Purification and nucleosome mapping analysis of native yeast plasmid chromatin.
There is much evidence indicating the importance in gene regulation of the positions of nucleosomes with respect to DNA sequence. Low resolution chromatin structures have been described for many genes, but there is a dearth of detailed high resolution chromatin structures. In the cases where they are available, high resolution maps have revealed much more complex chromatin structures, with multiple alternative nucleosome positions. The discovery that ATP-dependent chromatin remodelling machines are recruited to genes, with their ability to mobilise nucleosomes on DNA and to alter nucleosomal conformation, emphasises the necessity for obtaining high resolution nucleosome maps, so that the details of these remodelling reactions can be defined in vivo. Here, we describe protocols for purifying plasmid chromatin from cells of the yeast Saccharomyces cerevisiae and for mapping nucleosome positions on the plasmid using the monomer extension mapping method. This method requires purified chromatin, but is capable of mapping relatively long stretches of chromatin in great detail. Typically, it reveals very complex chromatin structures.
Kim
Yeonjung
Y
Laboratory of Cellular and Developmental Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Building 50 Room 3148, 50 South Drive MSC 8028, Bethesda, MD 20892-8028, USA.
Shen
Chang-Hui
CH
Clark
David J
DJ
D002843:Chromatin / Q000235:genetics / Q000302:isolation & purification*; D004247:DNA / Q000378:metabolism*; D005822:Genetic Vectors; D009707:Nucleosomes / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D002843:Chromatin; D009707:Nucleosomes; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase