Figure 4.

Efficient re-replication of G2 nuclei in geminin-depleted extract. Metaphase-arrested extracts were supplemented with sperm nuclei (10 ng DNA/μl) and released into interphase with CaCl₂. After incubation for 90 min, extracts were diluted with buffer and nuclei were centrifuged through a sucrose cushion. (A) Aliquots of the whole extract (total), the pelleted nuclei (pellet) and the supernatant (s/n) after centrifugation were immunoblotted for Orc1, Cdc6, Mcm7, geminin and nucleoplasmin. In the first three lanes, 2.5% of each sample was loaded; in the last lane, 50% of the pellet was loaded. (B) Isolated nuclei were optionally treated with lysolecithin and then transferred to fresh extract. After 1 or 20 min, chromatin was re-isolated and immunoblotted for the indicated proteins. (C) Nuclei were isolated as in (A), and were transferred to fresh interphase extracts containing aphidicolin and optionally supplemented with MG132. After a further 60 min, chromatin was isolated and immunoblotted for Mcm7. As a control, chromatin was isolated from the first extract at either 30 or 90 min. (D) G2 nuclei were prepared as in (A), except that the extract was supplemented with BrdUTP and [α-³²P]dATP. Intact nuclei were transferred to fresh untreated extract, extract immunodepleted with nonimmune antibodies, or extract immunodepleted of geminin plus or minus MG132. The second extract was also supplemented with BrdUTP. After a further 90 min incubation, DNA was isolated and fractionated on density gradients.