Shure M, Vinograd J
Cell. 1976 Jun;8(2):215-26. doi: 10.1016/0092-8674(76)90005-2.
By a method of overlapping the results obtained after agarose gel electrophoresis under two different sets of conditions, it has become possible to determine the number of superhelical turns in a given DNA by counting the bands present after partially relaxing the DNA (Keller and Wendel, 1974) with highly purified nicking-closing (N-C) enzyme from LA9 mouse cell nuclei. Because native supercoiled DNA is heterogeneous with respect to superhelix density, an average number of superhelical turns was determined. Virion SV40 DNA contains 26 +/- 0.5 superhelical turns, and native Minicol DNA contains 19 +/- 0.5 superhelical turns. The above are values at 0.2 M NaCl and at 37 degrees C, the condition under which the enzymatic relaxations were performed. The superhelix densities determined by the band counting method have been compared with superhelix densities determined by buoyant equilibrium in PDl-CsCl gradients. The Gray, Upholt, and Vinograd (1971) calculation procedure has been used for evaluating the superhelix densities by the latter method with the new statement, however, that relaxed DNA has zero superhelical turns. Comparison of the superhelix densities obtained by both methods permits a calculation of an unwinding angle for ethidium. The mean value from experiments with SV40 DNA is 23 +/- 3 degree. The average number of superhelical turns in SV40, 26, combined with the value, 21, obtained by both Griffith (1975) and Germond et al. (1975) for the average number of nucleosomes per SV40 genome, yields an average of 1.25 superhelical turns per 1/21 of the SV40 genome. If the regions of internucleosomal DNA are fully relaxed, 1.25 correesponds to the average number of superhelical turns with a nucleosome. When analyzed under identical conditions, the limit product generated by ligating a nicked circular substrate in the presence of 0.001 M Mg2+ at 37 degrees C (ligation conditions) is slightly more positively supercoiled than the limit product obtained when the N-C reaction is performed in 0.2 M NaCl at 37 degrees C. The difference in superhelix density as measured in gels between the two sets of limit products for both Minicol and SV40 DNAs is 0.0059 +/- 0.0005. This result indicates that the DNA duplex is overwound in the ligation solvent relative to its state in 0.2 M NaCl.
通过一种将在两组不同条件下进行琼脂糖凝胶电泳后获得的结果进行重叠的方法，利用从LA9小鼠细胞核中提取的高度纯化的切口封闭（N-C）酶，在部分松弛DNA（凯勒和温德尔，1974年）后通过计数出现的条带，就有可能确定给定DNA中超螺旋圈的数量。由于天然超螺旋DNA在超螺旋密度方面是异质的，所以确定了超螺旋圈的平均数。病毒粒子SV40 DNA含有26±0.5个超螺旋圈，天然小染色体DNA含有19±0.5个超螺旋圈。上述数值是在0.2M NaCl和37℃的条件下得到的，这是进行酶促松弛的条件。通过条带计数法确定的超螺旋密度与通过在PDl-CsCl梯度中的浮力平衡确定的超螺旋密度进行了比较。格雷、厄普霍尔特和维诺格拉德（1971年）的计算程序已被用于通过后一种方法评估超螺旋密度，但有新的说法，即松弛的DNA超螺旋圈数为零。两种方法获得的超螺旋密度的比较允许计算溴化乙锭的解旋角。用SV40 DNA进行实验得到的平均值为23±3度。SV40中超螺旋圈的平均数26，与格里菲思（1975年）和热尔蒙等人（1975年）得到的每个SV40基因组核小体平均数21相结合，得出每1/21的SV40基因组平均有1.25个超螺旋圈。如果核小体间DNA区域完全松弛，1.25对应于一个核小体的超螺旋圈平均数。在相同条件下分析时，在37℃、0.001M Mg2+存在下连接带切口的环状底物（连接条件）产生的极限产物比在37℃、0.2M NaCl中进行N-C反应时得到的极限产物超螺旋程度略高。对于小染色体和SV40 DNA，两组极限产物在凝胶中测得的超螺旋密度差异为0.0059±0.0005。这一结果表明，相对于其在0.2M NaCl中的状态，DNA双链在连接溶剂中过度缠绕。
