Abstract:
: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 degrees 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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Rodríguez-Campos A,Koop R,Faraudo S,Beato Mdoi
10.1093/nar/gnh107keywords:
subject
Has Abstractpub_date
2004-07-28 00:00:00pages
e111issue
13eissn
0305-1048issn
1362-4962pii
32/13/e111journal_volume
32pub_type
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