Spectrofluorometric measurement of the binding of ethidium to superhelical DNA from cell nuclei.
Spectrofluorometric measurement of the binding of ethidium to superhelical DNA from cell nuclei.
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荧光分光光度法测量乙锭与细胞核超螺旋 DNA 的结合。
DOI：
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发表时间：
1978
期刊：
影响因子：
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通讯作者：
I. Brazell
中科院分区：
文献类型：
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作者：
Peter R. Cook;I. Brazell
在含有非离子去污剂和高浓度盐的溶液中裂解HeLa细胞，可以释放保留许多细胞核形态特征的结构。这些类核含有很少的染色质蛋白。通过荧光分光光度法测定嵌入染料乙锭与未辐照和γ辐照的类核结合的量，我们证明了类核DNA是准环状和超螺旋的。乙锭以与超螺旋DNA结合的方式与类核结合：在低浓度下，与未辐照的类核结合的乙锭比与经γ辐照的DNA断裂的对应物结合的乙锭多，而在较高浓度下，与未辐照的类核结合的乙锭少。类核中的准环对γ射线的敏感性比纯PM2 DNA的环低22倍：它们必须含有约2.2 × 10（5）个碱基对。维持类核DNA准环状性的约束对极端温度和碱有很强的抵抗力;有些约束在双链体变性的条件下仍然存在。的约束是不稳定的乙锭，这表明他们是稳定的自由能的超螺旋。蛋白水解酶，而不是核糖核酸酶，消除了限制。约束机制的可能结构进行了讨论。
Structures retaining many of the morphological features of nuclei may be released by lysing HeLa cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain few chromatin proteins. We have shown that the DNA of nucleoids is quasicircular and supercoiled by measure spectrofluorometrically the amount of the intercalating dye, ethidium, bound to unirradiated and gamma-irradiated nucleoids. Ethidium binds to nucleoids in the manner characteristic of the binding to superhelical DNA: at low concentrations more ethidium binds to unirradiated nucleoids than to their gamma-irradiated counterparts with broken DNA, and at higher concentrations less ethidium binds to the unirradiated nucleoids. The quasi-circles in nucleoids are 22 times less sensitive to gamma-irradiation than are circles of pure PM2 DNA: they must contain about 2.2 X 10(5) base pairs. The constraints that maintain the quasi-circularity of nucleoid DNA are very resistant to extremes of temperature and alkali; some remain under conditions in which the duplex is denatured. The constraints are destabilised by ethidium suggesting that they are stabilised by free energy of supercoiling. Proteolytic enzymes, but not ribonucleases, remove the constraints. Possible structures for the constraining mechanism are discussed.