Transcriptionally competent chromatin assembled with exogenous histones in a yeast whole cell extract.
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.
Rodríguez-Campos
Antonio
A
CRG, Centre de Regulació Genòmica, Universitat Pompeu Fabra (UPF), Passeig Marítim, 37-49, 08003 Barcelona, Spain.
Koop
Ronald
R
Faraudo
Susanna
S
Beato
Miguel
M
D000818:Animals; D002457:Cell Extracts; D002843:Chromatin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly; D002875:Chromosomes / Q000737:chemistry; D006657:Histones / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D002457:Cell Extracts; D002843:Chromatin; D006657:Histones; D009707:Nucleosomes