Fig. 5. Mapping of Pax2-binding sites in the conserved 5' regions of Brn1. (A) Presence of high-affinity Pax2-binding sites in elements D and P. The binding of in vitro translated Pax2 protein to a labeled oligonucleotide containing the Pax2/5/8-binding site 1 of the CD19 promoter (Kozmik et al., 1992) was measured by EMSA in the absence (–) or presence of a 10- or 50-fold molar excess of the indicated competitor DNA. The competition strength of PCR fragments comprising elements C, D and P was compared with that of the high-affinity site 1 of the Blnk promoter (Schebesta et al., 2002). The protein-DNA complexes are shown together with a map, indicating the positions of the conserved elements relative to the Brn1-coding sequence. (B,C) Identification of the sites Dd (B) and Pc (C) as high-affinity Pax2-binding sequences. The same competition assay was used to evaluate the interaction of Pax2 with the indicated restriction fragments (P1-P3) or oligonucleotides containing putative Pax2/5/8-binding sites in element D (Da-Dd) and P (Pa-Pc). Substitution of two base pairs prevents binding of Pax2 to the mutant (m) sites Ddm (B) and Pcm (C). D, DdeI; T, TaqI. (D) Sequence alignment of sites Dd and Pc with the consensus Pax2/5/8 recognition sequence (Czerny and Busslinger, 1995). The positions relative to the Brn1 start codon and the nucleotide substitutions of the mutant sites are indicated.