title: Chromatin structure analysis of single gene molecules by psoralen cross-linking and electron microscopy.
abstract: Nucleosomes occupy a central role in regulating eukaryotic gene expression by blocking access of transcription factors to their target sites on chromosomal DNA. Analysis of chromatin structure and function has mostly been performed by probing DNA accessibility with endonucleases. Such experiments average over large numbers of molecules of the same gene, and more recently, over entire genomes. However, both digestion and averaging erase the structural variation between molecules indicative of dynamic behavior, which must be reconstructed for any theory of regulation. Solution of this problem requires the structural analysis of single gene molecules. In this chapter, we describe a method by which single gene molecules are purified from the yeast Saccharomyces cerevisiae and cross-linked with psoralen, allowing the determination of nucleosome configurations by transmission electron microscopy. We also provide custom analysis software that semi-automates the analysis of micrograph data. This single-gene technique enables detailed examination of chromatin structure at any genomic locus in yeast.
authors.affiliation: Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA, 95064, USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Brown
authors.forename: Julian A
authors.initials: JA
authors.lastname: Eskin
authors.forename: Stephan
authors.initials: S
authors.lastname: Hamperl
authors.forename: Joachim
authors.initials: J
authors.lastname: Griesenbeck
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Jurica
authors.forename: Hinrich
authors.initials: H
authors.lastname: Boeger
mesh_terms: D002498:Centrifugation; D002843:Chromatin / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000648:ultrastructure*; D042002:Chromatin Assembly and Disassembly; D002846:Chromatography, Affinity; D046508:Culture Techniques; D004271:DNA, Fungal / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D005363:Ficusin / Q000494:pharmacology*; D005656:Fungal Proteins / Q000032:analysis / Q000302:isolation & purification; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics*; D007091:Image Processing, Computer-Assisted; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D009691:Nucleic Acid Denaturation; D009707:Nucleosomes / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D012984:Software
chemical_list: D002843:Chromatin; D004271:DNA, Fungal; D005656:Fungal Proteins; D009707:Nucleosomes; D005363:Ficusin