Cloning and expression of a Xenopus short wavelength cone pigment.
The short wavelength visual pigment from Xenopus responsible for vision in the blue/violet portion of the spectrum was characterized by sequence spectroscopic analysis. The amino acid sequence was deduced by sequencing clones isolated by reverse transcription PCR, from retinal cDNA and genomic libraries. The gene contains 5 exons spanning 8.4 kb of genomic DNA and produces an mRNA of 2.4 kb in length. The deduced amino acid sequence predicts a protein of 347 amino acids with 76-78% identity to other short wavelength opsins. The mRNA encoding the Xenopus violet pigment was detected using in situ hybridization in cones, comprising a few percent of the total photoreceptors in the adult retina. The Xenopus violet opsin cDNA, modified to contain an epitope from the carboxyl terminus of bovine rhodopsin, was expressed in COS1 cells by transient transfection and analysed by UV-visible absorption spectroscopy. The protein expressed in COS1 cells migrated at 34 kD and was glycosylated at a single site in the amino terminus, exhibiting a diffuse pattern on SDS PAGE similar to bovine rhodopsin expressed in COS1 cells. Following incubation with 11-cis retinal, a light-sensitive pigment was formed with the lambdamax=425+/-2 nm. A Schiff base linkage between retinal and the violet opsin was demonstrated by acid denaturation. Xenopus violet opsin was sensitive to hydroxylamine in the dark, reacting with a half-time of 5 min at room temperature. This is the first group S pigment for amphibians. The pigment was expressed and purified from COS1 cells in a form that has permitted for the first time determination of the extinction coefficient, reactivity to hydroxylamine and presence of a Schiff base.
Starace
D M
DM
Department of Biochemistry and Molecular Biology, SUNY Health Science Center, 750 East Adams Street, Syracuse, NY 13210, USA.
Knox
B E
BE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D017949:Retinal Cone Photoreceptor Cells / Q000737:chemistry*; D012168:Retinal Pigments / Q000235:genetics*; D017299:Rod Opsins / Q000235:genetics / Q000302:isolation & purification; D013045:Species Specificity; D013056:Spectrophotometry, Ultraviolet; D014981:Xenopus / Q000235:genetics*
D018076:DNA, Complementary; D012168:Retinal Pigments; D017299:Rod Opsins