Functional analysis of the FR-β gene promoter. As described in “Materials and methods,” 293 cells were cotransfected with the wild-type or mutant FR-β promoter-luciferase construct and a β-galactosidase expression plasmid. Cells were harvested and tested for luciferase and β-galactosidase activity 48 hours after transfection. The luciferase activity was normalized to β-galactosidase activity. (A) Schematic of the FR-β gene promoter. The numbers indicate nucleotide positions in relation to the transcription start site (+ 1 nt). (B) Function of the Sp1 and ets elements in the FR-β gene. The FR-β gene promoter constructs were inserted in the pGL3 basic vector. The numbers in parentheses indicate the 5′ and 3′ ends of the promoter fragments in relation to the transcription start site (+ 1 nt). ΔSp1 indicates an internal deletion of the Sp1 site (– 78 nt to – 65 nt). ΔEBS indicates an internal deletion of the ets binding site (– 64 nt to – 43 nt). (C) Functional mapping of repressor elements in the FR-β promoter. The DNA sequences of the promoter constructs are numbered as described for panel B and also shown schematically. (D) Potential binding sites for transcription factors in the FR-β promoter sequence – 368 nt to – 308 nt. (E) Functional interaction between the repressor elements and the EBS in the FR-β promoter. The DNA sequences of the promoter constructs are numbered as described for panel B. Each experiment was repeated at least 4 times, and concordant results were obtained. Each bar is expressed as means of triplicate experiments. Error bar stands for SD.