Figure 4.

Intercalated banding of early and late replicating DNA sequences in metaphase chromosomes. (A and B) Metaphase spreads were obtained from synchronized cells grown in the presence of iododeoxyuridine (2.5 μM) during the initial 90–100 min of S phase followed by a second pulse with chlorodeoxyuridine (5 μM) starting 4 h after release into S phase until mitosis was reached. Controls showed that ∼90% of the cells had accumulated replication patterns 1 and 2 during the first pulse and that ∼80% had accumulated patterns 4 and 5 during the second pulse. Confocal imaging after differential immunolabelling of IdU-DNA (green) and CldU-DNA (red) shows that the early and late replicating domains intercalate in a banded pattern. (C) Confocal imaging of a metaphase spread obtained from cells labelled with BrdU and that accumulated patterns 4 and 5. The whole chromosome set is shown. Immunostaining of BrdU-DNA was assigned a green pseudocolor that appears yellow in the merged image and total DNA was immunolabeled with an anti–DNA autoimmune antiserum (red). (D–F) To control the specificity of the differential immunolabelling of CldU-DNA- and IdU-DNA-synchronized CHF cells were grown for a full S phase in the presence of CldU (5 μM) and then for a second cell cycle in the presence of IdU (2.5 μM). Metaphase spreads show that IdU-DNA (E) is present in both chromatids of each chromosome, whereas CldU-DNA (D), as expected, is restricted to one chromatid. Red and green confocal images were merged (F) and areas of overlap appear yellow; some sister chromatid exchanges are clearly visible. Bar, 10 μm.