Figure 3.

Kinetics of mitotic stability of the HAC carrying the construct tetR-VP64-IRES-DsRed2. The cells were cultured under following conditions: dox⁺ 3 days and dox⁻ 3 days, dox⁺ 4 days and dox⁻ 4 days, dox⁺ 7 days and dox⁻ 7 days, dox⁺ 14 days and dox⁻ 14 days. To quantify the HAC retention, the cells were analyzed by FISH. FISH analysis included detection of the HAC on metaphase spreads. At least 70–80 metaphase spreads were analyzed for each point. Addition of doxycycline, which prevents fusion of the tetracycline repressor from binding to tetO sequences of the HAC, blocks the inactivation of HAC kinetochore. Targeting the transcriptional transactivator tTA^VP64 into the HAC kinetochore in the absence of doxycycline induces HAC loss.