Figure 1. Failure to grow at 36°C and elongated cell shape in an S. pombe asf1 mutant.

(A) L972 (asf1⁺) and SKP605-33 (h⁻ leu1-32 ura4-D18 asf1-33-13myc-kan^r) were grown on YES plates containing phloxine B at 26°C or 36°C for 24 h. Cell morphology was observed by a microscope. (B) Cdc2 (Y15) was highly phosphorylated in SKP605-33 (asf1-33-13myc-kan^r) at the restrictive temperature. L972 (asf1⁺) and SKP605-33 (asf1-33-13myc-kan^r) were incubated at 26°C or 36°C for 6 h. Cells were collected by centrifugation and washed once with STOP buffer. Protein extraction was performed by the glass-beads method. Samples were suspended into SDS-sample buffer. Ten micrograms of total proteins was used for western blotting. The relative intensity of each band (Cdc2Y15P) relative to the control (Cdc2) was measured using ImageJ (http://rsb.info.nih.gov/ij/). (C) Schematic representation of the strategy to identify protein kinases responsible for activation of the cell cycle checkpoint in the asf1-33 mutant.