Characterization of chromatin samples in the presence of Drosophila embryo extract by quantitative agarose gel electrophoresis.
Recent studies have focused attention on chromatin as both a negative and positive regulator of specific nuclear events. The vast majority of this research has been centered on chromatin remodeling and histone post-translational modifications over the regulatory regions of specific genes. However, due the technical difficulties of such studies, the contribution of the higher-order structure of chromatin on the regulation of gene expression has not been as thoroughly investigated and the majority of the initial studies have used biophysical methods or microscopy. Until recent technical developments, the main hindrance for these biophysical investigations of chromatin has been an almost absolute requirement for large amounts of highly purified material. The development of an agarose gel electrophoresis method (quantitative agarose gel electrophoresis), initially designed for the analysis of the three-dimensional structure of purified and in vivo-assembled chromatin over a promoter region, has been expanded to include studies of chromatin in the presence of a Drosophila crude extract. The technique presented in the study reported here will help in paving the way for subsequent analyses of structural modifications of chromatin that are linked with the recruitment of various chromatin-associated factors present in the provided extract(s).
Adkins
Nicholas L
NL
Marshall University, Department of Biological Sciences, 1 John Marshall Drive, Huntington, WV 25755, USA.
Johnson
Cotteka N
CN
Georgel
Philippe T
PT
D000818:Animals; D001667:Binding, Competitive / Q000235:genetics; D015139:Blotting, Southern / Q000379:methods; D002457:Cell Extracts / Q000032:analysis; D002843:Chromatin / Q000737:chemistry*; D042002:Chromatin Assembly and Disassembly; D002847:Chromatography, Agarose / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004330:Drosophila / Q000196:embryology*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D009707:Nucleosomes / Q000378:metabolism
D002457:Cell Extracts; D002843:Chromatin; D004268:DNA-Binding Proteins; D009707:Nucleosomes