Fig. 6. SpGsc competes for binding at SpOtx cis elements. (A) SpGsc binds with specificity to an SpOtx site from the Spec2a promoter (contained on the CII fragment). Bacterially produced GST-GSC fusion protein purified by glutathione affinity chromatography formed a complex that was competable with the sequences containing an intact SpOtx cis element (lane 2 versus lanes 3 and 7), but not with those in which this element was altered by point mutation (lane 5) or by sequence replacement as defined by Yuh et al. (Yuh et al., 2001) (lane 6). Addition of GST antibody (lane 4) supershifted a significant fraction of this complex. GSC protein derived from GSC-GST also formed specific complexes (lane 8) as shown by competition reactions with the probe sequence (lane 9), and those containing mutated Otx elements (lanes 10,11). (B) SpGsc and SpOtx compete for binding to CII. GSC/CII complexes are shown in lanes 2 and 3. GST-Otx/CII complexes (lane 4) are supershifted with Otx antibody (lane 5). Reactions containing mixtures of GSC and GST-Otx were carried out under limiting probe concentrations. Lanes 6-8, constant amounts of GST-Otx were mixed with increasing quantities of GSC; Lanes 10-13, constant amounts of GSC were mixed with increasing amounts of GST-Otx. (C) SpGsc down regulates the activity of a promoter driven by SpOtx. Embryos (100) were injected with a promoter/CAT transgene construct and either no (–) or 2x10⁶ or 6x10⁶ molecules of SpGsc mRNA. St refers to a positive control reaction containing chloramphenicol acetyl transferase.