Fig. 5. Enhancer stimulated trans-activation in reconstituted transcription reactions. In vitro transcription reactions containing both promoter and enhancer plasmids were performed using a human nuclear extract that lacks GAGA, as a source of basal transcription factors. Transcription reactions containing 50 ng of the promoter plasmid were conducted in the absence or presence of recombinant GAGA, ΔPOZ, POZ-DBD and Gal4-VP16, as indicated. The promoter plasmid contained five GAGA sites upstream of a minimal core promoter, with the exception of lane 8, where the promoter lacked GAGA sites. Reactions also contained a 6-fold excess of 5×GAGA-5×Gal4-pBluescript enhancer plasmid, with the exception of lane 7, which contained equal amounts of 5×GAGA-pBluescript and 5×Gal4-pBluescript plasmids. Transcription products were detected by primer extension, separated on a 7% denaturing polyacrylamide gel and subjected to autoradiography. The results shown were quantified by phosphoimager analysis and revealed a minor reduction in transcription levels in the presence of the activators alone (∼0.8- to 1-fold basal levels), a 6- to 7-fold activation in the presence of both GAGA and Gal4-VP16, and 5-fold activation by POZ-DBD and Gal4-VP16.