Fig. 1

TetR-mediated repression of transcription initiation. (A) Schematic diagram of the tetracycline-regulatable single expression cassette and the expected bicistronic mRNA. The tetR probe for Northern blot analysis is underlined. (B) Northern Blot analysis. Mock-treated Vero cells and cells transfected independently with the empty vector, the pcDNAtetR plasmid, the 1Pc, and the 1Pi were harvested 2 days posttransfection. Total RNA was separated using the TRIzol reagent, followed by chloroform extraction and precipitation with isopropanol. Total RNA (20 µg) was run in denaturing conditions and blotted on Hybond-N membranes to detect the presence of specific mRNAs that hybridize with a ³²P-labelled tetR probe (XbaI-EcoRI DNA fragment indicated in A). A transcript of about 0.6 Kb corresponding to the tetR mRNA is shown. Tc regulation of the 2.0 Kb bicistronic mRNA expression from the 1Pi is observed. (C) Densitometric analysis of the radiolabeled 2-Kb bands is represented. Results obtained with the different constructs are expressed in arbitrary units.