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The cephalopod molluscs are a group of invertebrates that occupy a wide range of oceanic photic environments. They are an ideal group of animals, therefore, in which to study the evolution of rhodopsin. The cDNA sequence of the rhodopsin gene of the cuttlefish Sepia officinalis (L.) (Sub-class Coleoidea, Order Sepiida) is presented, together with an analysis of the structure of the gene. A proline-rich C terminus is present; this structure is characteristic of cephalopod rhodopsins. In common with all invertebrate opsins studied so far, the equivalent site to the counterion in vertebrate opsins is occupied by an aromatic amino acid. An intron is present that splits codon 107, in contrast to the intronless rhodopsin gene in two species of myopsid squid. A spectral tuning model involving substitutions at only three amino acid sites is proposed for the spectral shifts between the rhodopsins of Sepia officinalis, three species of squid and Paroctopus defleini.

Purpose: Salamander retinas are known to contain at least three cone pigments and two rod pigments. The purpose of this study was to clone and characterize the visual pigments from salamander cones., Methods: cDNA fragments of cone pigments were amplified from a salamander retina cDNA library by PCR using a pair of primers with consensus for visual pigments. These fragments were cloned and used as probes for library-screening. The full-length cDNAs were isolated from the retinal library using the cloned PCR products as probes. DNA sequences were determined by the dideoxynucleotide chain termination method., Results: Two pigment cDNAs were cloned and sequenced from the salamander library. The global GenBank search showed that they do not match any existing sequences but have significant sequence similarity to visual pigments. One of the pigment cDNAs showed a high sequence homology with red cone pigments from other species and thus, was designated as a red cone opsin. The other pigment was designated as a blue cone opsin as it is most homologous to the chicken and goldfish blue cone pigments. Both cDNAs contain a full-length coding region encoding 365 amino acids in the red and 363 amino acids in the blue cone pigment. Hydropathy analysis predicted that both pigments could form seven hydrophobic transmembrane helices. Both pigments retain the key amino acid residues critical for maintaining the structure and function of opsins and have similar G-protein interaction sequences which differ from that of rod opsin. Phylogenetic analysis indicates that the red opsin belongs to the L group and the blue opsin belongs to the M1 group of visual pigments., Conclusions: The salamander red and blue cone pigments share high sequence homology with the cone pigments of other species.

This paper describes the localization of newly identified visual pigment opsins in the tiered retina of the Japanese yellow swallowtail Papilio xuthus. We first cloned three cDNAs encoding visual pigment opsins, PxRh1, PxRh2 and PxRh3, and then carried out histological in situ hybridization to localize their mRNAs in the retina. By combining the present data with our previous electrophysiological results, we concluded that both PxRh1 and PxRh2 correspond to visual pigments expressed in photoreceptor cells sensitive in the green wavelength region (green receptors), whereas PxRh3 corresponds to a pigment in red receptors. The in situ hybridization studies showed that some photoreceptor cells express two opsin mRNAs. In the ventral half of the eye, all green receptors in the distal tier were labelled by both PxRh1 and PxRh2 probes. The labelling by the PxRh2 and PxRh3 probes was detected throughout the eye in the proximal tier; in 18 % of ommatidia, the probes labelled the same photoreceptor cell. These results suggest that the possible co-localization of two different visual pigments will broaden the sensitivity spectrum of the photoreceptor cells.

Purpose: Changes in retinal photopigments represent a fundamental step in the evolution of visual systems, in that addition of new pigment types or alterations in the spectral absorption properties of existing pigments modify visual capacities and thus open new visual worlds. To provide a tool that would allow direct examination of the changes caused by the presence of novel photopigments, this study was designed to determine whether a gene encoding a human cone photopigment introduced into the mouse genome would be expressed in a cone-specific manner and would support phototransduction., Methods: Mice transgenic for the human long wavelength-sensitive (L) photopigment were generated by microinjection of fertilized mouse eggs. RNA expression in different tissues was monitored by reverse transcription-polymerase chain reaction analysis. Photopigment protein was localized in retinal cross sections and wholemounts by antibody staining. Light transduction of the cone photopigments was assessed by flicker photometric electroretinography (ERG)., Results: The human transgene was expressed specifically in the mouse cones in quantities comparable to those of the mouse middle wavelength-sensitive (M) pigment gene. Immunocytochemical analysis showed that the human L pigment was abundantly synthesized in most mouse cones, was translocated to the outer segments, and caused no detectable cone degeneration. Electroretinographic spectral sensitivity analysis showed that the human L pigment was efficient in eliciting an electrical response. The degree of expression of the transgene in the two founders correlated well with the spectral responsivity of the ERG., Conclusions: The human L photopigment transduces light efficiently in mouse cones, implying that all protein domains necessary for efficient interaction with intracellular transport and signal transduction machineries in mouse cones have been conserved through evolution. The expression of the human L photopigment gene in both classes of cone of the mouse retina indicates that the transgene did not have the regulatory elements necessary for restricting its expression to mouse M cones or that such elements are not recognized in mouse UV-sensitive cones.

