A Novel Cone-like Pigment Expressed in Xenopus laevis Rod Cells

Abstract

Abstract: : . Purpose: The purpose of this study was to clone and characterize the green rod pigment in Xenopus laevis. Methods: The cDNA for the Xenopus "green rod", blue-sensitive pigment was cloned and sequenced from Xenopus retina mRNA by reverse transcriptase polymerase chain reaction (PCR) amplification using primers to conserved regions of amphibian blue-sensitive pigment and RACE PCR. Immunohistochemical studies in flat-mount retina using 1) a polyclonal antibody raised against the predicted N-terminal sequence of the Xenopus green rod pigment, 2)a monoclonal anti-bovine red rod opsin antibody, and 3) wheat germ agglutinin (which labels all Xenopus cone cells) determined the cellular localization of the Xenopus SWS2, P434 pigment. Spectral proerties of the expressed protein were determined by absorption spectroscopy. Results: A novel Xenopus opsin cDNA containing a full-length coding region has been cloned and sequenced. The deduced amino acid sequence predicts a protein of 362 amino acids. Sequence analysis indicates that it belongs firmly in the SWS2 class of visual pigments and has 89%, 80%, and 75% amino acid sequence identity with bullfrog, salamander and newt, SWS2 pigments, respectively. Staining of Xenopus retina with a Xenopus SWS2, P434 specific polyclonal antibody indicates that the blue-sensitive pigment is expressed in green rods. SWS2, P434 was expressed in COS cells, and the pigment formed with the A1 chromophore, 11-cis retinal, showed an absorption λ_maxof 434 nm. The pigment is sensitive to hydroxylamine, suggesting that it has cone-like properties. Conclusions: A novel blue-sensitive opsin cDNA has been cloned and sequenced from the retina of adult Xenopus laevis which encodes a protein belonging to the SWS2 group of opsins. The expressed opsin possesses cone-opsin-like properties yet was identified only in the Xenopus green rod cells.

Keywords: molecular biology • color pigments and opsins • photoreceptors