Elmore Steven, Müller Michiel, Vischer Norbert, Odijk Theo, Woldringh Conrad L
Section Molecular Cytology, Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands.
J Struct Biol. 2005 Sep;151(3):275-87. doi: 10.1016/j.jsb.2005.06.004.
DNA regions close to the origin of replication were visualized by the green fluorescent protein (GFP)-Lac repressor/lac operator system. The number of oriC-GFP fluorescent spots per cell and per nucleoid in batch-cultured cells corresponded to the theoretical DNA replication pattern. A similar pattern was observed in cells growing on microscope slides used for time-lapse experiments. The trajectories of 124 oriC-GFP spots were monitored by time-lapse microscopy of 31 cells at time intervals of 1, 2, and 3 min. Spot positions were determined along the short and long axis of cells. The lengthwise movement of spots was corrected for cell elongation. The step sizes of the spots showed a Gaussian distribution with a standard deviation of approximately 110 nm. Plots of the mean square displacement versus time indicated a free diffusion regime for spot movement along the long axis of the cell, with a diffusion coefficient of 4.3+/-2.6x10(-5) microm2/s. Spot movement along the short axis showed confinement in a region of the diameter of the nucleoid ( approximately 800 nm) with an effective diffusion coefficient of 2.9+/-1.7x10(-5) microm2/s. Confidence levels for the mean square displacement analysis were obtained from numerical simulations. We conclude from the analysis that within the experimental accuracy--the limits of which are indicated and discussed--there is no evidence that spot segregation requires any other mechanism than that of cell (length) growth.
通过绿色荧光蛋白（GFP）-乳糖阻遏物/乳糖操纵子系统观察到靠近复制起点的DNA区域。分批培养细胞中每个细胞和每个类核的oriC-GFP荧光斑点数量与理论DNA复制模式相符。在用于延时实验的显微镜载玻片上生长的细胞中也观察到了类似的模式。通过对31个细胞进行时间间隔为1、2和3分钟的延时显微镜观察，监测了124个oriC-GFP斑点的轨迹。沿着细胞的短轴和长轴确定斑点位置。对斑点的纵向移动进行了细胞伸长校正。斑点的步长呈高斯分布，标准差约为110 nm。均方位移与时间的关系图表明，斑点沿细胞长轴的移动处于自由扩散状态，扩散系数为4.3±2.6×10⁻⁵μm²/s。斑点沿短轴的移动局限在类核直径（约800 nm）的区域内，有效扩散系数为2.9±1.7×10⁻⁵μm²/s。均方位移分析的置信水平通过数值模拟获得。我们从分析中得出结论，在实验精度范围内（其限度已指明并讨论），没有证据表明斑点分离需要除细胞（长度）生长之外的任何其他机制。
