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faculty: "FNWI" and publication year: "2005"
| Authors||S.P. Elmore, M. Muller, N.O.E. Vischer, T. Odijk, C.L. Woldringh|
|Title||Single-particle tracking of oriC-GFP fluorescent spots during chromosome segregation in Escherichia coli.|
|Journal||Journal of Structural Biology|
|Faculty||Faculty of Science|
|Institute/dept.||FNWI: Swammerdam Institute for Life Sciences (SILS)|
|Keywords||DNA segregation; Escherichia cloi; Mean square displacement; Random and cinfined diffusion; Simulations|
|Classification||35.73 biochemistry: molecular genetics|
|Abstract||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 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 ? 2.6 ? 10?5 ?m2/s. Spot movement along the short axis showed confinement in a region of the diameter of the nucleoid (800 nm) with an effective diffusion coefficient of 2.9 ? 1.7 ? 10?5 ?m2/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.|
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