Purpose: To design ribozymes--catalytic RNA molecules--to cleave the P23H and S334Ter mutant mRNA selectively and to test them in vitro to determine their potential as therapeutic agents in the prevention of autosomal dominant retinitis pigmentosa., Methods: Synthetic RNA targets were used in cleavage assays to determine the catalytic efficiencies of the ribozymes in vitro. Cleavage products were analyzed by denaturing polyacrylamide gel electrophoresis. Total retinal RNA was also used as a substrate, and opsin mRNA cleavage was assayed by reverse transcription-polymerase chain reaction., Results: All three ribozymes cleaved the mutant target specifically. Substrate cleavage was seen in less than 5 mM magnesium and was detectable after 15 minutes of incubation. The most active ribozyme against the P23H target was the hammerhead (kcat:K(m) [Michaelis-Menton constant] ratio = 5 x 10(7) M/min), then the P23H hairpin ribozyme (kcat:K(m) ratio = 9 x 10(5) M/min) and the S334Ter hammerhead (kcat:K(m) ratio = 8 x 10(5) M/min). No cleavage activity was observed, when wild-type target sequences or inactive control ribozymes were used. The ribozymes bound and specifically digested the intact mutant opsin mRNA in the presence of all normal retinal RNA., Conclusions: Ribozymes can discriminate between the mutant and wild-type sequences of mRNA associated with autosomal dominant retinitis pigmentosa. The kinetics and specificity of ribozyme cleavage indicate that they should reduce the amount of aberrant rhodopsin in the rod cells and may have potential as therapeutic agents against genetic disease.

11Z-9-Demethyl-9-benzyl- and 9-phenyl-retinals were synthesized stereoselectively from the beta-ionone analog-tricarbonyliron complexes and their interaction with bovine opsin was investigated.

We report here the regeneration of the visual pigments of mouse, rat, goldfish and pigeon, which have wavelengths of maximal absorption at 359 nm, 358 nm, 359 nm, and 393 nm, respectively. The construction and functional assays of the ultraviolet or near-ultraviolet pigments from a wide range of vertebrate species will allow us to study the molecular bases of ultraviolet vision for the first time.

The absorption maxima of both rod and cone visual pigments of the bottlenose dolphin (Tursiops truncatus) are blue-shifted relative to those of terrestrial mammals. A comparison of the sequence of the dolphin rod photopigment gene with that of the bovine rod suggests that, fo the 28 nonidentical amino acids, three amino acid substitutions at positions 83, 292, and 299 in the dolphin rod pigment are responsible for the 10 nm blue shift in absorption maxima. A similar comparison of the dolphin long-wavelength sensitive (LWS) cone photopigment gene with those of the human LWS cones suggests that a single substitution at position 292 (using the convention of rhodopsin numbering) in the dolphin LWS cone pigment results in a blue shift in absorption maxima. A mutagenesis study reveals that the combination of the three dolphin specific substitutions in the bovine rod pigment (83D to 83N, 292A to 292S, and 299A to 299S) causes a blue shift from the wild-type lambdamax of 499 nm to 389 nm. The single substitution in the dolphin LWS cone pigment (292S to 292A) causes a red shift from the wild-type lambdamax of 524 nm to 552 nm. The interactions of the three amino acids identified in the rod pigment with the chromophore may be a general mechanism for blue shifting in rod visual pigments. Furthermore, the single substitution in the dolphin LWS opsin gene is a novel mechanism of wavelength modulation in mammalian LWS pigments.

We have identified an opsin, melanopsin, in photosensitive dermal melanophores of Xenopus laevis. Its deduced amino acid sequence shares greatest homology with cephalopod opsins. The predicted secondary structure of melanopsin indicates the presence of a long cytoplasmic tail with multiple putative phosphorylation sites, suggesting that this opsin's function may be finely regulated. Melanopsin mRNA is expressed in hypothalamic sites thought to contain deep brain photoreceptors and in the iris, a structure known to be directly photosensitive in amphibians. Melanopsin message is also localized in retinal cells residing in the outermost lamina of the inner nuclear layer where horizontal cells are typically found. Its expression in retinal and nonretinal tissues suggests a role in vision and nonvisual photoreceptive tasks, such as photic control of skin pigmentation, pupillary aperture, and circadian and photoperiodic physiology.

