ORGANIZATION OF HIGHER-LEVEL CHROMATIN STRUCTURES (CHROMOMERE, CHROMONEMA AND CHROMATIN BLOCK) EXAMINED USING VISIBLE LIGHT-INDUCED CHROMATIN PHOTO-STABILIZATION

The method of chromatin photo‐stabilization by the action of visible light in the presence of ethidium bromide was used for investigation of higher‐level chromatin structures in isolated nuclei. As a model we used rat hepatocyte nuclei isolated in buffers which stabilized or destabilized nuclear matrix. Several higher‐level chromatin structures were visualized: 100nm globules—chromomeres, chains of chromomeres—chromonemata, aggregates of chromomeres—blocks of condensed chromatin. All these structures were completely destroyed by 2M NaCl extraction independent of the matrix state, and DNA was extruded from the residual nuclei (nuclear matrices) into a halo. These results show that nuclear matrix proteins do not play the main role in the maintenance of higher‐level chromatin structures. Preliminary irradiation led to the reduction of the halo width in the dose‐dependent manner. In regions of condensed chromatin of irradiated nucleoids there were discrete complexes consisting of DNA fibers radiating from an electron‐dense core and resembling the decondensed chromomeres or the rosette‐like structures. As shown by the analysis of proteins bound to irradiated nuclei upon high‐salt extraction, irradiation presumably stabilized the non‐histone proteins. These results suggest that in interphase nuclei loop domains are folded into discrete higher‐level chromatin complexes (chromomeres). These complexes are possibly maintained by putative non‐histone proteins, which are extracted with high‐salt buffers from non‐irradiated nuclei.