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Purpose. We have previously reported the discovery of a partial cDNA clone encoding a putative novel opsin (Soni and Foster, Invest. Opihalmol. Vis. Sei. 1996 Feb;37(3):#4265). A phylogcnetic analysis of this opsin shows that it diverges before the evolution of other vertebrate opsins. Thus, it has been named Ancient opsin (A-opsin). Our aim is to analyze the structural characteristics of full length A-opsin cDNA. Additionally, we wish to investigate the function of A-opsin pigment using functional expression with recombinant baculovirus. Methods. The remaining coding sequences of A-opsin were amplified from salmon ocular cDNA using 5′ and 3′ RACE PCR. A-opsin cDNA for expression was modified by addition of a carboxyl terminal 6X-histidine tag, enabling simple protein purification. Recombinant baculovirus was generated by homologous recombination with a transfer vector containing modified A-opsin cDNA. A-opsin protein was generated by infection of SF-9 cells with recombinant baculovirus. A-opsin was detected using a his-tag directed antibody. Results. Within opsin families, opsins from different species show amino acid identity of 65%-95%. Between families, they show identity of 40-50%. Full length A-opsin cDNA shows 32-42% identity with all other vertebrate opsin families. The new cDNA sequences reveal that A-opsin has several additional modifications. A-opsin has a short carboxyl tail with a singular serine residue and lacks a N terminal glycosylation site. Western blot analysis of infected SF-9 cells shows a his-tagged protein of 32 kDa indicating that we have expressed full length A-opsin protein. Conclusions. Based upon a partial clone, we had previously proposed that A-opsin forms a novel, and phylogenetically distinct, opsin class. Our analysis of full length A-opsin cDNA agrees with this hypothesis. We are currently investigating the spectral characteristics of A-opsin pigment in order to resolve the functional role of this novel opsin family.

Type

Journal article

Journal

Investigative Ophthalmology and Visual Science

Publication Date

01/12/1997

Volume

38