Molecular cloning of a rhodopsin gene from salamander rods.

PURPOSE Salamander photoreceptor cells have been used widely as models in vision research. However, the salamander opsin genes had not been cloned. The purpose of this study was to clone a salamander rhodopsin and to determine its primary structure and cell type-specific expression. METHODS Using salamander retina RNA as a template and Xenopus rhodopsin-specific oligonucleotides as primers, reverse transcription and polymerase chain reaction (RT-PCR) were used to amplify and clone a rhodopsin cDNA fragment. This fragment was used as a probe to isolate a full-length cDNA of the rhodopsin from a cDNA library of salamander retina. The dideoxynucleotide chain termination method was used to determine the nucleotide sequence. Single rod and cone cells were isolated by micromanipulation, and the absorbance spectra of the rod outer segments were measured with a photon-counting microspectrophotometer. Individual rod and cone cells were lysed for RT-PCR and Southern blot analysis to detect cell-specific expression of this gene. RESULTS A 1.2 kb rhodopsin cDNA containing the full-length coding region of rhodopsin has been cloned and sequenced from the larval tiger salamander, Ambystoma tigrinum. This cDNA encodes 354 amino acids that, by hydropathy profile, could form seven transmembrane domains characteristic of other rhodopsins. Sequence identity was found with other amphibian rhodopsins at the nucleic acid (82% to 83%) and the amino acid (88% to 89%) levels. Key amino acids critical for structure and function of rhodopsin have been retained. The mRNA of this rhodopsin was identified in red rod cells (lambda max 506 nm). No expression of the gene was detected in cone cells. CONCLUSIONS The cloned rhodopsin is a newly isolated member of the G protein-coupled receptor superfamily. This protein is expressed in rods but not in cones.