Figure 2.

(A) The mitotic delay induced by Mps1 overexpression is independent of Bfa1 and Bub2. RLY700 (WT), RLY724 (Δbfa1), RLY727 (Δbub2), RLY729 (Δmad1), and RLY731 (Δmad2) strains, which all contain the same integrated copy of Gal-Mps1 (14), were cultured overnight in media without galactose (YPR) and then shifted to media containing 2% galactose (YPG) for 6 hours. A control stain, RLY569, which contains the vector alone, was also included. (a) Cells were fixed, stained with DAPI, and the percentage of large budded cells (bud size greater than 2/3 of the mother size) with undivided DNA mass were determined and shown as histograms. (b) Total cell extracts were prepared and analyzed by immunoblotting using anti-Clb2 and anti-actin antibodies. Lane 1, RLY569; lane 2, RLY700; lane 3, RLY724; lane 4, RLY727; lane 5, RLY729; lane 6, RLY731. (B) Overexpression of Bfa1 induces an anaphase cell cycle arrest that is independent of the other mitotic checkpoint proteins. (a) Strains bearing vector alone (RLY569) or Gal-Bfa1 were streaked onto a plate containing 2% galactose. The photograph was taken after 3 days at 30°C. Sector 1, RLY569; sector 2, RLY690 (WT); sector 3, RLY688 (Δbub2); sector 4, RLY691 (Δmad1); sector 5, RLY693 (Δmad2). (b) The strains in B, a were cultured overnight in media without galactose (YPR) and then shifted to media containing 2% galactose (YPG) for 6 hours. Cells were fixed and stained with DAPI and an anti-tubulin (MT) antibody. The images show the arrest morphology of RLY690 cells. The same arrest morphology was also observed for the other Gal-Bfa1 bearing strains. (c) The percentages of cells exhibiting the arrest morphology, as shown in b, were determined for each of the strains in the experiment described in B, b. (d) Total extracts were prepared from each of the cell cultures in the experiment described in B, b and analyzed by immunoblotting by using anti-Clb2 and anti-actin antibodies. [Bar = 10 μm(B, b).]