time (12-16 h) immediately following transfection markedly increased the DNase I digestion sensitivity of the newly assembled plasmid chromatin. Furthermore, minichromosomes isolated from such butyrate-treated cells are depleted in histone H1 and contain highly acetylated forms of histone H4. These findings are entirely consistent with our earlier speculation (Gorman et al., Nucleic Acids Res. 11, 1044; 1983) that appropriate butyrate treatment might stimulate transient expression of newly transfected genes by facilitating their assembly into an "active" type of chromatin structure.

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Selected References

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

  1. Camerini-Otero R. D., Zasloff M. A. Nucleosomal packaging of the thymidine kinase gene of herpes simplex virus transferred into mouse cells: an actively expressed single-copy gene. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5079–5083. doi: 10.1073/pnas.77.9.5079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Candido E. P., Reeves R., Davie J. R. Sodium butyrate inhibits histone deacetylation in cultured cells. Cell. 1978 May;14(1):105–113. doi: 10.1016/0092-8674(78)90305-7. [DOI] [PubMed] [Google Scholar]
  3. Cereghini S., Yaniv M. Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication. EMBO J. 1984 Jun;3(6):1243–1253. doi: 10.1002/j.1460-2075.1984.tb01959.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Choder M., Bratosin S., Aloni Y. A direct analysis of transcribed minichromosomes: all transcribed SV40 minichromosomes have a nuclease-hypersensitive region within a nucleosome-free domain. EMBO J. 1984 Dec 1;3(12):2929–2936. doi: 10.1002/j.1460-2075.1984.tb02234.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Flasar F. M. Oceans on titan? Science. 1983 Jul 1;221(4605):55–57. doi: 10.1126/science.221.4605.55. [DOI] [PubMed] [Google Scholar]
  6. Glikin G. C., Ruberti I., Worcel A. Chromatin assembly in Xenopus oocytes: in vitro studies. Cell. 1984 May;37(1):33–41. doi: 10.1016/0092-8674(84)90298-8. [DOI] [PubMed] [Google Scholar]
  7. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  8. Gorman C. M., Howard B. H., Reeves R. Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate. Nucleic Acids Res. 1983 Nov 11;11(21):7631–7648. doi: 10.1093/nar/11.21.7631. [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. Gurdon J. B., Melton D. A. Gene transfer in amphibian eggs and oocytes. Annu Rev Genet. 1981;15:189–218. doi: 10.1146/annurev.ge.15.120181.001201. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Innis J. W., Scott W. A. Chromatin structure of simian virus 40-pBR322 recombinant plasmids in COS-1 cells. Mol Cell Biol. 1983 Dec;3(12):2203–2210. doi: 10.1128/mcb.3.12.2203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lopata M. A., Cleveland D. W., Sollner-Webb B. 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 Res. 1984 Jul 25;12(14):5707–5717. doi: 10.1093/nar/12.14.5707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Loyter A., Scangos G. A., Ruddle F. H. Mechanisms of DNA uptake by mammalian cells: fate of exogenously added DNA monitored by the use of fluorescent dyes. Proc Natl Acad Sci U S A. 1982 Jan;79(2):422–426. doi: 10.1073/pnas.79.2.422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McClelland M. The effect of site specific methylation on restriction endonuclease cleavage (update). Nucleic Acids Res. 1983 Jan 11;11(1):r169–r173. doi: 10.1093/nar/11.1.235-c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McCutchan J. H., Pagano J. S. Enchancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J Natl Cancer Inst. 1968 Aug;41(2):351–357. [PubMed] [Google Scholar]
  18. Merril C. R., Switzer R. C., Van Keuren M. L. Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4335–4339. doi: 10.1073/pnas.76.9.4335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mertz J. E. Linear DNA does not form chromatin containing regularly spaced nucleosomes. Mol Cell Biol. 1982 Dec;2(12):1608–1618. doi: 10.1128/mcb.2.12.1608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Miller T. J., Mertz J. E. Template structural requirements for transcription in vivo by RNA polymerase II. Mol Cell Biol. 1982 Dec;2(12):1595–1607. doi: 10.1128/mcb.2.12.1595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mulligan R. C., Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2072–2076. doi: 10.1073/pnas.78.4.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Partington G. A., Yarwood N. J., Rutherford T. R. Human globin gene transcription in injected Xenopus oocytes: enhancement by sodium butyrate. EMBO J. 1984 Dec 1;3(12):2787–2792. doi: 10.1002/j.1460-2075.1984.tb02210.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Reeves R., Gorman C. M., Howard B. In vivo incorporation of Drosophila H2a histone into mammalian chromatin. Nucleic Acids Res. 1983 May 11;11(9):2681–2700. doi: 10.1093/nar/11.9.2681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Reeves R. Transcriptionally active chromatin. Biochim Biophys Acta. 1984 Sep 10;782(4):343–393. doi: 10.1016/0167-4781(84)90044-7. [DOI] [PubMed] [Google Scholar]
  25. Roginski R. S., Skoultchi A. I., Henthorn P., Smithies O., Hsiung N., Kucherlapati R. Coordinate modulation of transfected HSV thymidine kinase and human globin genes. Cell. 1983 Nov;35(1):149–155. doi: 10.1016/0092-8674(83)90217-9. [DOI] [PubMed] [Google Scholar]
  26. Rösl F., Waldeck W., Sauer G. Isolation of episomal bovine papillomavirus chromatin and identification of a DNase I-hypersensitive region. J Virol. 1983 May;46(2):567–574. doi: 10.1128/jvi.46.2.567-574.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shelton E. R., Wassarman P. M., DePamphilis M. L. Structure, spacing, and phasing of nucleosomes on isolated forms of mature simian virus 40 chromosomes. J Biol Chem. 1980 Jan 25;255(2):771–782. [PubMed] [Google Scholar]
  28. Simpson R. T. Structure of chromatin containing extensively acetylated H3 and H4. Cell. 1978 Apr;13(4):691–699. doi: 10.1016/0092-8674(78)90219-2. [DOI] [PubMed] [Google Scholar]
  29. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  30. Sussman D. J., Milman G. Short-term, high-efficiency expression of transfected DNA. Mol Cell Biol. 1984 Aug;4(8):1641–1643. doi: 10.1128/mcb.4.8.1641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Weintraub H., Groudine M. Chromosomal subunits in active genes have an altered conformation. Science. 1976 Sep 3;193(4256):848–856. doi: 10.1126/science.948749. [DOI] [PubMed] [Google Scholar]
  32. Wu C. The 5' ends of Drosophila heat shock genes in chromatin are hypersensitive to DNase I. Nature. 1980 Aug 28;286(5776):854–860. doi: 10.1038/286854a0. [DOI] [PubMed] [Google Scholar]

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