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RESEARCH PRODUCT

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

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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: 100 nm globules—chromomeres, chains of chromomeres—chromonemata, aggregates of chromomeres—blocks of condensed chromatin. All these structures were completely destroyed by 2 M 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.