Fig. 2. The san and vtn loci encode the zebrafish homologs of CCM1 and CCM2. (A) The zebrafish (Dr) San (CCM1) protein sequence is aligned with the C. elegans (Ce), mouse (Mm) and human (Hs) homologs, demonstrating strong identity among the three vertebrate genes. The N-terminal NPxY motif indicated by the single brown line (NPAY, residues 191-194 of the zebrafish protein and residues 192-195 of the mouse and human protein) is altered in the C. elegans homolog to NPAF. This motif is associated with interaction of san with both ICAP1 and vtn (Zawistowski et al., 2002; Zawistowski et al., 2005; Zhang et al., 2001). The other two NPxF/Y motifs, indicated by brown double lines, were not required for interaction of san with vtn (Zawistowski et al., 2005). The three blue double lines indicate the location of the ankyrin repeats and the single green line denotes the FERM domain. The red asterisk indicates the position of the Ystop mutation in the zebrafish san ty219c allele. The protein motifs are shown schematically below the sequence. The red asterisk again denotes the san ty219c mutation and the region of the protein shown in red corresponds to exon 14, deleted in the san m775 allele. (B) Real-time PCR analysis was used to examine the level of san mRNA message containing exon 14. mRNA isolated from m775 wild-type siblings, and both wild-type and mutant ty219c embryos show levels of exon 14-containing message 10-fold greater than in the san m775 mutant embryos (m775 -/- level set to 1 for comparison). (C) The Vtn (CCM2) protein is very well conserved among vertebrates [the zebrafish (Dr) Vtn protein sequence is aligned with the mouse (Mm) and human (Hs) homologs, no C. elegans homolog is detectable]. The single blue line indicates the sequence corresponding to the PTB domain and the red asterisk indicates the position of the Ystop mutation in the zebrafish vtn m201 allele. The Vtn protein shown schematically below the sequence illustrates the position of the PTB domain and site of the vtn m201 mutation.