Effects of doxycycline on the hygromycin sensitivity of two A. fumigatus strains that express tTA (p444) in conjunction with the tetO7-hph reporter gene (p482). (A): Ten thousand conidia from the tTA-1 or tTA-2 transformants were spotted onto the center of a plate containing 1 mg/ml hygromycin and the indicated concentrations of doxycycline (μg/ml) and colony diameter was measured with time. (B): Doxycycline regulation of hph RNA levels in the tTA-1 and tTA-2 strains by Northern blot analysis. Total RNA isolated from overnight cultures grown in the presence of 0–200 μg/ml doxycycline was fractionated by agarose gel electrophoresis, transferred to nylon membranes, and probed with a 32P-labeled hph probe. (C): Hybridization intensity in each lane of the Northern blot in (B) was normalized to levels of the SYBR-green II-stained rRNA by phosphorimager analysis and is shown as a percentage of the levels seen in the tTA-1 transformant in the absence of doxycycline.