Deletion and point mutation analysis of the Miwi promoter.
a), Schematic diagram of the CpG islands and E1, E2 and CCAAT boxes in the Miwi promoter. The short vertical bars represent the CpG dinucleotides. b), Promoter activities of a series of deleted constructs determined by luciferase assay. Left panel, schematic representation of the mutants linked with luciferase gene in pGL3 vector. The nucleotides are numbered from the potential transcriptional start site that was assigned +1. Right panel, the relative activities to the 5M4 construct of a series of mutant constructs determined by luciferase assays. c), Point mutation analysis in the E1, E2 and CCAAT boxes of the Miwi promoter by luciferase assay. The 5M4 construct of the promoter (303 bp) was used for these point mutation analyses by linkage to the luciferase gene. The relative activities to the 5M4 construct of the mutant constructs were determined by luciferase assays. The open box and open circle indicate the intact binding site of E1 and E2 or CCAAT box respectively. The filled box or circle indicates the corresponding mutations. Non modified pGL3 was used as a negative control. The firefly luciferase activity was normalized to the Renilla luciferase activity, and the data is shown as the fold increase/decrease over the luciferase activity of pGL3-5M4. The results are the mean ± S.D. of three independent experiments. *, P<0.05; **, P<0.001 compared to the 5M4 wild-type construct.