The kinetics of the dark-adapted salamander rod photocurrent response to flashes producing from 10 to 10(5) photoisomerizations (Phi) were investigated in normal Ringer's solution, and in a choline solution that clamps calcium near its resting level. For saturating intensities ranging from approximately 10(2) to 10(4) Phi, the recovery phases of the responses in choline were nearly invariant in form. Responses in Ringer's were similarly invariant for saturating intensities from approximately 10(3) to 10(4) Phi. In both solutions, recoveries to flashes in these intensity ranges translated on the time axis a constant amount (tauc) per e-fold increment in flash intensity, and exhibited exponentially decaying "tail phases" with time constant tauc. The difference in recovery half-times for responses in choline and Ringer's to the same saturating flash was 5-7 s. Above approximately 10(4) Phi, recoveries in both solutions were systematically slower, and translation invariance broke down. Theoretical analysis of the translation-invariant responses established that tauc must represent the time constant of inactivation of the disc-associated cascade intermediate (R*, G*, or PDE*) having the longest lifetime, and that the cGMP hydrolysis and cGMP-channel activation reactions are such as to conserve this time constant. Theoretical analysis also demonstrated that the 5-7-s shift in recovery half-times between responses in Ringer's and in choline is largely (4-6 s) accounted for by the calcium-dependent activation of guanylyl cyclase, with the residual (1-2 s) likely caused by an effect of calcium on an intermediate with a nondominant time constant. Analytical expressions for the dim-flash response in calcium clamp and Ringer's are derived, and it is shown that the difference in the responses under the two conditions can be accounted for quantitatively by cyclase activation. Application of these expressions yields an estimate of the calcium buffering capacity of the rod at rest of approximately 20, much lower than previous estimates.

The visual pigment rhodopsin is a prototypical G protein-coupled receptor. These receptors have seven transmembrane helices and are activated by specific receptor-ligand interactions. Rhodopsin is unusual in that its retinal prosthetic group serves as an antagonist in the dark in the 11-cis conformation but is rapidly converted to an agonist on photochemical cis to trans isomerization. Receptor-ligand interactions in rhodopsin were studied in the light and dark by regenerating site-directed opsin mutants with synthetic retinal analogues. A progressive decrease in light-dependent transducin activity was observed when a mutant opsin with a replacement of Gly121 was regenerated with 11-cis-retinal analogues bearing progressively larger R groups (methyl, ethyl, propyl) at the C9 position of the polyene chain. A progressive decrease in light activity was also observed as a function of increasing size of the residue at position 121 for both the 11-cis-9-ethyl- and the 11-cis-9-propylretinal pigments. In contrast, a striking increase of receptor activity in the dark-i.e., without chromophore isomerization-was observed when the molecular volume at either position 121 of opsin or C9 of retinal was increased. The ability of bulky replacements at either position to hinder ligand incorporation and to activate rhodopsin in the dark suggests a direct interaction between these two sites. A molecular model of the retinal-binding site of rhodopsin is proposed that illustrates the specific interaction between Gly121 and the C9 methyl group of 11-cis-retinal. Steric interactions in this region of rhodopsin are consistent with the proposal that movement of transmembrane helices 3 and 6 is concomitant with receptor activation.

We have cloned and sequenced the pineal gland-specific opsin (P opsin) gene, p(Pm), of marine lamprey, P. marinus. The deduced P opsin, P(Pm), consisting of 444 aa, is about 90 aa longer than the orthologous opsins of chicken, pigeon, and American chameleon. The evolution of P(Pm) is characterized not only by a significantly higher rate of aa replacement than the other P opsins but also by a larger number of unique aa replacements at highly conserved sites in the nonTM region. These changes in P(Pm) might have been important in achieving marine lamprey-specific interaction between P(Pm) and other proteins in the pineal gland.

We have found that clay acts as a novel model matrix for the amphiphilic protein-opsin to mimic the visible absorption spectrum of a protonated retinal Schiff base (RSB) in vivo. Without strong acids at ambient temperature, a visible broad absorption spectrum with a lambda max at 530 nm covering the range from 400 to 680 nm was achieved for the protonated RSB with cationic surfactant-modified montmorillonite clay. The interlayers of the dimethyloctadecylamine (DOA) modified clay were found to provide amphiphilic space allowing the amphiphilic RSB to be intercalated easily and sequentially and protonated by the DOA. It is proposed that the visible absorption spectrum at lambda max 530 nm was attributable to electrostatic effects, permitting the appropriate distance between the nitrogen of the protonated RSB and the negatively charged clay interlayers and also to the anisotropic orientation of the RSB molecules in the interlayers.

