Shure M, Pulleyblank D E, Vinograd J
Nucleic Acids Res. 1977;4(5):1183-205. doi: 10.1093/nar/4.5.1183.
Systems for gel electrophoresis in the presence of one of the intercalative unwinding ligands, ethidium or chloroquine, have been developed which permit the resolution of highly supercoiled closed circular DNA molecules differing by unit values of the topological winding number, alpha. All native closed circular DNAs examined, including the viral and intracellular forms of SV40 and polyoma DNA, bacterial plasmid DNAs, and the double stranded closed circular DNA genome of the marine bacteriophage, PM2, are more heterogeneous with respect to the number of superhelical turns present than are the thermal distributions observed in the limit products of the action of nicking-closing (N-C) enzyme on the respective DNAs. In the cases of SV40 and polyoma, where it has been shown that the supercoiling is a combined consequence of the binding of the four nucleosomal histones, H2a, H2b, H3 and H4, and the action of N-C enzyme, the breadth of the distributions within the form I DNAs poses specific problems since the work of other laboratories indicates that the number of nucleosomes on the respective minichromosomes falls within a narrow distribution of 21. If it is assumed that all nucleosomes have identical structures, and that the DNA within a nucleosome is not free to rotate, the native DNA would be anticipated to be less heterogeneous than the thermal equilibrium mixtures present in N-C enzyme relaxed SV40 and polyoma DNAs. The absolute number of superhelical turns (at 37 degrees C in 0.2 M NaCl) in virion polyoma DNA has been determined to be 26 +/- 1, which is the same value obtained for virion SV40 DNA. This is consistent with the observations that polyoma DNA has a higher molecular weight, a lower superhelix density, but the same number of nucleosomes as SV40 DNA. In addition, the distributions within the virion and intracellular form I DNAs of both SV40 and polyoma were found to be indistinguishable.Images
已经开发出在嵌入解旋配体（溴化乙锭或氯喹）之一存在下的凝胶电泳系统，该系统能够分离拓扑缠绕数α相差单位值的高度超螺旋闭环DNA分子。所有检测的天然闭环DNA，包括SV40和多瘤病毒DNA的病毒形式和细胞内形式、细菌质粒DNA以及海洋噬菌体PM2的双链闭环DNA基因组，与在各自DNA上切口封闭（N-C）酶作用的极限产物中观察到的热分布相比，其超螺旋匝数的数量更具异质性。在SV40和多瘤病毒的情况下，已表明超螺旋是四种核小体组蛋白H2a、H2b、H3和H4结合以及N-C酶作用的综合结果，I型DNA内分布的宽度带来了特定问题，因为其他实验室的工作表明，各自微型染色体上核小体的数量落在21的窄分布范围内。如果假设所有核小体具有相同的结构，并且核小体内的DNA不能自由旋转，那么天然DNA预计比N-C酶松弛的SV40和多瘤病毒DNA中存在的热平衡混合物的异质性更低。已确定病毒粒子多瘤病毒DNA中的超螺旋匝数绝对值（在37℃、0.2M NaCl中）为26±1，这与病毒粒子SV40 DNA获得的值相同。这与多瘤病毒DNA具有更高的分子量、更低的超螺旋密度但与SV40 DNA具有相同数量核小体的观察结果一致。此外，发现SV40和多瘤病毒的病毒粒子和细胞内I型DNA内的分布无法区分。图像
