Abstract
We have recently established a system for purifying minichromosomes in a native state from Saccharomyces cerevisiae. This system is extremely efficient, and a single-step purification yields samples with sufficient purity and quantity for mass spectrometry (MS) analysis of histones and non-histone proteins tightly associated with the minichromosome. The templates can also be used in various biochemical assays in vitro, such as transcription and recombination, and could be suitable for EM or other biophysical studies.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Ducker, C. E., and Simpson, R. T. (2000) The organized chromatin domain of the repressed yeast a cell-specific gene STE6 contains two molecules of the corepressor Tup1p per nucleosome, Embo J 19, 400–409.
Unnikrishnan, A., Gafken, P. R., and Tsukiyama, T. (2010) Dynamic changes in histone acetylation regulate origins of DNA replication, Nat Struct Mol Biol 17, 430–437.
Adams, A., Gottschling, D., and Stearns, T. (1997) Methods in yeast genetics, Cold Spring Harbor Laboratory Press, New York.
Thoma, F., Bergman, L. W., and Simpson, R. T. (1984) Nuclease digestion of circular TRP1ARS1 chromatin reveals positioned nucleosomes separated by nuclease-sensitive regions, J Mol Biol 177, 715–733.
Akiyoshi, B., Nelson, C. R., Ranish, J. A., and Biggins, S. (2009) Quantitative proteomic analysis of purified yeast kinetochores identifies a PP1 regulatory subunit, Genes Dev 23, 2887–2899.
Acknowledgments
This work was supported in part by grants from NIGMS and Leukemia & Lymphoma Society to S.B. and T.T and a Beckman Young Investigator Award to S.B.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Unnikrishnan, A., Akiyoshi, B., Biggins, S., Tsukiyama, T. (2012). An Efficient Purification System for Native Minichromosome from Saccharomyces cerevisiae . In: Morse, R. (eds) Chromatin Remodeling. Methods in Molecular Biology, vol 833. Humana Press. https://doi.org/10.1007/978-1-61779-477-3_8
Download citation
DOI: https://doi.org/10.1007/978-1-61779-477-3_8
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-476-6
Online ISBN: 978-1-61779-477-3
eBook Packages: Springer Protocols