Multiple sites of extraretinal photoreception are present in vertebrates, but the molecular basis of extraretinal phototransduction is poorly understood. This study reports the cloning of the first opsin specifically expressed in the directly photosensitive pineal and parapineal of cold-blooded vertebrates. This opsin, identified in channel catfish and termed parapinopsin, defines a new gene family of vertebrate photopigments and is expressed in a majority of parapinealocytes and a subset of pineal photoreceptor cells. Parapinopsin shows a caudal-rostral gradient of expression within the pineal organ. This study also reports the cloning of partial cDNAs encoding the channel catfish orthologues of rhodopsin and the red cone pigment-the full complement of retinal opsins in the species. In situ hybridization studies using probes derived from these retinal opsins, together with parapinopsin, reveal no expression of retinal opsins in pineal and parapineal organ and no expression of any opsin tested in the "deep brain," iris, or dermal melanophores. These data imply that phototransduction in these sites of extraretinal photoreception must be mediated by novel opsins.

Through low-temperature spectroscopy and G-protein (transducin) activating experiments, we have investigated molecular properties of chicken blue, the cone visual pigment present in chicken blue-sensitive cones, and compared them with those of the other cone visual pigments, chicken green and chicken red (iodopsin), and rod visual pigment rhodopsin. Irradiation of chicken blue at -196 degrees C results in formation of a batho intermediate which then converts to BL, lumi, meta I, meta II, and meta III intermediates with the transition temperatures of -160, -110, -40, -20, and -10 degrees C. Batho intermediate exhibits an unique absorption spectrum having vibrational fine structure, suggesting that the chromophore of batho intermediate is in a C6-C7 conformation more restricted than those of chicken blue and its isopigment. As reflected by the difference in maxima of the original pigments, the absorption maxima of batho, BL, and lumi intermediates of chicken blue are located at wavelengths considerably shorter than those of the respective intermediates of chicken green, red and rhodopsin, but the maxima of meta I, meta II, and meta III are similar to those of the other visual pigments. These facts indicate that during the lumi-to-meta I transition, retinal chromophore changes its original position relative to the amino acid residues which regulate the maxima of original pigments through electrostatic interactions. Using time-resolved low-temperature spectroscopy, the decay rates of meta II and meta III intermediates of chicken blue are estimated to be similar to those of chicken red and green, but considerably faster than those of rhodopsin. Efficiency in activating transducin by the irradiated chicken blue is greatly diminished as the time before its addition to the reaction mixture containing transducin and GTP increases, while that by irradiated rhodopsin is not. The time profile is almost identical with those observed in chicken red and green. Thus, the faster decay of enzymatically active state is common in cone visual pigments, independent of their spectral sensitivity.

We have cloned and sequenced the red opsin gene, rCh, of goat (Capra hircus). When rCh is expressed in cultured cells and reconstituted with 11-cis retinal, the resulting visual pigment has a wavelength of maximal absorption (lambda max) of 553 nm. This result and the deduced aa sequence of the goat red opsin suggest that the lambda max values of the red pigments of goat and human are based mainly on Y277 and T285. The slightly lower lambda max value of the red pigment in goat than in human (approximately 560 nm) can be explained by a single aa difference between the goat (A180) and human (S180) red pigments.

Three distinct opsin-encoding cDNAs, designated MANOP1, MANOP2 and MANOP3, were isolated from the retina of the sphingid moth Manduca sexta. MANOP1 codes for a protein with 377 amino acid residues. It is similar in sequence to members of a phylogenetic group of long-wavelength-sensitive arthropod photopigments, most closely resembling the opsins of ants, a praying mantis, a locust and the honeybee. MANOP2 and MANOP3 opsins have 377 and 384 residues respectively. They belong to a related group of insect visual pigments that include the ultraviolet-sensitive rhodopsins of flies as well as other insect rhodopsins that are also thought to absorb at short wavelengths. The retina of Manduca sexta contains three rhodopsins, P520, P450 and P357, with absorbance peaks, respectively, at green, blue and ultraviolet wavelengths. There is evidence that MANOP1 encodes the opsin of P520. We suggest that MANOP2 encodes P357 and that MANOP3, representing a class of blue-sensitive insect photopigments, encodes P450.

Resonance Raman spectra of recombinant human green and red cone pigments have been obtained to examine the molecular mechanism of color recognition by visual pigments. Spectra were acquired using a 77 K resonance Raman microprobe or preresonance Raman spectroscopy. The vibrational bands were assigned by comparison to the spectra of bovine rhodopsin and model compounds. The C=NH stretching frequencies of rhodopsin, the green cone pigment, and the red cone pigment in H2O (D2O) are found at 1656 (1623), 1640 (1618), and 1644 cm(-1), respectively. Together with previous resonance Raman studies on iodopsin [Lin, S. W., Imamoto, Y., Fukada, Y., Shichida, Y., Yoshizawa, T., & Mathies, R. A. (1994) Biochemistry 33, 2151-2160], these values suggest that red and green pigments have very similar Schiff base environments, while the Schiff base group in rhodopsin is more strongly hydrogen-bonded to its protein environment. The absence of significant frequency and intensity differences of modes in the fingerprint and the hydrogen out-of-plane wagging regions for all these pigments does not support the hypothesis that local chromophore interactions with charged protein residues and/or chromophore planarization are crucial for the absorption differences among these pigments. However, our data are consistent with the idea that the Schiff base group in blue visual pigments is stabilized by protein and water dipoles and that the removal of this dipolar field shifts the absorption maximum from blue to green. A further red shift of the lambda(max) from the green to the red pigment is successfully modeled by the addition of hydroxyl-bearing amino acids (Ser164, Tyr261, and Thr269) close to the ionone ring that lower the transition energy by interacting with the change of dipole moment of the chromophore upon excitation. The increased hydrogen bonding of the protonated Schiff base group in rhodopsin is predicted to account for the 30 nm blue shift of its absorption maximum compared to that of the green pigment.

