Site-directed mutagenesis of the Sp1 binding site within the α₂ integrin core promoter altered activity. (A) The diagram shows the sequence of the 35-bp region of the α₂ integrin core promoter extending from bp −30 to −65 containing the intact Sp1 and AP2 binding sites and the sequence of the three mutant constructs prepared by PCR containing either a mutated 5′ Sp1 site, a mutated 3′ Sp1 site, or mutations of both Sp1 recognition sites. The shorter double-stranded DNA fragment extending from bp −30 to −65 that included the two tandem Sp1 sites as well as the AP2 site, but lacked 27 bp at the 5′ end (bp −65 to −92), was created by PCR and placed upstream of CAT structural sequences to form the p α₂65-CAT construct (shown in B). Mutations of the 5′ Sp1 site, the 3′ Sp1 site, or both sites were made by site-directed mutagenesis of the p α₂65-CAT construct. (B) The activity of the intact or mutant α₂ integrin core promoter. The promoter activity of the original construct pα₂92-CAT; the mutant constructs pα₂Δ 5′ Sp1-CAT, pα₂Δ 3′ Sp1-CAT, and pα₂Δ dbl Sp1-CAT; and pCAT Basic was compared with the activity of the pα₂ 65-CAT in induced K562 cells after transient transfection of the constructs 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 by thin-layer chromatography and differential extraction. The mean and standard deviation of CAT activity of the mutant constructs from four separation electroporations was determined relative to the pα₂65-CAT construct in induced K562 cells that was assigned a value of 1.0.