THE ANOMALOUS GEL MIGRATION OF A STABLE CRUCIFORM - TEMPERATURE AND SALT DEPENDENCE, AND SOME COMPARISONS WITH CURVED DNA
THE ANOMALOUS GEL MIGRATION OF A STABLE CRUCIFORM - TEMPERATURE AND SALT DEPENDENCE, AND SOME COMPARISONS WITH CURVED DNA
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DOI：
10.1093/nar/15.14.5765
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发表时间：
1987-07-24
影响因子：
14.9
通讯作者：
LILLEY, DMJ
中科院分区：
文献类型：
--
作者：
DIEKMANN, S;LILLEY, DMJ
我们对含有稳定十字形的线性DNA分子--假十字形片段的凝胶电泳性进行了分析。在50°C至55°C的不同温度下，以及在氯化钠、氯化镁或溴化乙锭存在的情况下，在聚丙烯酰胺凝胶中分析了该结构的迁移。异常迁移(延迟)的大小在40℃以下几乎与温度无关，但在这一点之后急剧下降，在70℃时外推到正常迁移。这表现为在添加高达60 mm的氯化钠时，异常迁移持续减少，而在添加氯化镁时，异常急剧减少到一个恒定值，达到10 mm。此外，在这些条件下，碎片的迁移在整个范围内几乎与温度无关。这些结果被解释为反映了四向结离子结合对十字臂相对位置的影响。伪十字的详细的电泳性与序列导向的弯曲DNA片段的电泳性形成了鲜明的对比。特别是，对添加1μ/ml溴化乙锭的反应提供了一种方便的方法来区分由曲率引起的异常延迟(大大减少的异常)或十字形连接(增强的异常)。
We have made an analysis of the gel electrophoretic properties of a pseudo-cruciform fragment, a linear DNA molecule containing a stable cruciform. The migration of this construct was analysed in polyacrylamide gels at various temperatures in the range 50° to 55°C, and in the presence of NaCl, MgCl2or ethidium bromide. The magnitude of the anomalous migration (retardation) was almost temperature independent up to 40°C, but decreased strongly beyond this point, extrapolating to normal migration at 70°C. Addition of salts reduced the anomaly. This took the form of a continuous reduction in anomalous migration with the addition of NaCl up to 60 mM, while with MgCl2there was a sharp reduction in the anomaly to a constant value which is reached by 10 mM. Under these conditions, moreover, the migration of the fragment became almost temperature-independent over the entire range. These results have been interpreted to reflect the influence of ion binding at the four-way junction on the relative disposition of the cruciform arms. The detailed electrophoretic properties of the pseudo-cruciform are in marked contrast to those of sequence-directed curved DNA fragments. In particular, the response to the addition of 1 μ/ml ethidium bromide offers a convenient method for distinguishing between anomalous retardation arising from curvature (greatly reduced anomaly) or a cruciform junction (enhanced anomaly).