We have previously cloned the opsins coding for the long-wavelength visual pigments from the Saharan silver ant and carpenter ant. Here we report two new cDNA clones isolated from cDNA libraries which also code for opsin proteins. These cDNAs code for deduced proteins with 369 amino acids which are 91% identical to each other, but only 38% identical to the previously cloned opsins. Phyletic comparisons suggest that these opsins are likely the ultraviolet sensitive visual pigments, a conclusion that is supported by the presence of a phenylalanine at the counterion position in the third transmembrane segment.

The color vision of mammals is controlled by photosensitive proteins called opsins. Most mammals have dichromatic color vision, but hominoids and Old World (OW) monkeys enjoy trichromatic vision, having the blue-, green-, and red-sensitive opsin genes. Most New World (NW) monkeys are either dichromatic or trichromatic, depending on the sex and genotype. Trichromacy in higher primates is believed to have evolved to facilitate the detection of yellow and red fruits against dappled foliage, but the process of evolutionary change from dichromacy to trichromacy is not well understood. Using the parsimony and the newly developed Bayesian methods, we inferred the amino acid sequences of opsins of ancestral organisms of higher primates. The results suggest that the ancestors of OW and NW monkeys lacked the green gene and that the green gene later evolved from the red gene. The fact that the red/green opsin gene has survived the long nocturnal stage of mammalian evolution and that it is under strong purifying selection in organisms that live in dark environments suggests that this gene has another important function in addition to color vision, probably the control of circadian rhythms.

Premature termination of protein synthesis by nonsense mutations is at the molecular origin of a number of inherited disorders in the family of G protein-coupled seven-helix receptor proteins. To understand how such truncated polypeptides are processed by the cell, we have carried out COS-1 cell expression studies of mutants of bovine rhodopsin truncated at the first 1, 1.5, 2, 3, or 5 transmembrane segments (TMS) of the seven present in wild-type opsin. Our experiments show that successful completion of different stages in the cellular processing of the protein [membrane insertion, N-linked glycosylation, stability to proteolytic degradation, and transport from the endoplasmic reticulum (ER) membrane] requires progressively longer lengths of the polypeptide chain. Thus, none of the truncations affected the ability of the polypeptides to be integral membrane proteins. C-terminal truncations that generated polypeptides with fewer than two TMS resulted in misorientation and prevented glycosylation at the N terminus, whereas truncations that generated polypeptides with fewer than five TMS greatly destabilized the protein. However, all of the truncations prevented exit of the polypeptide from the ER. We conclude that during the biogenesis of rhodopsin, proper integration into the ER membrane occurs only after the synthesis of at least two TMS is completed. Synthesis of the next three TMS confers a gradual increase in stability, whereas the presence of more than five TMS is necessary for exit from the ER.

The photoreceptor cells of the Drosophila compound eye are precisely organized in elementary units called ommatidia. The outer (R1-R6) and inner (R7, R8) photoreceptors represent two physiologically distinct systems with two different projection targets in the brain (for review see Hardie, 1985). All cells of the primary system, R1-R6, express the same rhodopsin and are functionally identical. In contrast, the R7 and R8 photoreceptors are different from each other. They occupy anatomically precise positions, with R7 on top of R8. In fact, there are several classes of R7/R8 pairs, which differ morphologically and functionally and are characterized by the expression of one of two R7-specific opsins, rh3 or rh4. Here, we describe the identification of a new opsin gene, rhodopsin 5, expressed in one subclass of R8 cells. Interestingly, this subclass represents R8 cells that are directly underneath the R7 photoreceptors expressing rh3, but are never under those expressing rh4. These results confirm the existence of two subpopulations of R7 and R8 cells, which coordinate the expression of their respective rh genes. Thus, developmental signaling pathways between R7 and R8 lead to the exclusive expression of a single rhodopsin gene per cell and to the coordinate expression of another one in the neighboring cell. Consistent with this, rh5 expression in R8 disappears when R7 cells are absent (in sevenless mutant). We propose a model for the concerted evolution of opsin genes and the elaboration of the architecture of the retina.

