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Fig. 2. Characterization of nucleotide sequences recognized by GST/ZicL(ZF) fusion protein. (A) SDS-PAGE of purified GST/Ci-ZicL(ZF) fusion protein (lane 2) and GST protein (lane 3). The molecular mass relative to the Marker (lane 1) shows the successful production of the fusion protein. (B) Compilation of the GST/Ci-ZicL(ZF) and GST/Cs-ZicL(ZF) binding sequences. DNA sequences that bound to the fusion proteins were selected and determined as described in Materials and methods. Cloned random sequences, after eight or 10 rounds of selection for GST/Ci-ZicL(ZF) and eight rounds for GST/Cs-ZicL(ZF), were aligned. The bottom sequence in each table represents the compiled most favored sequence at each nucleotide position. The underlined ggatc is identical to the 3' end of primer F1. (C,D) A mutation analysis of the binding sequence of GST/Ci-ZicL(ZF). Eleven types of oligonucleotides (C) were examined by a gel-shift assay (D). The boxed uppercase letters of the ZicL-b oligonucleotides indicate the consensus ZicL-binding sequence. The shaded lowercase letters in the µA-µJ oligonucleotides indicate mutated nucleotides in each mutant oligonucleotide. 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 with GST protein (data not shown).

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