Merlin S, Polisky B
Department of Biology, Indiana University, Bloomington 47405.
J Mol Biol. 1993 Mar 5;230(1):137-50. doi: 10.1006/jmbi.1993.1131.
The replication regulatory mechanisms by which the small, multicopy plasmid ColE1 maintains a constant steady-state copy number have been extensively characterized by a combination of in vivo genetics and in vitro biochemistry. We have extended the analysis of replication control into the "establishment" phase of replication, when ColE1-directed replicons replicate more than once per cell generation and the intracellular concentrations of plasmid-encoded replication regulatory elements are changing. To study establishment phase replication, in which plasmid-directed replicons amplify from an initially low concentration to the characteristic, steady-state concentration, bacteriophage-plasmid hybrids, termed phasmids, were constructed. Phasmids were shown to exhibit stability, segregation, and incompatibility properties similar to those of the parent plasmid. Establishment phase replication was analyzed by measuring the number of phasmids per cell as a function of time after infection. We observed a linear increase in phasmid concentration until the steady-state concentration characteristic of the ColE1 plasmid component of the hybrid was reached. The number of cell doublings required for the phasmid concentration to reach steady-state was inversely related to cell growth rate. The observed amplification kinetics imply that the frequency of replication initiation per phasmid continually decreases until steady-state is reached. Kinetics of establishment phase amplification were sensitive to rate of expression of RNA II. A phasmid containing an up mutation in the RNA II promoter amplified at a 15-fold faster rate than the wild-type phasmid. Concentration of the ColE1 replication negative regulator (RNA I) was proportional to phasmid concentration throughout the amplification phase. These results suggest that the same elements that regulate steady-state replication also control establishment phase replication.
小型多拷贝质粒ColE1维持恒定稳态拷贝数的复制调控机制，已通过体内遗传学和体外生物化学相结合的方法得到了广泛的表征。我们将复制控制的分析扩展到了复制的“建立”阶段，即在每个细胞世代中，由ColE1指导的复制子复制不止一次，且质粒编码的复制调控元件的细胞内浓度正在发生变化。为了研究建立阶段的复制，即质粒指导的复制子从初始低浓度扩增到特征性的稳态浓度，构建了被称为噬菌粒的噬菌体 - 质粒杂种。噬菌粒表现出与亲本质粒相似的稳定性、分离性和不相容性。通过测量感染后每个细胞中噬菌粒的数量随时间的变化来分析建立阶段的复制。我们观察到噬菌粒浓度呈线性增加，直到达到杂种中ColE1质粒成分的稳态浓度特征。噬菌粒浓度达到稳态所需的细胞分裂次数与细胞生长速率呈负相关。观察到的扩增动力学表明，每个噬菌粒的复制起始频率持续下降，直到达到稳态。建立阶段扩增的动力学对RNA II的表达速率敏感。在RNA II启动子中含有上调突变的噬菌粒，其扩增速度比野生型噬菌粒快15倍。在整个扩增阶段，ColE1复制负调控因子（RNA I）的浓度与噬菌粒浓度成正比。这些结果表明，调节稳态复制的相同元件也控制建立阶段的复制。
