Lyamichev V, Panyutin I, Mirkin S
Institute of Molecular Genetics, USSR Academy of Sciences, Moscow.
J Biomol Struct Dyn. 1984 Oct;2(2):291-301. doi: 10.1080/07391102.1984.10507568.
We extracted pAO3 plasmid DNA from E. coli cells, having "frozen" the transition between cruciform and double-helical conformations in DNA. The characteristic feature of the DNA isolation procedure is that all steps were carried out at temperature between 0 and 4 C and no phenol deproteinization was used, since it has been discovered that phenol destabilizes cruciform structures in pAO3 DNA. Two-dimensional gel electrophoresis has revealed no cruciform structures in the pAO3 DNA preparations obtained this way, although the superhelical density of DNA was sufficient for them. Cruciform structures are absent from intracellular pAO3 DNA at all growth stages of the bacterial culture: stationary and logarithmic, and under the induction of pAO3 DNA replication in chloramphenicol-treated cells.
我们从大肠杆菌细胞中提取了pAO3质粒DNA，使DNA中十字形和双螺旋构象之间的转变“冻结”。DNA分离过程的特点是所有步骤均在0至4摄氏度之间的温度下进行，且未使用苯酚脱蛋白，因为已发现苯酚会破坏pAO3 DNA中的十字形结构。二维凝胶电泳显示，以这种方式获得的pAO3 DNA制剂中不存在十字形结构，尽管DNA的超螺旋密度足以形成这些结构。在细菌培养的所有生长阶段（稳定期和对数期）以及氯霉素处理的细胞中pAO3 DNA复制诱导下，细胞内pAO3 DNA均不存在十字形结构。
