Site-directed mutagenesis of tandem AP1 consensus binding sites. (A) The diagram demonstrates the sequence of the 40-bp region of the ₂ enhancer extending from bp −1530 bp to −1570 containing the two intact but inversely oriented AP1 binding sites. The sequence of the three mutant constructs prepared by PCR containing mutations of the 5′ AP1 site, the 3′ AP1 site, or both AP1 binding sites is demonstrated. (B) To determine the role of one or both AP1 sites in enhancer activity, mutations of either the 5′ AP1 site, the 3′ AP1 site, or both sites were introduced into the shorter construct −1503/−1578 SV40pCAT. This shorter construct includes both AP1 binding sites but eliminates 77 bp of the 5′ region including the GATA and AP2 site and 77 bp of 3′ sequence. The enhancer activity of the original −1426/−1655 SV40pCAT construct was compared with the shorter intact construct −1503/−1578 SV40pCAT and the three mutant constructs, 5′ AP1m −1503/−1578 SV40pCAT, 3′ AP1m −1503/−1578 SV40pCAT, and Dbl AP1m −1503/−1578 SV40pCAT in uninduced and induced K562 cells after transient transfection. 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 the −1426/−1655 SV40pCAT construct in uninduced K562 cells, which was assigned a value of 1.0.