Analyzing chromatin structure and transcription factor binding in yeast.
The study of chromatin, once thought to be a purely structural matrix serving to compact the DNA of the genome into the nucleus, is of increasing value for our understanding of how DNA functions in the cell. This article provides two basic procedures for the study of chromatin in vivo. The first is a DNase I-based method for the treatment of isolated nuclei to resolve the chromatin structure of a particular region; the second employs dimethyl sulfate footprinting of whole cells in vivo to determine the binding of factors to cis elements in the locus of interest. Specific examples illustrating the techniques described are given from our work on the regulation of the yeast PHO8 gene, but have also been successfully and reliably applied to the study of many other yeast loci. These procedures make it possible to correlate the binding of a transactivator with an altered or perturbed chromatin organization at a specific locus.
Gregory
P D
PD
Institut für Physiologische Chemie, Universität München, Germany.
Barbaric
S
S
Hörz
W
W
D001665:Binding Sites; D001671:Biochemistry / Q000379:methods*; D002467:Cell Nucleus / Q000737:chemistry; D002843:Chromatin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000302:isolation & purification; D018983:DNA Footprinting / Q000379:methods*; D017931:DNA Primers; D003850:Deoxyribonuclease I / Q000378:metabolism; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism; D013463:Sulfuric Acid Esters / Q000737:chemistry; D019914:Taq Polymerase / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism
D002843:Chromatin; D017931:DNA Primers; D009707:Nucleosomes; D013463:Sulfuric Acid Esters; D014157:Transcription Factors; D004247:DNA; D019914:Taq Polymerase; D003850:Deoxyribonuclease I; C007482:dimethyl sulfate