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首页> 外文期刊>Thermochimica Acta: An International Journal Concerned with the Broader Aspects of Thermochemistry and Its Applications to Chemical Problems >ETHIDIUM BROMIDE INTERCALATION AND CHROMATIN STRUCTURE - A THERMAL ANALYSIS
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ETHIDIUM BROMIDE INTERCALATION AND CHROMATIN STRUCTURE - A THERMAL ANALYSIS

机译：溴化乙锭嵌入和染色质结构-热分析

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Differential Scanning Calorimetry has been performed in the temperature range 310 K - 410 K on intact thymocytes and physiologically isolated chromatin following Ethidium bromide intercalation. Native thymocytes exhibited four main thermal transitions (at 339 K, 347 K, 362 K and 375 K) that were assigned to the melting of different cellular components. At increasing dye concentrations an enthalpy redistribution became evident between the thermal transition at 362 K related to the melting of nucleosome organized in the 10 nm filament, and the transition at 375 K related to the melting of nucleosome organized in the 30 nm (or more) fiber. In correlation with increasing concentrations of Ethidium bromide, the disappearance and the subsequent reappearance of the highest temperature transition seem to be related to the unwrapping and subsequent wrapping of the chromatin fiber. Under similar condition, free DNA and digested chromatin do not show any enthalpy redistribution in their calorimetric profiles following Ethidium bromide intercalation. On the contrary, physiologically isolated chromatin displayed similar enthalpy redistribution between transitions assigned to chromatin DNA melting. An interesting difference appeared in the calorimetric profile of isolated chromatin with respect to the in situ material after chromatin extraction. In fact, a transition at 354 K, probably related to the melting of linker DNA became apparent (the transition at 362 K was assigned to the melting of DNA around the core particle). Selective digestions with different enzymes (micrococcal nuclease, proteinase K and DNase I) were carried out on thymocytes to verify the assignment of the main thermal transitions. In order to clarify the nature of the high temperature transitions native thymocytes were treated with topoisomerase I that removes superhelical turns from topologically closed DNA molecules. A comparison of calorimetric data with thermal denaturation profiles obtained by spectropolarimetric measurements on physiologically isolated chromatin gave further confirmation to the peak assignment by distinguishing the thermal transitions related to protein denaturation from the ones assigned to chromatin-DNA. (C) 1997 Elsevier Science B.V. [References: 32]
机译：在溴化乙锭插入后，在完整的胸腺细胞和生理分离的染色质上，在310 K-410 K的温度范围内进行了差示扫描量热法。天然胸腺细胞表现出四个主要的热转变（分别为339 K，347 K，362 K和375 K），这些转变与不同细胞成分的融化有关。随着染料浓度的增加，在与10 nm长丝组织的核小体融化有关的362 K处的热转变与与在30 nm（或更高波长）组织的核小体融化有关的在375 K的跃迁之间，焓重新分布变得很明显。纤维。与溴化乙锭浓度的增加相关，最高温度转变的消失和随后的重新出现似乎与染色质纤维的展开和随后的包裹有关。在类似条件下，溴化乙锭嵌入后，游离DNA和消化的染色质在量热曲线中没有显示任何焓的重新分布。相反，从生理上分离的染色质在分配给染色质DNA熔解的跃迁之间显示出相似的焓重分布。相对于染色质提取后的原位材料，分离的染色质的量热曲线出现了一个有趣的差异。实际上，在354 K处的转变可能很明显，这可能与接头DNA的熔解有关（在362 K处的转变被分配为核心颗粒周围DNA的熔解）。在胸腺细胞上用不同的酶（微球菌核酸酶，蛋白酶K和DNase I）进行选择性消化，以验证主要热转变的分配。为了阐明高温转变的本质，用拓扑异构酶I处理了天然胸腺细胞，该拓扑异构酶I从拓扑封闭的DNA分子中去除了超螺旋转角。通过对生理学上分离的染色质进行分光极化测定获得的热变性曲线与量热数据的比较，通过区分与蛋白质变性相关的热转变与染色质-DNA的热转变，进一步确认了峰的分配。 （C）1997 Elsevier Science B.V. [参考：32]

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