Figure 4.

Centromeres replicate with euchromatin in tetraploid Kc cells and in larval diploid neuroblasts. (A) Mitotic X chromosomes from cells pulsed with dig-dUTP nucleotide analog (green) and then chased for 4 h show heavy labeling in the heterochromatin surrounding centromeres (Cid, red), as expected for incorporation during late S phase. (B) Mitotic X chromosomes from cells pulsed with dig-dUTP and then chased for 10 h were in early S phase at the time of the pulse, because they show heavy labeling in the euchromatic arms. There are also foci of incorporation corresponding to both sister centromeres. (C) Pulse-labeling and imaging of interphase Kc cells shows that centromeres replicate in the early S-phase period when euchromatin is also replicating. We tracked cell survival and S-phase progression over a 5-h period, and mitotic index over a 25-h period in all labeling experiments. These parameters were indistinguishable from control, untreated cultures. In labeled cultures after 7 h we observed ≈98% labeling of mitotic figures, indicating that virtually all cells in S phase at the time of the pulse received the nucleotide analog. (D) Neuroblast centromeres are contained within one to three heterochromatic chromocenters (H3K9me, blue). Pulse-labeling with dig-dUTP reveals foci of DNA replication in two centromeric spots and in euchromatin. Cultured cells and dissected larval brains were labeled and prepared as described (8).