Cell cultures were started from at least three independent clones and grown until the late exponential phase. Cells were collected by centrifugation, frozen in liquid nitrogen and stored at À70 C. Bacterial cells were lysed by sonication (3 · 15 s), and yeasts by vortexing with glass beads (0.5 vol) in +4 C for 30 min. Lysates were cleared by centrifugation, and enzymatic activities were determined immediately. Total protein concentrations were measured by using a Bradford protein assay reagent (Bio-Rad). b-Galactosidase (LacZ) and firefly or Renilla luciferase (LUC or RUC) activities were measured with commercial kits from Promega according to the manufacturer's instructions. LacZ activity was determined as the colour intensity at A414 nm. Luciferase activities were measured as relative light units (RLUs) with luminometer (Biohit or ThermoLabsystems). À1 PRF efficiencies were calculated from normalized firefly luciferase activities with the following formula: [(LUC activity from the test construct)/(LacZ or RUC activity from the test construct)]/[(LUC activity from the inframe control)/(LacZ or RUC activity from the inframe control)] · 100%.