Figure 4 .
The cdc13-1 and stn1-63 mutations behave like hypomorphic alleles. (A) Steady-state protein levels of the wild-type Cdc13-(myc)18 and mutant Cdc13-1-(myc)18 proteins, expressed from a CEN vector; extracts were prepared from strains grown at 23° or at 36° for 3.5 hr and analyzed by anti-myc Western analysis on 8% SDS-PAGE. The strain also expresses an Est1-(myc)13 protein (as an integrated tagged construct), which provided an internal control for protein levels. (B) Comparison of stn1-Δ/pCEN STN1 vs. stn1-Δ/pCEN stn1-63, as assessed by single-colony propagation on rich media (Left panels) or cell-cycle progression of liquid cultures (Right panels) grown at 23° and 36°; arrows indicate that cell division still occurs at 36°. (C) Viability of RAD24 and rad24-Δ versions of the stn1-Δ/pCEN stn1-63 strain at the indicated temperatures; serial dilutions were plated on prewarmed rich media plates and photographed after 2.5 days (for ≥28° incubations) or after 4 days (for 23° and 25° incubations). (D) Steady-state protein levels of the Stn1-63-(myc)7 protein, expressed from a CEN vector, assessed as described in A. (E) Telomere length of the stn1-Δ/pCEN stn1-63 strain compared to the isogenic stn1-Δ/pCEN STN1 strain at 23°.