A critical electrolyte concentration (CEC) method based on binding of toluidine blue molecules under Mg2+ competitive conditions has been proposed for discriminating differences in DNA-protein complexes in vitro (8) and associated with different states of chromatin supra-organization (1, 4). In the method proposed by Vidal and Mello (8), Mg2+ ions are added to the experimental staining solutions and the Mg2+ concentration at which metachromasy is prevented has been considered to be the CEC value.When studying variants of this technique, it was found that ribonucleoprotein complexes could be metachromatically highlighted after the CEC value of DNA-protein complexes was attained. This report describes a variant of the CEC technique in which the nucleolus is significantly discriminated. MATERIALS AND METHODSRoot tip cells of Allium ceps, salivary gland cells of Drosophila aracataca, Balb/3T3 cells, and NIH/3T3 cells transformed by transfection with DNA from carcinogen-treated human cells (5) were used.All the materials were fixed in an ethanol-acetic acid (3 : 1, v/v) mixture for 1 min, with the exception of the onion root tips, which lasted 15 min in the fixative. The glands of D, aracataca were squashed in a drop of 45% acetic acid after fixation, and frozen for removal of the coverslips, after which the preparations were rinsed in 70% ethanol and air dried.In the other materials, fixation was immediately followed by a 5-min bath in 70% ethanol. The onion root tips were subsequently processed for paraffin wax embedding and cut into 12-pm thick sections. The cultured cells were air dried immediately after the 70% ethanol treatment.Staining was performed with a 0.025% toluidine blue (Merck) solution in McIlvaine buffer at pH 4. 1, for 15 min. Immediately after staining, the preparations were treated with 0.02 M to 0.10 M aqueous MgC12 solutions for 15 min, rapidly rinsed in distilled water (5s), air dried, cleared in xylene and mounted in Canada balsam.Some preparations were treated with a 0.1 % RNase III (Sigma) aqueous solution at 37°C for 1 hr prior to staining.Spectral absorption curves were obtained for the nucleolus and the chromatin of the onion cell nuclei in order to detect their absorption peak wavelengths, and consequently, metachromasy loss at the chromatin level. Microspectrophotometry was carried out with Zeiss equipment (EMI 6256 photomultiplier, photometer 01, Planapo 100/1.25 objective, optovar 2, measuring diaphragm diameter of 0.
The binding of toluidine blue molecules to nucleic acid phosphates under conditions of competition with Mg2+ ions was studied in Trichosia pubescens polytene chromosome regions differing in composition and organization (RNA-rich areas and puffs, DNA puffs, heterochromatin). The aim was to find variability in the Mg2+ concentration at which metachromasy was completely prevented (= critical electrolyte concentration; CEC) and that could reflect differences at the level of nucleoprotein complexes in particular chromosome regions and developmental phases. Although high CEC values have been found in tightly packed chromatin such as that of heterochromatin zones, the CEC values for the other regions have proven to be affected not only by availability and proximity of DNA phosphates but also by RNA richness and other factors. Among these, changes in DNA geometry and packing state have been assumed for explaining increased values after RNA removal in DNA puffs and RNA-rich zones. Based on CEC values it has been suggested that alterations at the level of nucleoprotein complexes may occur in puffs before they are morphologically detectable. CEC results for polytene chromosomes were thus revealed to vary considerably with slight variations in nucleoprotein composition and organization. However, since a complex and apparently contradictory rationale has to be used for explaining part of the results, CEC is not recommended as a useful tool for extensive and comparative studies of this particular model.
A ligação de moléculas de azul de toluidina a grupamentos fosfatos de ácido nucléico em condições de competição com íons Mg2+ foi estudada em regiões de cromossomos politênicos de Trichosia pubescens que diferem em composição e organização (áreas ricas em RNA, pufes de RNA e DNA, heterocromatina). O objetivo foi encontrar variabilidade na concentração de Mg2+ na qual a metacromasia fosse completamente abolida (= concentração crítica de eletrólitos; CEC) e que refletisse diferenças ao nível dos complexos nucleoprotéicos em regiões cromossômicas e fases do desenvolvimento particulares. Embora valores altos de CEC tenham sido encontrados na cromatina altamente empacotada, como nas zonas heterocromáticas, os valores de CEC para outras regiões mostraram-se afetados não apenas pela disponibilidade e proximidade de fosfatos do DNA mas também pela riqueza em RNA e outros fatores. Entre estes, mudanças na geometria e no estado de empacotamento do DNA foram consideradas para explicar valores aumentados após remoção de RNA em pufes de DNA e zonas ricas em RNA. Baseando-se em valores de CEC foi sugerido que possam ocorrer alterações ao nível dos complexos nucleoprotéicos em pufes antes que estes sejam detectáveis morfologicamente como tal. Os valores de CEC nos cromossomos politênicos revelaram-se, pois, altamente variáveis com as variações em composição e organização nucleoprotéica do substrato. Contudo, uma vez que um raciocínio complexo e aparentemente contraditório tem que ser utilizado para a interpretação de parte dos resultados, ...
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