Disintegration of nucleoskeletal elements by metrizamide/2 M salt isopyknic centrifugation

Supramolecular structures that remain bound to chromosomal DNA under high salt conditions are believed to anchor DNA in the interphase nuclear skeleton. In order to identify these anchorage structures, the non-DNA materials that remain firmly bound to chromosomal DNA under conditions that disintegrate the high salt-stable architecture of nuclei were investigated. Nuclei of Ehrlich ascites cells were histone-depleted by treatment with 2 M salt. The residual halo structures were gently sheared and subjected to metrizamide isopyknic centrifugation in the presence of 2 M salt. By this combined treatment the high salt stable nuclear skeleton becomes disintegrated and three main fractions are resolved. A light fraction comprises the DNA which appears to be essentially depleted of other nuclear components. The only non-DNA material that could be identified in the DNA band is a fraction of (nascent) RNP. No other materials which could reflect nucleoskeletal elements (e.g. lamina proteins) were found together with DNA. A peak of intermediate density comprises RNA/RNP dissociated from DNA. The heavy fraction contains the proteins that become dissociated from DNA by high-salt and/or centrifugal forces, e.g. histones and the major nuclear lamina proteins. The results indicate that nascent RNP is more tightly bound to chromosomal DNA than other components that may be involved in nuclear skeletons. This suggest that transcription complexes represent at least one type of anchorage structure of DNA, which is consistent with results indicating that nascent RNA and actively transcribed DNA sequences are preferentially retained in high-salt-treated nuclei.