Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

Mitotic stability of an episomal vector containing a human scaffold/matrix-attached region is provided by association with nuclear matrix

Nature Cell Biology volume 2, pages 182–184 (2000)Cite this article

DNA replication occurs in tight association with the nuclear matrix, where binding of the replication origin to the nuclear matrix must precede the onset of S phase1,2,3,4. We have shown previously that the origin of replication of the simian virus 40 (SV40) genome linked to a human scaffold/matrix-attached region (S/MAR) allows sustained episomal replication (where an episome is autonomous, self-replicating DNA) that is independent of the expression of the virally encoded large T-antigen5. A vector with this combination of SV40 origin and potential for matrix association is maintained in cultured cells for at least 100 cell generations, in the absence of selection5. Here we show, by in situ hybridization and nuclear-fractionation procedures, that there is a specific interaction of this vector with the nuclear matrix and the chromosome scaffold, presumably through proteins that both structures have in common. This interaction correlates with replication of the vector as an episome. These observations allow a mechanistic explanation for the episomal replication and mitotic stability of this new type of vector.

To prevent chromosomal rearrangements from occurring during fractionation, we added a crosslinking step to the protocol. Cis-DDP is a reagent that links matrix proteins to endogenous S/MAR with high specifity8. Cis-DDP can be applied to the living cell and activated by reducing the concentration of chloride ions. In these experiments, we replaced digestion with DNaseI during the fractionation procedure by digestion with six restriction enzymes that do not cut pEPI-1, together with either one restriction enzyme that linearizes the vector (EcoRI or BglII, Fig. 1a) or two enzymes that excise the S/MAR element (EcoRI and BglII, Fig. 1a). After nuclear fractionation, the crosslinked complexes were treated with proteinase K and the purified DNA was amplified by the polymerase chain reaction (PCR) to trace the complete vector and its S/MAR-depleted variant. Vectors without the S/MAR were found in fraction 3 and, to a lesser extent, in fraction 4 (Fig. 2c, PCRII) whereas S/MAR-containing vectors were found in the nuclear-matrix fraction 5 and to a lesser extent in fraction 4 (Fig. 2c, PCRI).

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: In situ hybridization of S/MAR–origin-containing vector (pEPI-1) and its S/MAR-depleted version (pGFP-C1) to CHO metaphase chromosomes.
Figure 2: Nuclear fractionation and identification of the S/MAR-containing vector by southwestern and PCR analysis.

References

  1. Dijkwell, P. A. & Hamlin, J. L. Int. Rev. Cyt. 162A, 455–484 (1995).

  2. Cook, P. Science 284, 1790–1795 ( 1999).

    Article  CAS  Google Scholar 

  3. Cossons, N. et al. J. Cell. Biochem. 67, 439– 450 (1997).

    Article  CAS  Google Scholar 

  4. Bode, J., Benham, C., Knopp, A. & Mielke, C. Crit. Rev. Eukar. Gene Expr. (in the press).

  5. Piechaczek, C., Fetzer, C., Baiker, A., Bode, J. & Lipps, H. J. Nucleic Acids Res. 27, 426– 428 (1999).

    Article  CAS  Google Scholar 

  6. Robins, D. M., Ripley, S., Henderson, A. S. & Axel, R. Cell 23, 29–39 ( 1981).

    Article  CAS  Google Scholar 

  7. Nickerson, J. A., Krockmalnic, G. & Penman, S. in Cell Biology, a Laboratory Handbook (ed. Celis, J. E.) 184–192 (Academic, London, 1998).

  8. Ferraro, A., Cervoni, L., Eufemi, M., Altieri, F. & Turano, C. J. Cell. Biochem. 62, 495– 595 (1996).

    Article  CAS  Google Scholar 

  9. Wegner, M. et al. Nucleic Acids Res. 17, 9909– 9931 (1989).

    Article  CAS  Google Scholar 

  10. Mielke, C., Kohwi, Y., Kohwi-Shigematsu, T. & Bode, J. Biochemistry 29, 7475–7485 (1990).

    Article  CAS  Google Scholar 

  11. Kay, V. & Bode, J. Methods Mol. Cell Biol. 5, 186–194 (1995).

    Google Scholar 

  12. Benham, C., Kohwi-Shigematsu, T. & Bode, J. J. Mol. Biol. 274, 181–196 (1997).

  13. Calos, M. P. Proc. Natl Acad. Sci. USA 95, 4084– 4085 (1998).

    Article  CAS  Google Scholar 

  14. Lehman, C. W. & Botchan, M. R. Proc. Natl Acad. Sci. USA 95, 4338–4343 (1998).

    Article  CAS  Google Scholar 

  15. Ballestas, M. E., Chatis, P. A. & Kaye, K. M. Science 284, 641– 644 (1999).

    Article  CAS  Google Scholar 

  16. Fanning, E. & Knippers, R. Annu. Rev. Biochem. 61, 55–85 (1992).

    Article  CAS  Google Scholar 

  17. Liu, C. G., Maercker, C., Castanon, M. J., Hauptmann, R. & Wiche, G. Proc. Natl Acad. Sci. USA 93, 4278–4283 (1996).

    Article  CAS  Google Scholar 

  18. Laemmli, U. K. Nature 227, 680–685 ( 1970).

    Article  CAS  Google Scholar 

  19. Miskimis, W. K., Roberts, M. P., McClelland, A. & Ruddle, F. H. Proc. Natl Acad. Sci. USA 82, 6791– 6794 (1985).

    Google Scholar 

  20. Saiki, R. K. et al. Science 239, 487–491 (1988).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Alfried Krupp von Bohlen und Halbach foundation (H.J.L.), by the EC (grant BIO4-CT98-0203) and DFG (grant Bo 419/5-3) (J.B.) and by the NSF (grant DBI99-04549) and NIH (grant ROI-GM47012) (C.B.). We thank F. Grummt for providing the NTS-1 and NTS-2 sequences and C. Fetzer for his help with the graphs.

Correspondence and requests for material should be addressed to H.J.L.

Supplementary information is available on Nature Cell Biology’s World-Wide Web site (http://www.nature.com/ncb) or as paper copy from the London editorial office of Nature Cell Biology.

Author information

Authors and Affiliations

  1. Institut für Zellbiologie, Universität Witten/Herdecke, Stockumer, Strasse 10, Witten , D-58448, Germany

    A. Baiker, C. Maercker, C. Piechaczek, S. B. A. Schmidt & H. J. Lipps

  2. Gesellschaft für Biotechnologische Forschung mbH, Mascheroder, Weg 1, Braunschweig , D-38124, Germany

    J. Bode

  3. Department of Biomathematical Sciences, Mount Sinai School of Medicine, New York, 10029, , New York, USA

    C. Benham

Authors
  1. A. Baiker
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C. Maercker
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C. Piechaczek
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. S. B. A. Schmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J. Bode
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C. Benham
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. H. J. Lipps
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to H. J. Lipps.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Baiker, A., Maercker, C., Piechaczek, C. et al. Mitotic stability of an episomal vector containing a human scaffold/matrix-attached region is provided by association with nuclear matrix. Nat Cell Biol 2, 182–184 (2000). https://doi.org/10.1038/35004061

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35004061

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing