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6.  Postface

The subnuclear localization of DNMT1 throughout the cell cycle was determined by following the distribution of GFP-DNMT1 fusion protein by live cell microscopy. The specific cell cycle stages in live cells were identified by co-expressing DNA Ligase I fused to DsRed fluorescent protein (RFP-Ligase). Normally only mitosis and interphase can be identifed in live cells (Fig. I-A). Expression of RFP-Ligase allows identification of all the cell cycle stages in live cells. During S phase RFP-Ligase associates with RF and, thus, by following a cell through S phase the transition to G2 can be identified followed by mitosis and the transition to G1 (Fig. I-B). We observe that DNMT1 shows a diffused nuclear distribution in G1 phase, associates with RF during S phase and is bound to pericentric heterochromatin in G2 cells. Thus, the combination of both, the subnuclear distribution of GFP-DNMT1 and RFP-Ligase can be used as a marker to identify the cell cycle stage in fixed cells, or in a snapshot of live cells without the need for following the transition through different cell cycle stages (Fig. I-C). In other words, at any given time the cell cycle phase can clearly be deduced from the distribution of these two markers. Therefore, these markers should be valuable for the study of cell cycle dependent processes.


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Fig. I. Cell cycle marker for live and fixed cells. (A) Only mitosis (M) and interphase can be identified in live cells based on the cellular and nuclear morphology. (B) Expression of RFP-Ligase allows identification of the different cell cycle stages by live cell microscopy. In live cells expressing RFP-Ligase, G2 phase can be identified by following the transition of a cell from S phase (punctate RFP-Ligase pattern corresponding to replication sites) into G2 when RFP-Ligase is diffused in the nucleus. The progression through S phase can be followed by the different patterns of RF. During M phase, the nuclear membrane is broken down and RFP-Ligase is excluded from the chromatin. Cells in G1 phase can be identified by following a cell through mitosis into G1 when RFP-Ligase is diffused in the nucleus. (C) Expression of both GFP-DNMT1 and RFP-Ligase allows identification of the cell cycle stage in fixed as well as live cells. Colocalization of RFP-Ligase and GFP-DNMT1 at RF during S phase is shown in yellow. During G2, only GFP-DNMT1 is bound to pericentric heterochromatin (shown as green "donut" shaped structures) while RFP-Ligase is diffused. During mitosis GFP-DNMT1 is at the chromatin. In G1, both RFP-Ligase and GFP-DNMT1 are diffused (shown as yellow).


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