Figure 4

Replication timing of human artificial chromosomes in line 17-B12. BrdU detection (red) in cells that have been blocked with colcemid in mitosis following BrdU pulses during S phase (see Materials and methods). Artificial chromosome (small arrows; enlarged artificial chromosomes are shown in inserts) and chromosome 17 (large arrow) locations in each spread were confirmed by FISH analyses using a D17Z1 probe (data not shown). (a-d) Images from different periods in S phase. (a) Early in S phase, at 0-2 h, the two artificial chromosomes present in this spread are not replicating. Some incorporation of BrdU on chromosome 17 is detectable. (b) In the middle of S phase, at 2-4 h, two of four artificial chromosomes are replicating. (c) Later, at 4-6 h, all three artificial chromosomes are being coordinately replicated. Some BrdU incorporation within chromosome 17 arms is detectable. (d) Late in S phase, at 6-8 h, artificial chromosomes are not replicating. The centromere region on chromosome 17 is replicating (large arrow). Because of the A-rich sequence composition of satellite III on Yq, BrdU is preferentially incorporated into one strand, producing an asymmetrical staining pattern on Yq (arrowheads) [84].