Characterization of a putative novel opsin from atlantic salmon

Purpose. To investigate the structural characteristics of a putative novel opsin family. Methods. We have initiated a screen of salmon ocular opsins. Degenerate PCR primers were designed against evolutionarily conserved structural domains of opsins. cDNA was synthesized from ocular RNA. Opsin cDNAs were amplified using opsin specific degenerate primers. cDNAs were subsequently subcloned and sequenced. 5′ and 3′ RACE PCR was used to isolate the complete coding region. Phylogenetic trees of cloned opsins were generated using commercially available software (PAUP). Results. Within opsin families, opsins from different species show amino acid identity of 65-95%. Between families, they show amino acid identity of 40-50%. During our screen we isolated an opsin cDNA partial clone (tentatively called salopsin) whose deduced translation (153 amino acids) showed approximately 40% identity to all other vertebrate opsin families, including pinopsin. Salopsin contains a Schiff base lysine for chromophore binding, a cysteine involved in an intramolecular disulfide bond, and three hydrophobic domains corresponding to the expected transmembrane domains of other opsins. Additionally, salopsin contains differences in conserved residues within suggested transducin interacting domains, and a deletion of two amino acids within the second intradiskal loop. In the various phylogenetic trees generated, salopsin was placed as either a third clade at the long wavelength/short wavelength duplication event, or as a separate clade preceding it. Salopsin was never placed within another opsin family. Conclusions. Based upon the amino acid identity of salopsin (≈ 40%) to all other vertebrate opsin families, we propose that it may form a novel opsin family. Salopsin has several structural features common to vertebrate opsins yet contains differences in functionally important residues. Finally, phylogenetic analysis places salopsin as a separate early clade in vertebrate opsin evolution. We have begun further studies to determine the structural features and functional importance of salopsin.