Abstract
Abstract: :
Purpose: The short-wave-sensitive (SWS1) cone pigment of the guinea pig (Cavia porcellus) has λmax at about 400 nm as determined by microspectrophotometry. This falls between the two wavelength groups common in mammals; about 370 nm (e.g. rat, mouse) and 420-440 nm (e.g. primate, bovine). We have therefore determined the sequence of this opsin to examine the tuning sites involved. Methods: Sequencing was performed on the amplified DNA obtained by RT-PCR and RACE of retinal cDNA, using primers designed to conserved regions of mammalian short-wave opsin sequences. The full-length gene sequence was cloned into the mammalian expression vector pMT4, allowing transient expression in HEK293T cells following transfection using GeneJuice. Harvested cells were incubated with retinal and the pigment solubilized from the cell membranes and purified by immunoaffinity chromatography using immobilized anti-1D4 antibody. Results: Expression data confirms this pigment to be violet-sensitive rather than ultraviolet-sensitive. Translation of the guinea pig short-wave-sensitive opsin sequence shows a serine residue at site 90 (bovine rod numbering system), in common with other mammalian sequences. The adjacent potential tuning sites at positions 86 and 93 are valine and alanine respectively, unique amongst mammalian short-wave sequences. Other tuning candidates are a cysteine at site 49 in helix I instead of leucine or phenylalanaine and phenylalanine-alanine at sites 203-204 in helix V as opposed to tyrosine-threonine in all other sequences. Conclusions: The guinea pig short-wave opsin does not show the expected residues at previously identified tuning sites and thus novel residues may contribute to its spectral tuning. Site-directed mutagenesis and expression of the generated mutants is being used to investigate the relative contributions of these residues.
Keywords: color pigments and opsins • molecular biology • protein structure/function