The P56S mutation causes a tTA-like phenotype. The activity of wild-type and P56S-mutated rtTA was measured in C33A cells transfected with a reporter plasmid carrying the firefly luciferase gene under the control of the viral LTR-2ΔtetO promoter (LTR-2ΔtetO; A) or under the control of a minimal CMV promoter coupled to an array of seven tetO elements (CMV-7tetO; B). Furthermore, rtTA activity was measured in HeLa X1/6 cells  that contain chromosomally integrated copies of the CMV-7tetO luciferase construct (CMV-7tetO-integrated; C). Cells were transfected with the indicated rtTA expression plasmid (both rtTA variants contain the F86Y and A209T mutations ) or pBluescript as a negative control (-), and a plasmid constitutively expressing Renilla luciferase to correct for differences in transfection efficiency. Cells were cultured with different dox concentrations (0–1000 ng/ml). The ratio of the firefly and Renilla luciferase activities measured two days after transfection reflects the rtTA activity. All values are relative to the wild-type rtTA activity at 1000 ng/ml dox, which was arbitrarily set at 100%.