Fig. 6. Ppa-LIN-39 and its Extradenticle-like cofactor Ppa-CEH-20 can bind to the Ppa-pax-3 promoter in vitro. (A) Putative HOX-PBC-binding sites in the Ppa-pax-3 promoter. SITEBLAST analysis of the Ppa-pax-3 promoter including 3 kb of upstream sequence revealed one putative binding site (arrowhead). Beneath, we show the nucleotide sequence of the predicted binding site `a' with the core binding site TGATGAATCG (wild type, wt). For electrophoretic mobility shift assays this core binding site has been mutated (mt) to TtgcGAcgCG. (B) In electrophoretic mobility shift assays, Ppa-LIN-39 binds to a Drosophila Antennapedia control oligonucleotide alone and in conjunction with Ppa-CEH-20. Ppa-LIN-39 does not bind Ppa-pax-3 oligonucleotides on its own (lane 6), but strongly binds together with Ppa-CEH-20 (lane 7). Ppa-LIN-39 and Ppa-CEH-20 do not bind to the mutated site (SaM, lane 9). (C) Phylogenetic footprint of the HOX-PBC-binding site in the Ppa-pax-3 promoter. The sequence from position -2216 to -2132 of Ppa-pax-3 is shown. Comparison of P. pacificus (P.pa), Pristionchus sp. 11 (P.11) and Pristionchus maupasi (P.ma). The consensus HOX-PBC-binding site is indicated by a shaded box, other types of consensus binding sites are boxed. Boxes 1 and 2 are HOX monomer binding sites; box 3 is a HMG-binding site. (Bottom) The tree shows the phylogenetic relationship of these three species.