Organization of chromosomes in HeLa cells: isolation of histone-depleted nuclei and nuclear scaffolds

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Abstract

Histone-depleted nuclei were prepared from isolated HeLa nuclei by extracting the histones and other proteins wiht 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 results of the experiments is that the DNA in the histone-depleted nuclei is highly organized, and that this residual-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 12000 S, when 2 M NaCl is used to extract the histones, or 6000 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 or 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 around 65000-70000 Daltons, while the second group has molecular weights of 50000-55000 Daltons. The major species of this latter group of proteins have mobilities that are similar to the proteins of the metaphase chromosomal scaffold.

Original languageEnglish (US)
Pages (from-to)291-304
Number of pages14
JournalJournal of cell science
VolumeVOL.42
StatePublished - Jan 1 1980

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