Figure 1
Analysis of the cloned zebrafish β1 subunit gene and novel splice variants. A) Alignment of cloned human, rat, and zebrafish β1 amino acid sequences. Black = identical in all three species; grey = identical in 2/3 species or conserved substitution. Shown for zebrafish is the most conserved β1 splice form (variant D). Hs = Homo sapiens, Rn = Rattus norvegicus, z = zebrafish; DS* = cysteine residue predicted to participate in a disulfide bridge, based on the myelin P0 protein crystal structure; DS = predicted second disulfide bridge; N = predicted N-linked glycosylation site (N1 = human/rat, N2 = zebrafish); M1 = site of epilepsy-causing deletion (I70_E74del) in Hsβ1; M2 = site of second epilepsy-causing mutation (C121W) in Hsβ1; M3/4 = site of third and fourth epilepsy-causing mutations (R85C, R85H); S1 = nonsynonymous Hsβ1 single nucleotide polymorphism (SNP, G/A > R85H); S2 = nonsynonymous Hsβ1 SNP (C/T > T189M); P = phosphorylation site (tyrosine Y181) that regulates ankyrin recruitment (NOTE: Y200 = Y181 following cleavage of 19 amino acid signal peptide); IN = putative internalization sequence. Consensus sequence for V-type IG domain is depicted beneath the alignment: G = glycine, x = any residue, ^ = hydrophobic residues, C = cysteine, - = gap in alignment with consensus sequence, W = tryptophan, * = basic residue, L = leucine, D = aspartic acid, & = glycine, alanine, or aspartate, and Y = tyrosine. Red indicates zebrafish residues that deviate from the consensus sequence. See Results for references supporting sequence annotation. B) 5' and 3' RLM-RACE PCR and RT-PCR identified four distinct splice variants expressed from zβ1 locus on zebrafish chromosome 16 (Ensembl). C) Splice donor and acceptor sites of zebrafish β1 splice variants, derived from comparing cloned cDNA against genomic DNA sequences (Ensembl). Consensus GT-AG splice sites are labeled in red. A splice-site deviating from the consensus appears in grey. D) Schematic diagram of β1 splice variants A, B, and D, whose predicted proteins differ only in the length of their intracytoplasmic C-terminal tail. S-S = disulfide bridge. NH3 = 5' amino terminus, CO2 = 3' carboxyl terminus, β = putative N-linked glyosylation site. Alignment of C-terminal tail of variants zβ1A, zβ1B, and zβ1D (below). zβ1C is not shown as it is predicted to lack both extracellular IG and transmembrane domains.