We describe the identification of a novel opsin gene isolated from the eyes of Atlantic salmon. The cDNA sequence predicts a protein that has the key features of an opsin, but shows only 32-42% amino acid identity to the known opsin families. Phylogenetic analysis suggests that this opsin is a member of a hitherto unrecognised opsin family that diverged early in the evolution of vertebrate photopigments. We have tentatively called this opsin family the vertebrate ancient (VA) opsins. The identification of VA opsin may ultimately help to resolve some of the uncharacterised photoreceptor functions of the eye, which include the regulation of circadian rhythms, pupil size and corneal pigmentation.

The chicken pineal gland has an endogenous circadian oscillator that controls the diurnal oscillation of N-acetyltransferase activity responsible for melatonin rhythm. It has been speculated that the chicken pineal cell contains a photoreceptive molecule that receives the environmental light signal and transmits the signal to the oscillator for resetting the phase. In spite of several lines of evidence suggesting the similarity between retinal and pineal photon-signal transducing proteins, the identity of the photoreceptive molecule had been an open question. In 1994, we isolated a pineal cDNA encoding a novel photoreceptive molecule and named it "pinopsin." The protein expressed in 293EBNA cells bound 11-cis-retinal to form a blue-sensitive pigment with an absorption maximum at about 470 nm. A putative G-protein interaction site of pinopsin shared a relatively high similarity in amino acid sequence to that of rhodopsin, implying that pinopsin functionally couples with transducin or transducin-like G-protein(s) in the pineal cells. We have cloned a cDNA for chicken pineal transducin alpha-subunit, and the deduced amino acid sequence contained a potential site to be ADP-ribosylated by pertussis toxin (PTX). Therefore, the transducin-mediated pathway could be blocked by PTX, though previous studies showed that treatment of the cultured chicken pineal cells with PTX had no effect on the light-induced phase-shift of the oscillator. Accordingly, it is unlikely that transducin mediates the light-input pathway to the oscillator, which may involve PTX-insensitive G-protein(s) or some unidentified component(s). The G-protein coupled receptor-mediated signaling processes regulating melatonin synthesis are discussed.

The sequences of the entire blue opsin gene in the squirrel monkey (Saimiri boliviensis) and the five introns of the human blue opsin gene were obtained. Intron 3 of these genes contains an Alu sequence and intron 4 contains a partial mer13 sequence. A comparison of the squirrel monkey opsin sequence with published mammalian opsin sequences shows that features believed to be functionally critical are all conserved. However, the blue opsin has evolved twice as fast as rhodopsin and is only as conservative as the beta globin, which has evolved at the average rate of mammalian proteins. Interestingly, the interhelical loops are, on average, actually more conservative than the transmembrane alpha helical regions. The introns of the blue opsin gene have evolved at the average rate of introns in primate genes.

The sequences of two locust opsins have been determined by dideoxy nucleotide sequencing of PCR products from cDNA derived from eyecup tissue. The opsins (Lo1 and Lo2) are encoded by 381 and 380 amino acid residues, respectively, with hydropathy profiles and placement of key amino acid residues suggestive of a typical seven-transmembrane rhodopsin structure. The sequence alignment of Lo1 reveals significant homology to mantid opsin. These opsins contain retinal as their visual chromophore and have similarity to the Rh1 type sequences from Drosophila and Calliphora which use 3-hydroxy retinal. Lo2 is most closely related to the Rh3/4 type of visual pigments from Drosophila. The retinal-based opsins show reduced numbers of charged amino acids in the loop region connecting transmembrane segments V and VI compared to the 3-hydroxy retinal opsins. Sequence alignment of all the known insect visual pigments has shown that only those with maximal sensitivity in the blue/UV spectral range, Lo2 and the Rh3/4 opsins of Drosophila, have three charged amino acids in transmembrane segments II, IV and VII. The charged residue in transmembrane VII is two helical turns away from the positively charged Schiff base and could act directly as a counterion to it. From the secondary structure analysis of opsin, the two charged residues in transmembrane II and IV would be in close proximity to form a dipole. These polar motifs in Lo2 and Rh3/Rh4 could act in wavelength modulation of short wavelength sensitive pigments and substantiate the proposed external two-point charge model which accounts for the spectral sensitivity of visual pigments [Honig, B., Dinur, U., Nakanishi, K., Balogh-Nair, V., Gawinowicz, M.A. and Motto, M. (1979). Journal of the American Chemical Society, 101, 7084-7086].

