Figure 6

Mitochondrial replication. Cells with YFP-tagged mitochondria were grown in BrdU for 1–6 h, fixed, and Br-DNA immunolabelled. (A,B) On growth in BrdU, progressively more mitochondrial foci containing Br-DNA are seen. Bar: 2 μm. (C) Changes in the fraction of DNA foci containing Br-DNA (assuming cells contain 468 foci/cell; Table 1, line 3); essentially all foci contain Br-DNA within 3 h. (D) Integrated intensity of Br-DNA labelling per focus (in arbitrary units, with the value at 1 h set to unity). The average intensity per focus is constant between 1 and 2 h, and then rises; extrapolating the line from 2–6 h to 22 h (the length of the cell cycle) gives an intensity of 10 (square). (E) Three models for replication. Two DNA foci, each initially containing 10 unreplicated genomes (open circles), are shown. (a) Here, a focus is a unit of replication. In the first hour, all genomes in one focus replicate together (red circles) and half the foci become labelled (as in Figure 6C); in the second hour, all genomes in a second focus replicate together so that all genomes are now labelled (again as in Figure 6C). However, during the third hour, the intensity of foci continues to increase (Figure 6D), so we would have to assume that genomes re-replicate (dark red circles) and some would presumably have to be degraded to maintain genome numbers. (b) All genomes replicate independently of all others. The kinetics in (D) and (E) are consistent with this model. (c) Here, a polymerizing complex transfers from genome to genome in one focus before going to another focus; then genomes in one focus would replicate successively, before replication switched to another focus. This would give the progressive increase seen in Figure 6D, but many foci would remain unlabelled after 2 h (which is inconsistent with Figure 6E).