Fig. 1. Characterization of the tetO₇:ROT1 mutant strain. (A) Rot1 protein levels in extracts from cultures of the ROT1-HA (MCY10) and the tetO₇:ROT1-HA (MCY192) strains grown on YPD and incubated in the presence of 5 µg/ml doxycycline were examined by western analysis. A control extract from the wild-type strain (CML240) is included (no tag). A non-specific band that crossreacts with the antibody is shown as loading control (^*). (B) Tenfold serial dilutions from exponentially growing cultures of the wild-type strain (CML240) and the tetO₇:ROT1 (JCY216) strain transformed with a control vector or a centromeric plasmid containing the ROT1 gene, were spotted onto YPD medium with or without 5 µg/ml doxycycline (dox) and incubated at 28°C for 3 days. (C) Exponentially growing cultures of the wild-type (CML240) and the tetO₇:ROT1 (JCY216) strains were incubated in the presence of 5 µg/ml doxycycline for 8 hours. Graph shows the distribution of cells at different cell cycle stages. Pictures show DIC images, DAPI staining of DNA and staining of spindle by indirect immunofluorescence using anti-tubulin antibody. A collection of rebudded cells is shown in for the tetO₇:ROT1 strain (see text). Values are the mean+s.d. of three experiments.