Figure 1.
Distance constraints for the transcriptional activation property of HS2, HS3, and the miniLCR. The three activating elements (A) HS2, (B) HS3, and (C) miniLCR, were subcloned upstream of a γ-globin promoter linked to a luciferase gene. Variable-length phage λ DNA fragments were subcloned between the activating element and promoter. The test constructs and a selection construct were linearized and cotransfected into K562 cells. Stably transfected pools of cells were isolated and assayed for luciferase activity as a measure of γ-globin promoter activity (mean ± SEM). The number of pools analyzed for the various constructs was: HS2γluc, 7; HS2(2.2)γluc, 7; HS2(3.4)γluc, 6; HS2(5.1)γluc, 7; HS2(7.3)γluc, 4; HS3γluc, 7; HS3(2.2)γluc, 8; HS3(3.4)γluc, 8; HS3(5.1)γluc, 5; HS3(8.5)γluc, 7; miniLCRγluc, 16; miniLCR(2.2)γluc, 12; miniLCR(2.3)γluc, 12; miniLCR(5.1)γluc, 12; miniLCR(7.3)γluc, 11. (A) The activity of a construct containing only the γ-globin promoter fused to the luciferase gene (pγluc) are shown by ○, whereas in A–C the activity of constructs containing activating elements are shown by •. The luciferase activity of pγluc was 0.43 ± 0.49 light units per s/μg × 10−3 (n = 3).