Because over expression of Fob1 also increases rDNA recombination rates [67], we generated a weaker version of the tet-activator by replacing the VP16 activation domain with activation domain A of Gcn4 [27], [68]. By carefully titrating Fob1 expression levels using a single copy of both TETOP2-Fob1 and the tTA-GCN4A activator in diploid cells, we could express FOB1 at normal levels in the absence of doxycycline, while effectively suppressing expression in the presence of 20 µg/ml doxycycline (Figure 5A).
