Fig. 3. ZicL-binding sequences in the cis-regulatory region of the Ci-Bra gene. (A) DNA sequence of the minimal 483-bp Ci-Bra enhancer (see Corbo et al., 1997; Fujiwara et al., 1998). Solid unfilled boxes, dotted unfilled boxes, filled boxes and the dotted line indicate Ci-Snail-binding sites, Su(H)-binding sites, E box sequences and a TATA element, respectively. The arrow represents the presumptive transcription start site. Solid lines represent elements with sequence similarity to the consensus ZicL-binding sequence. (B,C) A gel shift assay of the binding of GST/Ci-ZicL(ZF) to oligonucleotides corresponding to various locations in the Ci-Bra transcription regulatory region. Oligonucleotide sequences shown in (B) were examined in a gel-shift assay (C). The boxed letters of ZicL-b indicate the consensus ZicL-binding sequence. The shaded letters in Bra-123 to Bra-1996 indicate nucleotides identical to those in the consensus ZicL-binding sequence. The binding seemed specific because it was not detected under conditions of zinc-removed incubation (lane 1), pre-incubation with x100 molar excess of unlabeled competitor DNA or replacement by GST protein (data not shown). (D) Comparison of the Brachyury upstream sequences between Ciona intestinalis (upper) and C. savignyi (lower). Boxes in the C. intestinalis sequence indicate presumptive ZicL-binding sites, and solid lines in the C. savignyi sequence indicate sequences that resemble the consensus ZicL-binding sequence.