Figure 2.

DNA synthesis on replication foci attached to the nuclear matrix in vivo and in vitro. (A) Exponentially growing CHO cells were labeled uniformly with [¹⁴C]thymidine and then pulse labeled with 50 µCi/ml [³H]thymidine for 3 min at 37°C. Replication foci attached to the nuclear matrix were prepared with increasing concentrations of DNase I. DNA was isolated and counted. The percentage of ³H counts (filled circle) and ¹⁴C counts (open circle) were plotted versus DNase I concentration. (B) CHO cells grown on coverslips were labeled uniformly with [¹⁴C]thymidine, replication foci attached to the nuclear matrix were prepared by digestion with 20 U/ml DNase I and incubated for 0 (1), 30 (2) and 60 min (3) in DNA synthesis medium containing 1 µCi/ml [³H]dTTP; another sample was incubated for 1 h in the same medium containing 100 µg/ml aphidicolin (4). As a control, cells were permeabilized with Triton X-100 and incubated for 1 h in DNA synthesis medium containing 1 µCi/ml [³H]dTTP in the absence (5) and presence (6) of 100 µg/ml aphidicolin. Cells were collected by trypsinization, DNA was TCA precipitated and its specific radioactivity determined as the ratio of ³H to ¹⁴C counts. In both (A) and (B) each result is the mean of three independent experiments. Error bars show standard deviations.