A schematic diagram of the ₂ promoter-CAT construct and the megakaryocytic enhancer in the irrelevant promoter construct SV40pCAT. (A) The construct p₂2592-CAT contains the entire 5′ flanking region from bp −2592 to +109 of the 5′ untranslated region of the ₂ integrin gene upstream to the CAT structural gene. The construct −1426/−2592 SV40pCAT consists of the megakaryocyte enhancer region extending from bp −1426 to −2592 in the irrelevant promoter construct SV40pCAT. Constructs −1842/−2592 SV40pCAT, −1426/−1842 SV40pCAT, −1426/−1655 SV40pCAT, and −1426/−1542 SV40pCAT were derived from the construct −1426/−2592 SV40pCAT. (B) The 1,166-bp region of the distal 5′ flank serves as a megakaryocytic enhancer. The constructs p₂2592-CAT, −1426/−2592 SV40pCAT, −1842/−2592 SV40pCAT, −1426/−1842 SV40pCAT, −1426/−1655 SV40pCAT, and −1655/−1842 SV40pCAT were transfected in parallel with the SV40pCAT plasmid that contains the strong viral promoter SV40 without an enhancer into either uninduced (⊠) or induced (▪) K562 cells. Cotransfection with RSV-luciferase was used to control for transfection efficiency. After 48 hours of incubation, cell extracts were assayed. After normalization for transfection efficiency, CAT activity of the constructs was determined using thin-layer chromatography and differential extraction. The mean and standard deviation of CAT activity of the mutated constructs in uninduced or induced K562 cells from three separate experiments was determined relative to the activity of SV40pCAT in uninduced K562 cells, which was assigned a value of 1.0.