CLONING AND EXPRESSION OF A XENOPUS SHORT-WAVELENGTH CONE PIGMENT

The short wavelength visual pigment from Xenopus responsible for visio n in the blue/violet portion of the spectrum was characterized by sequ ence spectroscopic analysis. The amino acid sequence was deduced by se quencing clones isolated by reverse transcription PCR, from retinal cD NA and genomic libraries. The gene contains 5 exons spanning 84 kb of genomic DNA and produces an mRNA of 2.4 kb in length. The deduced amin o acid sequence predicts a protein of 347 amino acids with 76-78% iden tity to other short wavelength opsins. The mRNA encoding the Xenopus v iolet pigment was detected using in situ hybridization in cones, compr ising a few percent of the total photoreceptors in the adult retina. T he 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 spectrosc opy. The protein expressed in COS1 cells migrated at 34 kD and was gly cosylated at a single site in the amino terminus, exhibiting a diffuse pattern on SDS PAGE similar to bovine rhodopsin expressed in COS1 cel ls. Following incubation with Il-cis retinal, a light-sensitive pigmen t was formed with the lambda(max) = 425 +/- 2 nm. A Schiff base linkag e between retinal and the violet opsin was demonstrated by acid denatu ration. Xenopus violet opsin was sensitive to hydroxylamine in the dar k, 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 p urified from COS1 cells in a form that has permitted for the first tim e determination of the extinction coefficient, reactivity to hydroxyla mine and presence of a Schiff base. (C) 1998 Academic Press.