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. 2012 Jul 20;31(16):3468–3479. doi: 10.1038/emboj.2012.194

Figure 4.

Figure 4

Sister chromatid colocalization and SCI are not equivalent measures of sister chromatid cohesion. (A) The SSB-Ypet fusion was used to monitor replication parameters. A representative picture of the MG1655-SSB-YPet strain grown in Minimal Medium A+casamino acids+glycerol. (B) histogram of the amount of cells present in each cell size category according to the number of SSB-YPet foci observed in the cells. (C) Representation of the cell cycle in the MG1655 strain grown in Minimal Medium A+casamino acids+glycerol at 30°C. The percentage of cells in the population according to the number of SSB-Ypet foci is indicated. The cells with two foci were split into two categories: the small cells represent a size below the median size of the cells with 1 focus, and the large cells represent a size above the median size of the cells with 1 focus. The generation time (τ) is 80 min, the C period is 56 min (brown arrows) and the D period is 45 min (cyan arrow). The timing of replication (black vertical bars), the estimated timing of the loss of SC cohesion (red vertical bars) and the estimated timing of the loss of SC colocalization (green vertical bars) are indicated for each locus. (D) Representative pictures of the parS/ParBP1-GFP tag inserted at the Ori-1, Ori-3 Ter-1 and Terdif locus. The MG1655 and MG1655matP cells were grown in minimal medium A supplemented with casamino acids and glycerol (Green parS/ParBP1-GFP, Red Phase contrast microscopy). For each strain the average number of foci per cell is indicated. (E) Measure of the colocalization of the sister parS/ParBP1GFP foci. The cell cycle parameters (generation time, length of the C period and D period) measured in Figure 4A–C were used to estimate the average number of copies of each locus in the cells (Nbloc). The average number of foci per cell (Nbfoc) was measured from (Figure 4D). The left axis represents the colocalization index, defined as (Nbloc−Nbfoc)/Nbloc. The right axis represents the estimated lifespan of SC colocalization. (F) Measure of SCI with the LacloxP system for the Ori-1, Ori-3, Ter-1, Terdif and Ter-6 loci in the MG1655matP strain. The relative normalized recombination frequency is defined as (Inter SC matP/Intra SC matP)/(Inter SC wt/Intra SC wt).