Fig. 6. Tor2 interferes with Mei2 function. (A) h+/h– tor2+/tor2+ and h+/h– nmt-tor2/tor2+ diploid cells were grown in the presence or absence of thiamine for 18 hours, and were then spotted onto malt extract plates with or without thiamine. Plates were incubated for 48 hours at 25°C and then stained with iodine vapour (left panel). Alternatively, a sample from each spot was resuspended in water at the same optical density and treated overnight with glusulase. Equal volumes were then plated on YES medium and the number of colonies was counted and represented as a plot (right panel). (B) The indicated strains were plated on minimal medium or minimal medium supplemented with thiamine and incubated at 32°C for three days. Photographs of the plates are shown. Lower panel, the indicated strains were grown at 25°C in minimal medium in the presence or absence of thiamine for 18 hours to mid-exponential phase (OD₅₉₅=0.8-1), diluted to OD₅₉₅=0.1, and then incubated in the same medium at 32°C. Growth curves at 32°C are shown. (C) Crude extracts were prepared from exponentially growing cells expressing tagged proteins, as indicated. They were subjected to IgG pull-down (TAP pull-down) with IgG-Sepharose beads. HA-Tor2 was detected by western blot (WB). Immunoblots of the crude extracts are also shown.