Fig. 3. TAF_IIs directly regulate the RNR genes. (A) Analysis of the MMS-induced phosphorylation of Crt1. Wild-type (SW87), taf90-1 (LY20) and taf145-2 (YSW93) cells transformed with pMH190 (GAL-3MYC-CRT1, ARS/CEN, URA3) were grown in SC-URA plus raffinose, and Crt1 was induced for 2 h by the addition of galactose (Huang et al., 1998). Cultures were then incubated for 1 h at 37°C, followed by a 2 h treatment with MMS (0.1%). Crt1 was detected by western blotting using anti-myc monoclonal antibodies. TAF_II90p served as a loading control. (B) Effects of the inactivation of TAF_II mutants on active RNR genes. Cells were treated with 0.03% MMS for 2.5 h at 23°C and then shifted to 37°C. Aliquots of cells were withdrawn at the times indicated in the figure. A separate culture of wild-type cells was treated with 100 µg/ml cycloheximide at the time of temperature shift (+CHX). (C) Northern blot of RNR2 and RNR3 mRNA.