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.
Sheval
E V
EV
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119899, Russia.
Prusov
A N
AN
Kireev
I I
II
Fais
D
D
Polyakov
V Yu
VY
D000818:Animals; D002021:Buffers; D002467:Cell Nucleus / Q000235:genetics / Q000528:radiation effects* / Q000648:ultrastructure; D002843:Chromatin / Q000235:genetics / Q000528:radiation effects* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000235:genetics / Q000528:radiation effects / Q000648:ultrastructure; D004996:Ethidium; D022781:Hepatocytes / Q000378:metabolism / Q000528:radiation effects / Q000648:ultrastructure; D007399:Interphase / Q000235:genetics / Q000528:radiation effects; D008027:Light; D046529:Microscopy, Electron, Transmission; D034521:Nuclear Matrix-Associated Proteins / Q000235:genetics / Q000528:radiation effects* / Q000648:ultrastructure; D010777:Photochemistry / Q000379:methods*; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000528:radiation effects; D011829:Radiation Dosage; D051381:Rats; D013347:Subcellular Fractions
D002021:Buffers; D002843:Chromatin; D004268:DNA-Binding Proteins; D034521:Nuclear Matrix-Associated Proteins; D004996:Ethidium