FIG. 1.

Overexpression of Myt1 causes a G₂ cell cycle delay. (A) HeLa cells synchronized at the G₁/S border by a double thymidine block protocol were infected with recombinant adenoviruses encoding either β-GAL or the kinase-active (Myt1) or -inactive (Myt1N238A) form of Myt1 tagged with a Myc epitope. Cells were harvested at various times after release from the block, and the cellular DNA content was analyzed by flow cytometry. (B) Mitotic index measurements were made to assess whether cells were arresting in the G₂ or M phase of the cell cycle. Cells were synchronized and infected with adenoviruses as described above. Nocodazole was added to trap cells in mitosis. At various times after release from the block, mitotic chromosome spreads were prepared and scored. At least 600 nuclei were counted in each experiment. (C) Kinase-active Myt1 (HisMyt1) or kinase-inactive Myt1 (HisMyt1N238A) was purified from Sf9 insect cells as a hexahistidine-tagged fusion protein. Glutathione S-transferase–cyclin B1-Cdc2 (K33R) complexes were also purified from insect cells as described previously (40). Kinase reactions were performed in vitro in the presence of substrate alone (left panel, lane 1) or substrate in the presence of either kinase-active (left panel, lane 2) or -inactive (left panel, lane 3) Myt1. Reaction mixtures were resolved by SDS-PAGE and subjected to autoradiography. Levels of kinase-active (right panel, lane 1) and kinase-inactive (right panel, lane 2) Myt1 in the reaction mixtures were assessed by Western blotting. (D) HeLa cells synchronized by a double thymidine block protocol were infected with recombinant adenoviruses encoding either β-GAL or the kinase-active (Myt1) or -inactive (Myt1N238A) form of Myt1 tagged with a Myc epitope. Cells were harvested at 14 h after release from the block, and the cellular DNA content was analyzed by flow cytometry (left panel). Levels of Myt1 and Myt1N238A were determined by immunoblotting (right panel).