Gruzdev A D, Lezzi M
Institute for Cytology and Genetics, Russian Academy of Sciences, Siberian Department, Novosibirsk.
Chromosome Res. 1998 Aug;6(5):367-78. doi: 10.1023/a:1009296404879.
The torsional tension of unconstrained double-helical DNA was determined in transcriptionally hyperactive Balbiani ring 2 (BR2) and in inactive polytene chromosome bands of Chironomus tentans. The method used is based on the dual ability of small intercalating ligands to (a) sense, by differential binding, twists that deviate from that of regular B-form DNA and (b) create positive torsional tension in closed double-stranded DNA, thereby compensating for any negative torsional tension that existed before intercalation. Isolated nuclei of salivary glands were stained with the intercalating fluorescent dye ethidium bromide (EtBr) at various concentrations, and the temporal fluorescence intensity changes (deltaI/I per min) occurring in BR2 and in inactive bands were monitored under a confocal laser scanning microscope during the process of DNA nicking by laser irradiation or DNAase I. From the EtBr concentration at which deltaI/I per min was neither positive nor negative after nicking (i.e. at the equivalence point), the relative twist difference (RTD) was calculated. In bands, it was found to be very small, suggesting that their unconstrained DNA is under low torsional stress. In contrast, the RTD of DNA in highly expanded areas of BR2 was estimated to be negative and of a significant magnitude in absolute terms. This indicates that transcriptionally hyperactive DNA is under considerable negative torsional tension.
在转录活跃的巴尔比亚尼环2（BR2）和摇蚊无活性多线染色体带中，测定了无约束双螺旋DNA的扭转张力。所采用的方法基于小嵌入配体的双重能力：（a）通过差异结合感知偏离规则B型DNA的扭曲；（b）在封闭的双链DNA中产生正扭转张力，从而补偿嵌入前存在的任何负扭转张力。用不同浓度的嵌入荧光染料溴化乙锭（EtBr）对唾液腺分离核进行染色，并在共聚焦激光扫描显微镜下监测激光照射或DNA酶I切割DNA过程中BR2和无活性带中发生的时间荧光强度变化（每分钟ΔI/I）。根据切割后每分钟ΔI/I既非正也非负的EtBr浓度（即等效点），计算相对扭曲差异（RTD）。在染色体带中，发现RTD非常小，这表明其无约束DNA处于低扭转应力下。相比之下，BR2高度扩展区域中DNA的RTD估计为负，绝对值相当大。这表明转录活跃的DNA处于相当大的负扭转张力下。
