FIGURE 2.

Analysis of human UGDH gene promoter activity in orbital fibroblasts. A, sequences of the promoter spanning −1858 to +71 nt (Fragment 9) and the deletion mutants indicated were generated as described under “Experimental Procedures.” They were fused to the fruit fly luciferase gene in pGL2 basic vector and transfected into orbital fibroblasts concurrently with an internal control pRLTK vector. Deletion constructs are diagramed schematically. Rectangular boxes represent the regions upstream from the UGDH coding sequence. Luciferase activity was normalized to that of pRLTK used as a transfection efficiency control and are shown as bar graphs on the right. B, the sequence spanning −1436 to −1388 bp of the UGDH gene promoter exhibits repressor activity. The schematic diagram of the chimeric construct including the putative repressor fused to an SV40 promoter driven vector is shown. This was transfected into orbital fibroblasts. C, the sequence extending from −598 to −148 nt of Fragment 3 was deleted, resulting in Fragment 13, removing several Sp1 binding sites. D, Fragment 3 was subjected to the deletions indicated. Data represent the mean ± S.D. of two experiments, each performed in triplicate.