Flagellate green algae such as Chlamydomonas and related genera are guided by their eyes to places where light conditions are optimal for photosynthetic growth. These eyes constitute the simplest and most common visual system found in nature. The eyes contain optics, photoreceptors and the elementary components of a signal-transduction chain. Rhodopsin serves as the photoreceptor, as it does in animal vision. Upon light stimulation, its all-trans-retinal chromophore isomerizes into 13-cis and activates a photoreceptor channel which leads to a rapid Ca2+ influx into the eyespot region. At low light levels, the depolarization activates small flagellar currents which induce in both flagella small but slightly different beating changes resulting in distinct directional changes. In continuous light, Ca2+ fluxes serve as the molecular basis for phototaxis. In response to flashes of higher energy the larger photoreceptor currents trigger a massive Ca2+ influx into the flagella which causes the well-known phobic response. The identification of proteins contributing to this signalling system has just begun with the isolation and cloning of the opsins from Chlamydomonas and Volvox. These plant opsins are highly charged, are not typical seven-helix receptors, and are believed to form a protein complex with the photoreceptor channel. In Spermatozopsis, a G-protein has been found which interacts either directly with the rhodopsin or with the rhodopsin-ion channel complex. By using insertional mutagenesis, genes coding for proteins that are involved in signalling have been tagged. One of them is connected to the flagellar channel and crucial for the flagellar action potential. Elucidation of photoreception in flagellated algae will provide deeper insight into the development of visual systems, starting from single-celled organisms and moving up through higher animals.

The chicken gene encoding pineal gland-specific opsin (P-opsin) has been previously characterized. We report here the orthologous pigeon (Columba livia) P-opsin gene. The deduced pigeon P-opsin lacks a potential N-glycosylation site in the N-terminus, but has multiple phosphorylation sites in the C-terminus, which are opposite from the characteristics of the chicken P-opsin.

The yeast Saccharomyces cerevisiae has been investigated for expression of mammalian opsin as an alternative to the currently used expression in COS-1 mammalian cells. The synthetic opsin gene was placed under the control of the inducible promoter GAL1 in the multicopy yeast/ Escherichia coli shuttle vector YEpRF1. Transformation of a GAL+ S. cerevisiae strain with the vector and growth of galactose-induced cultures to saturation showed the production of 2.0 +/- 0.5 mg of opsin from about 10(10) cells by ELISA. The addition of 11-cis-retinal to either cell spheroplasts or lysed cells showed that a fraction (2-4%) of the total expressed opsin reconstituted to rhodopsin. This fraction was purified to homogeneity and was shown to be fully functional and indistinguishable from bovine rhodopsin by the following criteria: (i) UV-visible absorption spectra, (ii) the formation of metarhodopsin II and its rate of decay, and (iii) initial rate of transducin activation as measured by the formation of a complex between transducin (alpha subunit) and guanosine 5'-[gamma-[35S]thio]triphosphate. The purified fraction was homogeneously glycosylated. However, glycosylation was distinct from that of bovine rhodopsin as judged by mobility on SDS/PAGE and endoglycosidase H sensitivity.

The vertebrate opsins have been classified into four major phylogenetic groups. One of them, a short wavelength-sensitive (SWS) opsin group, is further divided into two subgroups, SWS-I and SWS-II, having the wavelengths of maximal absorption of about 420 and 450 nm, respectively. Here we report the DNA sequences of the SWS-I and SWS-II genes from the lizard Anolis carolinensis. The shorter wavelengths of absorption by the two SWS subgroup opsins seem to be achieved by different sets of amino acid replacements in the transmembrane regions.

The conger eel (Conger conger) is a nocturnal fish that can be found living in shallow coastal water and deep water down to 1000 m. The conger eel has a pure rod retina with a visual pigment maximally sensitive to blue light around 487 nm. We have cloned and sequenced the opsin cDNA which is presumed to code for this visual pigment and have found it to be highly homologous to the form of opsin that is expressed in mature, deep-living European eels (Anguilla anguilla). The opsin sequence information presented here provides additional evidence that specific amino acid sites are involved in the spectral tuning of this class of blue-sensitive visual pigments.

A mutation in the gene for the rod photoreceptor molecule rhodopsin causes congenital night blindness. The mutation results in a replacement of Gly90 by an aspartic acid residue. Two molecular mechanisms have been proposed to explain the physiology of affected rod cells. One involves constitutive activity of the G90D mutant opsin [Rao, V. R., Cohen, G. B., & Oprian, D. D. (1994) Nature 367, 639-642]. A second involves increased photoreceptor noise caused by thermal isomerization of the G90D pigment chromophore [Sieving, P. A., Richards, J. E., Naarendorp F., Bingham, E. L., Scott, K., & Alpern, M. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 880-884]. Based on existing models of rhodopsin and in vitro biochemical studies of site-directed mutants, it appears likely that Gly90 is in the immediate proximity of the Schiff base chromophore linkage. We have studied in detail the mutant pigments G90D and G90D/E113A using biochemical and Fourier-transform infrared (FTIR) spectroscopic methods. The photoproduct of mutant pigment G90D, which absorbs maximally at 468 nm and contains a protonated Schiff base linkage, can activate transducin. However, the active photoproduct decays rapidly to opsin and free all-trans-retinal. FTIR studies of mutant G90D show that the dark state of the pigment has several structural features of metarhodopsin II, the active form of rhodopsin. These include a protonated carboxylic acid group at position Glu113 and increased hydrogen-bond strength of Asp83. Additional results, which relate to the structure of the active G90D photoproduct, are also reported. Taken together, these results may be relevant to understanding the molecular mechanism of congenital night blindness caused by the G90D mutation in human rhodopsin.

