Fig. 3. Sequence of zebrafish Gli1 and identification of point mutations in the three dtr alleles. (A) The deduced amino acid sequence of zebrafish Gli1 (zfGli1) aligned with mouse Gli1 (mGli1) and zebrafish Gli2 (zfGli2). The entire coding region of gli1 was sequenced in each of the three ENU-induced dtr alleles (dtr^tm276, dtr^te370and dtr^ts269) and point mutations were found for each allele (boxes). The altered amino acid in dtr^tm276is shown above the box while nonsense mutations are indicated by red hexagons. Gli2 mutations found in you-too are from Karlstrom et al. (Karlstrom et al., 1999). The five zinc finger regions are indicated by lines and potential sites for phosphorylation by protein kinase A (PKA) are indicated by asterisks. A putative VP-16 activator-like domain is indicated by a blue box. Colored sections indicate regions of homology schematized in C. (B) Sequencing ferograms showing point mutations in the three dtr alleles. In dtr^tm276U 1633 is mutated to G, changing tyrosine 440 (UAC: Y) into an aspartic acid (GAC: D). In dtr^ts269 C 2956 is mutated to U, changing glutamine 881 (CAG: Q) into a stop codon (UAG). In dtr^te370C 3073 is mutated to U, changing glutamine 920 (CAG: Q) into a stop codon (UAG). (C) Schematic representation of zebrafish and mouse Gli1 and Gli2 protein sequences showing the positions of the stop codons (arrowheads) in the zebrafish mutant alleles. The position corresponding to the site of cleavage that results in a repressor form of Ci is shown by an arrow. Red boxes indicate regions shared among all three sequences, green boxes indicate sequences shared in mouse and zebrafish Gli1 (with percentage amino acid identity indicated), while gray boxes show sequences shared between zebrafish and mouse Gli2 (with percentage amino acid identity indicated). The zinc finger region is marked by ZnFn. Blue box shows region of homology to the VP-16 activator domain, asterisks indicate potential PKA phosphorylation sites. (D) Cladogram showing similarity of mouse (m), frog (Xn) and zebrafish (zf) Gli sequences. Tree is based on ClustalW alignment of amino acid sequences. A search of zebrafish EST databases and genomic trace sequences using mouse Gli1 sequence did not reveal a sequence more similar than the zebrafish Gli1 sequence shown above.