Genome-wide in vitro reconstitution of yeast chromatin with in vivo-like nucleosome positioning
| Autor: | Nils, Krietenstein, Christian J, Wippo, Corinna, Lieleg, Philipp, Korber |
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| Rok vydání: | 2012 |
| Předmět: |
Genomic Library
Titrimetry DNA Restriction Enzymes Saccharomyces cerevisiae Sodium Chloride Chromatin Assembly and Disassembly Chromatin Nucleosomes Histones Adenosine Triphosphate Electroporation Escherichia coli Animals Micrococcal Nuclease Nucleic Acid Conformation Drosophila Electrophoresis Polyacrylamide Gel Genome Fungal DNA Fungal Dialysis Plasmids |
| Zdroj: | Methods in enzymology. 513 |
| ISSN: | 1557-7988 |
| Popis: | Recent genome-wide mapping of nucleosome positions revealed that well-positioned nucleosomes are pervasive across eukaryotic genomes, especially in important regulatory regions such as promoters or origins of replication. As nucleosomes impede access to DNA, their positioning is a primary mode of genome regulation. In vivo studies, especially in yeast, shed some light on factors involved in nucleosome positioning, but there is an urgent need for a complementary biochemical approach in order to confirm their direct roles, identify missing factors, and study their mechanisms. Here we describe a method that allows the genome-wide in vitro reconstitution of nucleosomes with very in vivo-like positions by a combination of salt gradient dialysis reconstitution, yeast whole cell extracts, and ATP. This system provides a starting point and positive control for the biochemical dissection of nucleosome positioning mechanisms. |
| Databáze: | OpenAIRE |
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