FIG. 4.

Tor1 is required for a normal response to HU. (A and B) The response of tor1 mutants to HU is delayed. Wild-type and Δtor1 cells were grown to log phase and shifted to medium containing 12 mM HU. (A) Samples were taken every hour, and nuclei were isolated and subjected to FACS analysis. (B) Total RNA was prepared from samples taken at the indicated time points (hours) after a shift to 12 mM HU. Northern blots were probed with cdt2⁺ and cdc18⁺ (MBF targets) and with act1⁺ (loading control). (C) Loss of Tor1 rescues the mitotic catastrophe of Δrad3 mutants. Cells were incubated with or without 12 mM HU for 6 h at 30°C and then stained with DAPI and calcofluor to visualize nuclear DNA and septa, respectively. Percentages indicate abnormal mitosis, scoring for the “cut” phenotype in which the septum is formed despite the absence of chromosome replication. (D) The rapid loss of viability of the Δrad3 or Δcds1 mutant strain is rescued by Δtor1. Cells were grown to log phase and shifted to 12 mM HU for 6 h, and samples were taken every hour to determine cell viability by assessing plating efficiency on rich medium. WT, wild type. (E and F) Loss of Tor1 is epistatic over loss of Cds1. Strains were grown to log phase and shifted to 12 mM HU. The percentage of cells with septa was measured at the indicated times by staining with calcofluor and DAPI and visualized by fluorescence microscopy.