Fig. 1. mezzo encodes a paired-like homeobox transcription factor related to the Mix-like family. (A) Sequence alignment between zebrafish Mezzo, mouse Sebox and human Sebox. The homeodomain is overlined. Identical amino acids are printed on a black background, similar residues are printed on a grey background. (B) Nucleotide and deduced amino acid sequence of the 5' end of the mezzo transcript. The likely potential translation initiation codon is boxed (lower) and the sequence targeted by the morpholino oligonucleotide used in this study is shown. Note that the 5' end of the cDNA contains another in frame potential initiation codon TGGATGC (top). However, the sequence surrounding this ATG fits poorly with the consensus sequence defined by Kozak ACCATGG. The second potential initiator codon (lower): ACTATGG located 57 bp 3' from the first in the ORF, is in good agreement with Kozak's rules with the most critical residues -3 and +4 being conserved. Furthermore, attempts to isolate longer cDNA clones by rescreening the library with a probe derived from the 5' end of the cDNA gave only clones that started at about the same position as the cDNA described above. Mapping of the 5' end of the mezzo transcript by primer extension revealed that the 5' end of the mezzo mRNA is located a just a few base pairs from the 5' end of the mezzo cDNA we isolated. Finally, in the course of overexpression studies, we observed that only the protein initiated at this second ATG has a biological activity (data not shown). (C) Comparison of the Mezzo, Bon/Mixer, Mml, Mix-1 and C-Mix homeodomains. (D) Schematic representation of the mezzo gene structure. The homeodomain is shaded and the position of three introns is indicated.