Defining TLC1 transcription enhancers and repressors using a transcription reporter system. (A) Schematic representation of the different TLC1 promoter regions cloned upstream of the lacZ reporter gene. Gray boxes indicate the sequence near the TLC1 5′-end, while black boxes indicate the region encompassing snR161 and the region upstream. (B) Northern blot analysis of the lacZ RNA expressed from different TLC1 promoter regions. Total RNA was extracted from cells carrying the different TLC1 promoter–lacZ fusions, separated on Agarose gel, and visualized using an end-labeled oligonucleotide probe complementary to the lacZ sequence. TLC1 RNA abundance was determined as in Figure 1 and is an average of three experiments with a SD of ±39% or less. (C) Schematic representation of different CYC1-TLC1 promoter regions cloned upstream of the lacZ reporter. The CYC1 promoter was used as a test promoter to verify repressing and enhancing activity of the different TLC1 promoter elements. The CYC1 promoter is represented by two empty boxes: One refers to the core element (TATACYC1); the other, to the upstream activating sequence of CYC1 (UASCYC1). Constructs containing the TLC1 promoter regions E and D are indicated by UASTLC1E and URSTLC1D, respectively. URSACT1 indicates a construct carrying a fragment of the internal coding sequence of ACT1 gene as a negative control. The known repressor sequence upstream of the CAR1 gene was used as a positive control (URSCAR1). (D) Northern blot analysis of lacZ RNA expressed from the different CYC1-TLC1 constructs. The RNA was prepared and detected as in B. TLC1 RNA abundance is an average of three experiments with a SD of ±88% or less. nd stands for nondetected RNA.
