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Analysis of Reconstituted Chromatin Using a Solid-Phase Approach

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Chromatin Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 523))

Abstract

Complex experimental strategies involving in vitro reconstituted chromatin or simple chromatin interaction studies are much facilitated by immobilizing the nucleosomal arrays to paramagnetic beads. Chromatin-containing beads can be retrieved from a reaction mix solution on a magnet fast and quantitatively, effectively separating bound, loosely attached and unbound components efficiently. This chapter details a convenient strategy for immobilization of linear plasmid DNA on streptavidin-coated beads, the reconstitution of chromatin on such beads and some fundamental handling procedures.

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References

  1. Almouzni, G. and Méchali, M. (1988) Assembly of spaced chromatin. Involvement of ATP and DNA topoisomerase activity. EMBO J. 7, 4355–4365.

    PubMed  CAS  Google Scholar 

  2. Becker, P.B. and Wu, C. (1992) Cell-free system for assembly of transcriptionally repressed chromatin from Drosophila embryos. Mol. Cell. Biol. 12, 2241–2249.

    PubMed  CAS  Google Scholar 

  3. Tremethick, D. (1999) Preparation of chromatin assembly extracts from Xenopus oocytes. In Methods in Molecular Biology, Vol. 119 Chromatin Protocols, Ed. P.B. Becker, Humana Press, Chapter 12, 175–186.

    Google Scholar 

  4. Bonte, E. and Becker, P.B. (1999) Preparation of chromatin assembly extracts from preblastoderm Drosophila embryos. In Methods in Molecular Biology, Vol. 119 Chromatin Protocols, Ed. P.B. Becker, Humana Press, Ch. 13, 187–194.

    Google Scholar 

  5. Gaillard, P.H. and Roche, D. (1999) Nucleotide excision repair coupled to chromatin assembly. In Methods in Molecular Biology, Vol. 119 Chromatin Protocols, Ed. P.B. Becker, Humana Press, Ch. 17, 231–244.

    Google Scholar 

  6. Heald, H., Tournebize, R., Blank, T.A., Sandaltzopoulos, R., Becker, P.B., Hyman, A. and Karsenti, K. (1996) Self organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature 382, 420–425.

    Article  PubMed  CAS  Google Scholar 

  7. Sandaltzopoulos, R. and Becker, P.B. (1995) Analysis of protein-DNA interaction by solid-phase footprinting. Meth. in Mol. and Cell Biol. 5, 176–181.

    Google Scholar 

  8. Sandaltzopoulos, R., Blank, T., and Becker, P.B. (1994) Transcriptional repression by nucleosomes but not H1 in reconstituted preblastoderm Drosophila chromatin. EMBO J. 13, 373–379.

    PubMed  CAS  Google Scholar 

  9. Sandaltzopoulos, R. and Becker, P.B. (1994) Solid phase footprinting: Quick and versatile. Nucleic Acids Res. 22, 1511–1512.

    Article  PubMed  CAS  Google Scholar 

  10. Sandaltzopoulos, R. and Becker, P.B. (1998). Heat shock factor increases the reinitiation rate from potentiated chromatin templates. Mol. Cell. Biol. 18, 361–367.

    PubMed  CAS  Google Scholar 

  11. Sandaltzopoulos, R. and Becker, P.B. (2003). Analysis of activator-dependent transcription reinitiation in vitro. Meth. Enzymol. 370, 487–501.

    Article  PubMed  CAS  Google Scholar 

  12. Fletcher, T.M., Ryu, B.-W., Baumann, C.T., Warren, B.S., Fragoso, G., John, S. and Hager, G.L. (2000). Structure and dynamic properties of a glucocorticoid receptor-induced chromatin transition. Mol. Cell. Biol. 20, 6466–6475.

    Article  PubMed  CAS  Google Scholar 

  13. Acevedo, M.L. and Kraus, W.L. (2003) Mediator and p300/CBP-steroid receptor coactivator complexes have distinct roles, but function synergistically, during estrogen receptor α-dependent transcription with chromatin templates. Mol. Cell. Biol. 23, 335–348.

    Article  PubMed  CAS  Google Scholar 

  14. Neubauer, B. and Hörz, W. (1989) Analysis of nucleosome positioning by in vitro reconstitution. Meth. Enzymol. 170, 630–644.

    Article  PubMed  CAS  Google Scholar 

  15. Luger, K., Rechsteiner, T. and Richmond T. (1999) Expression and purification of recombinant histones and nucleosome reconstitution. In Methods in Molecular Biology, Vol. 119 Chromatin Protocols, Ed. P.B. Becker, Humana Press, Ch. 1, 1–16.

    Google Scholar 

  16. Flaus, A. and Richmond, T. (1999) Base-pair resolution mapping of nucleosomes in vitro. In Methods in Molecular Biology, Vol. 119 Chromatin Protocols, Ed. P.B. Becker, Humana Press, Ch. 4, 45–60.

    Google Scholar 

  17. Blank, T.A. and Becker, P.B. (1996) The effect of nucleosome phasing sequences and DNA topology on nucleosome spacing. J. Mol. Biol. 260, 1–8.

    Article  PubMed  CAS  Google Scholar 

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Sandaltzopoulos, R., Becker, P.B. (2009). Analysis of Reconstituted Chromatin Using a Solid-Phase Approach. In: Chellappan, S. (eds) Chromatin Protocols. Methods in Molecular Biology, vol 523. Humana Press. https://doi.org/10.1007/978-1-59745-190-1_2

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  • DOI: https://doi.org/10.1007/978-1-59745-190-1_2

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-873-7

  • Online ISBN: 978-1-59745-190-1

  • eBook Packages: Springer Protocols

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