Department of Cell and Molecular Biology, Uppsala University, S-751 24 Uppsala, Sweden.
Plasmid. 2012 Mar;67(2):191-8. doi: 10.1016/j.plasmid.2012.01.007. Epub 2012 Jan 20.
The classical Meselson-Stahl density-shift method was used to study replication of pOU71, a runaway-replication derivative of plasmid R1 in Escherichia coli. The miniplasmid maintained the normal low copy number of R1 during steady growth at 30°C, but as growth temperatures were raised above 34°C, the copy number of the plasmid increased to higher levels, and at 42°C, it replicated without control in a runaway replication mode with lethal consequences for the host. The eclipse periods (minimum time between successive replication of the same DNA) of the plasmid shortened with rising copy numbers at increasing growth temperatures (Olsson et al., 2003). In this work, eclipse periods were measured during downshifts in copy number of pOU71 after it had replicated at 39 and 42°C, resulting in 7- and 50-fold higher than normal plasmid copy number per cell, respectively. Eclipse periods for plasmid replication, measured during copy number downshift, suggested that plasmid R1, normally selected randomly for replication, showed a bias such that a newly replicated DNA had a higher probability of replication compared to the bulk of the R1 population. However, even the unexpected nonrandom replication followed the copy number kinetics such that every generation, the plasmids underwent the normal inherited number of replication, n, independent of the actual number of plasmid copies in a newborn cell.
经典的 Meselson-Stahl 密度偏移法被用于研究 pOU71 的复制，pOU71 是质粒 R1 的失控复制衍生物，在大肠杆菌中。在 30°C 的稳定生长过程中，该小型质粒保持了 R1 的正常低拷贝数，但当生长温度升高到 34°C 以上时，质粒的拷贝数增加到更高水平，在 42°C 时，它以失控复制的方式复制，对宿主产生致命后果。随着拷贝数的增加，质粒的潜伏期（同一 DNA 连续复制之间的最短时间）在升高的生长温度下缩短（Olsson 等人，2003 年）。在这项工作中，在 pOU71 复制 39°C 和 42°C 后，其拷贝数下降时，测量了质粒的潜伏期，分别导致细胞中质粒拷贝数比正常情况下高出 7 倍和 50 倍。在拷贝数下降期间测量的质粒复制潜伏期表明，通常随机选择用于复制的质粒 R1 存在偏向性，即新复制的 DNA 比 R1 群体的大部分具有更高的复制概率。然而，即使是非随机复制也是如此，它遵循着拷贝数动力学，因此每一代，质粒都经历了正常的遗传复制次数 n，与新生细胞中质粒拷贝数的实际数量无关。
