Figure 1.—

Conditional alleles of SIR2. (A) Levels of α1 and ACT1 mRNA were measured by RT–PCR in strains bearing different alleles of SIR2. Cultures were maintained in log phase for >10 generations at the listed temperatures, and then RNA was collected and analyzed as described in materials and methods. α1 indicates steady-state levels of message transcribed from the HML locus; ACT1 message is shown as a control. RNA was analyzed from a wild-type strain, a strain lacking the SIR2 gene, and strains bearing the indicated alleles of SIR2. (B) Protein extracts made from cells bearing the indicated SIR2 alleles were subjected to a Western blot using an antibody specific for the Sir2 protein, as described in materials and methods. For the cycling cells, parallel cultures were grown to log phase at either 23° or 37°. G₁/early S-blocked cells were grown to early log phase at 23° and then blocked with α-factor. Half the culture was maintained at 23°, while the other half was shifted to 37° for 3 hr. G₂/M-blocked cells were grown to early log phase at 23° and then blocked with nocodazole. Half the culture was maintained at 23°, while the other half was shifted to 37° for 3 hr. (C) For each of the experiments shown in B, duplicate gels were run and stained with Coomassie to confirm consistent loading of samples from lane to lane.