Figure 5.

Cyclin B-induced mitotic events occur normally in the absence of RanBP1 and RCC1. (A) The activation of histone H1 kinase is not affected by the depletion of RanBP1 and RCC1. Nuclear assembly reactions were reconstituted with untreated (row 1), codepleted (row 2), or mock-depleted (row 3) cytosol. Nuclear formation was allowed to proceed for 60 min before addition of nondegradable cyclin B to initiate mitosis in half of the reaction mix (+ Cyclin B). The remainder of the sample was allowed to continue in the absence of cyclin B (− Cyclin B). At the times after cyclin B addition indicated (minutes), samples from each reaction were assayed for the H1 kinase activity. We observed that histone H1 kinase activity was induced synchronously in all of the reactions containing cyclin B. (B) The induction of NEBD and chromosome condensation are not affected by the depletion of RanBP1 and RCC1. Nuclear assembly was allowed to proceed for 60 min in extracts reconstituted with untreated (top), codepleted (middle), or mock-depleted (bottom) cytosol. Nondegradable cyclin B was added to half (left) of each nuclear assembly reaction, and buffer was added to the other half of the reaction (right). Nuclear breakdown occurred synchronously in the samples with cyclin B, and the nuclei remained intact in the samples without cyclin B. Each sample was stained with Hoechst 33258 DNA dye 90 min after cyclin B addition and photographed by fluorescence microscopy. Bar, 2.6 × 10 ⁻⁶ m.