Analysis of chromatin reconstitution

Publisher Website
Google Scholar
Abstract
The ability of high MW chicken erythrocyte chromatin to spontaneously self-assemble to native-like material, after dissociation by high ionic strength and reassociation by salt gradient dialysis, was critically examined. The native conformational state of the reassembled nucleoprotein complex was regenerated to the extent reflected by circular dichroism spectra and the thermally induced helix-coil transition of the nucleoprotein DNA. The internucleosomal packing of .apprx. 205 base pairs of DNA/repeating unit, as probed by digestion with micrococcal nuclease, was not regenerated upon reassembly and was replaced by a packing of .apprx. 160 base pairs/repeating unit. HMW chromatin containing only lysine-rich histones (H1 and H5) and core histones (H2A, H2B, H3 and H4) is not a true self-assembling system in vitro using the salt gradient dialysis system used here. Circular dichroism and thermal denaturation studies on core chromatin (lysine-rich histones removed) showed that core histones alone are not capable of reassembling high MW DNA into native-like core particles at low temperature (4.degree. C). Reassembly at 21.degree. C restored the circular dichroism but not the thermal denaturation properties to those characteristic of undissociated core chromatin. Nonetheless, micrococcal nuclease digestions of both reassembled core chromatin products were identical with undissociated native core chromatin. Reassembly in the presence of the complete complement of histones, followed by removal of the lysine-rich histones, did regenerate the thermal denaturation properties of undissociated native core particles. These results indicated multiple functions of the lysine-rich histones in the in vitro assembly of high MW chromatin.