Figure 1.

S phase replication labeling patterns are preserved. Cells ([a and b] Hv, human diploid fibroblasts; [c and d] SH-EP N14 human neuroblastoma cells; [e and f] HeLa cells; [g and h] CHO cells; and [i and j] C2C12 mouse myoblasts) were replication-labeled with BrdU (a–d, i, and j; 30-min pulses) or Cy3-dUTP (e–h). Cells were fixed immediately after BrdU labeling or 30 min after microinjection of Cy3-dUTP to obtain labeled S phase cells (left, a, c, e, g, and i). S phase cells display the typical replication labeling patterns (indicated on the left, for classification see text): (a) type I, (c) type II, (e) type III, (g) type IV, and (i) type V. Similarly labeled cells were grown for 1–5 d after labeling and fixed after this growth period (right, b, d, f, h, and j). The presence of similar patterns (types indicated on the right) several days after S phase labeling suggests that DNA replicating with a particular timing during S phase always occupies similar nuclear positions. Arrowheads in a mark the nucleoli that are always unlabeled. Arrowheads in c–e mark perinucleolar label. Arrows in b and d indicate unlabeled chromosome territories. The presence of unlabeled territories demonstrates that cells do not display similar patterns because they stopped cycling, but rather that they went through at least two mitoses after labeling (compare Fig. 3). Note also the two very close nuclei in f displaying similar type III patterns, suggesting that similar patterns were conserved in sister nuclei after cell division (compare Fig. 4, a–c). All pictures display single light optical sections through midnuclear planes except i and j, which show epifluorescence images. Bar in a is similar for all images.