a: Sequence comparison of the Ets site, the CD5X region, and the CD5Y region between human and mouse. Partial sequence alignment of the homologous regions on the mouse and human CD5 promoter, as determined by sequence analysis program from Intelligenetics Corporation. The consensus sequences for the Ets, CD5X, and CD5Y regions are shown in boxes.
b: Site-directed mutagenesis analysis showed that CD5Y region is important for basal CD5 transcription. Three areas in CD5X region (Xmut1, Xmut2, and Xmut3) and two in CD5Y region (Ymut1 and Ymut2) were mutated by site-directed mutagenesis on HincII B construct as described in Materials and Methods. Each mutant construct was cotransfected with pON405 LacZ construct in EL4 cells. Wild-type HincII B and HinfI B constructs were also transfected as controls. Each bar value represents the average of three independent transfections in one experiment, with standard deviation as error bars.
c: Site-directed mutagenesis analysis revealed that both the CD5X and CD5Y regions affect overall CD5 expression. Three sites in the CD5X region (Xmut1, Xmut2, and Xmut3) and two in the CD5Y region (Xmut1 and Xmut2) on the StuI B construct were mutated. Each X and Y mutant StuI B construct was co-transfected into EL4 cells along with pON405 LacZ construct. Wild-type StuI B and HincII constructs were also transfected as controls.