Organization of chromosomes in HeLa cells: isolation of histone-depleted nuclei and nuclear scaffolds.
Histone-depleted nuclei were prepared from isolate HeLa nuclei by extracting the histones and other proteins with polyanions (dextran sulphate and heparin) or with high salt concentrations as used previously. The particles were characterized by sucrose density gradient sedimentation, thin sectioning and electron microscopy, and by polyacrylamide gel electrophoresis. The general result of the experiments is that the DNA in the histone-depleted nuclei is highly organized, and that this residual, higher-order structure is maintained by a reproducible subset of nuclear proteins, and perhaps by RNA. Furthermore, the residual proteins remain associated, in some conditions, as rapidly sedimenting structures even when the DNA is digested with nucleases. These nuclear scaffolds can resemble extracted nuclei. Histone-depleted HeLa nuclei sediment in sucrose density gradients as well defined peaks with sedimentation coefficients of around 12 000 S, when 2M NaCl is used to extract the histones, or 6 000 S, when dextran sulphate is used. The rate of sedimentation is drastically decreased by treating the particles with trypsin, and reduced to a lesser extent with RNase A. Thin sectioning and electron microscopy show that histone-depleted nuclei possess the nuclear periphery and that internal material is also present. These general features are also seen in thin sections of nuclear scaffolds, which are prepared by treating the nuclei with micrococcal nuclease of DNase I in addition to extracting the histones. Two groups of major proteins are associated with histone-depleted HeLa nuclei and the nuclear scaffolds: One group has molecular weights of 50 000-55 000 Daltons. The major species of this latter group of proteins have mobilities that are similar to the proteins of the metaphase chromosomal scaffold.
D002467:Cell Nucleus / Q000032:analysis / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D002875:Chromosomes / Q000648:ultrastructure*; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000648:ultrastructure; D006657:Histones; D006801:Humans; D008854:Microscopy, Electron; D011506:Proteins / Q000032:analysis
D006657:Histones; D011506:Proteins; D004247:DNA