Fig. 4. Identification of nucleotides important for SO-DNA (soAE) interaction. (A) SO protein is shifted by soAE (SO + hot probe) in EMSA. Double-stranded probes bearing a single point mutation (6-22) or a stretch of mutations (2-5) were used as cold competitors (100 x molar excess) and compared to soAE (1) for their ability to compete for SO-binding. Nucleotides important for protein-DNA interaction are highlighted in red (very important) and orange (important) according to their competing potential. As a control, mock transfected reticulocyte lysate was incubated with p³² marked soAE (TNT mock). (B) DNA probes of sequences resembling soAE taken from other genes were used as cold competitors as well. A sequence out of the eye-specific enhancer of the ey gene is a strong competitor. The fragment loses its binding property when the GAT sequence is mutated to CCC (eyeless, eyeless mut). Out of the lozenge gene, only one of the previously described SO-binding sites shows a strong competition potential in EMSA (lz first, lz second). Strong competing sequences are also found in the first intron of the hh gene (hh first, hh second). SO binding is furthermore strongly competed by the well-described AREC3/Six4-binding site. (C) Upper half: sequences of the probes that were used as cold competitors in Fig. 4B. Based on these sequences and the results shown in A, a consensus binding sequence for SO was proposed. Lower half: previously described binding sites for the vertebrate Six1,2,4,5. These sequences appear to be related to the SO-binding sequence.