Site-directed mutagenesis of the AP2 and GATA consensus sites. (A) A diagram demonstrates the sequence of a 32-bp region within the megakaryocytic enhancer extending between bp −1623 and −1655 containing the intact AP2 and GATA binding sites and the sequence of 3 mutant constructs prepared by PCR to produce mutations of either the AP2 site, the GATA site, or both AP2 and GATA recognition sites. (B) Mutations in either the AP2 site, the GATA site, or both AP2 and GATA sites were incorporated into the construct −1426/−1655 SV40pCAT. The enhancer activity of the mutant constructs containing either a single mutation of the AP2 site, GATA site, or the AP2 and GATA sites was compared with the activity of the intact −1426/−1655 SV40pCAT in uninduced and 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 by thin-layer chromatography and differential extraction. The mean and standard deviation of CAT activity of the mutant constructs from at least three separate electroporations was determined relative to −1426/−1655 SV40pCAT in uninduced K562 cells, which was assigned the value of 1.0.