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Nucleic Acids Research 2004 32(13):e111; doi:10.1093/nar/gnh107
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Published online 28 July 2004

Nucleic Acids Research, Vol. 32 No. 13 © Oxford University Press 2004; all rights reserved

Transcriptionally competent chromatin assembled with exogenous histones in a yeast whole cell extract

Antonio Rodríguez-Campos, Ronald Koop1, Susanna Faraudo and Miguel Beato*

CRG, Centre de Regulació Genòmica, Universitat Pompeu Fabra (UPF), Passeig Marítim, 37-49, 08003 Barcelona, Spain and 1 Institut für Molekularbiologie und Tumorforschung, Philipps-Universitt, Emil-Mankopff-Strasse 2, 35037 Marburg, Germany

* To whom correspondence should be addressed. Tel: +34 93 224 09 01; Fax +34 93 224 08 99; Email: miguel.beato{at}crg.es

Received January 30, 2004; Revised April 13, 2004; Accepted July 2, 2004

We describe a cell-free chromatin assembly system derived from the yeast Saccharomyces cerevisiae, which efficiently packages DNA into minichromosomes in a reaction dependent on exogenous core histones and an ATP-regenerating system. Both supercoiled and relaxed plasmid DNA serve as templates for nucleosomal loading in a gradual process that takes at least 6 h for completion at 30°C. Micrococcal nuclease digestion of the assembled minichromosomes displays an extended nucleosomal ladder with a repeat length of 165 bp. The purified minichromosomes contain the four core histones in stoichiometric proportion and exhibit phased nucleosomes over the mouse mammary tumour virus (MMTV) promoter. The progesterone receptor and NF1 synergize on these minichromosomes resulting in efficient cell-free transcription. The ease of manipulation and the potential use of yeast strains carrying mutations in the chromatin handling machinery make this system suitable for detailed mechanistic studies.


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