Determination of the number of superhelical turns in simian virus 40 DNA by gel electrophoresis.
Determination of the number of superhelical turns in simian virus 40 DNA by gel electrophoresis.
复制标题
通过凝胶电泳测定猿病毒 40 DNA 中的超螺旋圈数。
DOI：
--
复制
发表时间：
1975
影响因子：
11.1
通讯作者：
W. Keller
中科院分区：
文献类型：
--
作者：
W. Keller
在增加溴化乙锭(EtdBR)存在的情况下，用过量的DNA松弛酶处理来自猴病毒40的闭合环状超螺旋DNA(SV40 DNA I)。去除乙基后，每个样品由一组紧密的环状DNA分子组成，它们的超螺旋转数(Tau)在tau的平均值附近呈高斯分布。DNA样品在琼脂糖凝胶中进行分析，在此条件下，电泳迁移率是超螺旋转数的函数。由于不同样品的DNA分子在tau附近的分布重叠，因此可以确定样品之间的平均超螺旋转数的差异，并用它来测量天然SV40 DNA I的平均数量(Tau)。根据这个标准，SV40 DNA I包含的分子呈高斯型分布，在37℃的平均值tau=-24+/-2附近有整数差异[在0.2M NaCI，10 mM Tris-HCl(pH 7.9)和0.2 mM EDTA中]。Tau的异质性可能是由于体内最后一个磷脂酸酯键关闭时DNA螺旋的热波动造成的。当与完全松弛的SV40 DNA在CsC1-二碘丙烷梯度中的浮移相关联时，超螺旋与松弛DNA的增量tau=24+/-2意味着当一个EtdBR分子插入时DNA螺旋解旋26-28度。
Closed-circular, superhelical DNA from simian virus 40 (SV40 DNA I) was treated with an excess of DNA-relaxing enzyme in the presence of increasing amounts of ethidium bromide (EtdBr). After removal of the ethidium, each sample consisted of a group of close-circular DNA molecules differing in their number of superhelical turns (tau) around a mean value of tau in a Gaussian-like distribution. The DNA samples were analyzed by electrophoresis in agarose gels under conditions where the electrophoretic mobility was a function of the number of superhelical turns. Since the distributions around tau of DNA molecules of different samples overlappped, the difference in the mean number of superhelical turns from sample to sample, delta tau, could be determined and used to measure the mean number (tau) for native SV40 DNA I. By this criterion, SV40 DNA I contains a Gaussian-like distribution of molecules differing by integral numbers around a mean value of tau = -24 +/- 2 at 37 degree C [in 0.2 M NaC1, 10 mM Tris-HC1 (pH 7.9), and 0.2 mM EDTA]. The heterogeneity in tau is probably a consequence of thermal fluctuations in the DNA helix at the time when the last phospholiester bond is closed in vivo. When correlated to the buoyant shift of completely relaxed SV40 DNA in a CsC1-propidium diiodide gradient, the number of delta tau = 24 +/- 2 of superhelical versus relaxed DNA implies an unwinding of the DNA helix by 26-28 degree upon intercalation of one molecule of EtdBr.