Live-cell imaging reveals assembly of replicons at replication factories. (A) Three yeast strains were constructed to analyze assembly of three different replicon pairs at an individual factory. In strains #1 (T7278), #2 (T9184), and #3 (T7277) with TetR-3xCFP and GFP-LacI, the indicated two chromosomal loci were marked by integration of tetO×224 and lacO×256. The replication timing profile (top) was obtained from Yabuki et al. (2002); 0 min represents the time of release from α-factor arrest. (B) Models for replication of two replicons at the same factory and at different factories. (C and D) Representative examples of strain #1 showing replication of CFP and GFP dots at the same factory (C) and at different factories (D). Cells were released from α-factor treatment. CFP and GFP images were acquired every 1 min for 65 min. The intensity of CFP/GFP dots and their distance were measured in 23 cells of strain #1 in single experiments. The change in intensity of each dot was fitted by a sigmoidal dose–response curve (see Materials and methods; see R², representing fitness, below) in which a midpoint in the increase of its intensity was defined as its replication time. Then 0 min was defined as the mid-replication time of two dots. R² = 0.70 (C, CFP), 0.81 (C, GFP), 0.78 (D, CFP), and 0.74 (D, GFP). Bars, 1 µm.