Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
9138962 | Journal of Structural Biology | 2005 | 13 Pages |
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 â¼110 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 Ã 10â5 μm2/s. Spot movement along the short axis showed confinement in a region of the diameter of the nucleoid (â¼800 nm) with an effective diffusion coefficient of 2.9 ± 1.7 Ã 10â5 μm2/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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Authors
Steven Elmore, Michiel Müller, Norbert Vischer, Theo Odijk, Conrad L. Woldringh,