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

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 Res

journal_title

Nucleic acids research

authors

Rodríguez-Campos A,Koop R,Faraudo S,Beato M

doi

10.1093/nar/gnh107

keywords:

subject

Has Abstract

pub_date

2004-07-28 00:00:00

pages

e111

issue

13

eissn

0305-1048

issn

1362-4962

pii

32/13/e111

journal_volume

32

pub_type

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