The retinal pigment epithelium (RPE) contains an abundant opsin that is distinct from rhodopsin and cone visual pigments and is able to bind the retinaldehyde chromophore. The putative retinal G protein-coupled receptor (RGR) was isolated in digitonin solution from bovine RPE microsomes and copurified consistently with a minor 34-kDa protein. The absorption spectrum of RGR revealed endogenous pH-sensitive absorbance in the blue and near-ultraviolet regions of light. Membrane-bound RGR was incubated with exogenously added all-trans-retinal and formed two long-lived pH-dependent photopigments with absorption maxima of 469 +/- 2.4 and 370 +/- 7.3 nm. The effects of hydrogen ion concentration suggest that the blue and near-UV photopigments are tautomeric forms of RGR, in which an all-trans-retinal Schiff base is protonated or unprotonated, respectively. The RPE pigment was also demonstrable by its reactivity to hydroxylamine in the dark. The retinaldehyde-RGR conjugate at neutral pH favors the near-UV pigment and is a novel light-absorbing opsin in the vertebrate eye.

Lake Baikal in Eastern Siberia is the deepest and one of the largest and most ancient lakes in the world. However, even in the deepest regions, oxygenation levels do not fall below 75-80% of the surface levels. This has enabled a remarkable flock of largely endemic teleost fish of the sub-order Cottoidei to colonize all depth habitats. We have previously shown that species that occupy progressively deeper habitats show a blue shift in the peak wavelength of absorbance (lambda max) of both their rod and cone visual pigments; for the rod pigments, a number of stepwise shifts occur from about 516 nm in littoral species to about 484 nm in abyssal species. By sequencing the rod opsin gene from 11 species of Baikal cottoids that include representatives from all depth habitats, we have been able to identify four amino acid substitutions that would account for these shifts. The effect of each substitution on lambda max is approximately additive and each corresponds to a particular lineage of evolution.

Previous work on the expression of bovine opsin fragments separated in the cytoplasmic region has allowed the identification of specific polypeptide segments that contain sufficient information to fold independently, insert into a membrane, and assemble to form a functional photoreceptor. To further examine the contributions of these and other polypeptide segments to the mechanism of opsin folding and assembly, we have constructed 20 additional opsin gene fragments where the points of separation occur in the intradiscal, transmembrane, and cytoplasmic regions. Nineteen of the fragments were stably expressed in COS-1 cells. A five-helix fragment was stably produced only after coexpression with its complementary two-helix fragment. Two fragments composed of the amino-terminal region and the first transmembrane helix were not N-glycosylated and were only partially membrane integrated. One of the singly expressed fragments, which is truncated after the retinal attachment site, bound 11-cis-retinal. Of the coexpressed complementary fragments, only those separated in the second intradiscal and third cytoplasmic regions formed noncovalently linked rhodopsin. Both of the pigments showed reduced transducin activation. Therefore, while many opsin fragments contain enough information to fold and insert into a membrane, only those separated at specific locations assemble to a retinal-binding opsin.

In order to find optimal light conditions for photosynthetic growth, the green alga Chlamydomonas uses a visual system. An optical device, a rhodopsin photoreceptor and an electrical signal transduction chain that mediates between photoreceptor and flagella comprise this system. Here we present an improved strategy for the preparation of eyespot membranes. These membranes contain a retinal binding protein, which has been proposed to be the apoprotein of the phototaxis receptor. The retinal binding protein, which we named chlamyopsin, was purified and opsin-specific antibodies were raised. Using these antibodies, the opsin was localized in the eyespot region of whole cells during growth and cell division. The opsin cDNA was purified and sequenced. The sequence reveals that chlamyopsin is not a typical seven helix receptor. It shows some homology to invertebrate opsins but not to opsins from halobacteria. It contains many polar and charged residues and might function as a light-gated ion channel complex. It is likely that this lower plant rhodopsin diverged from animal opsins early in opsin evolution.

Mutation of Gly90, Glu113, Ala292, and Lys296 in the visual pigment rhodopsin constitutively activates the protein for activation of the G protein transducin. Three of these mutations have been shown to cause two different human diseases. Mutation of Gly90 and Ala292 results in complete night blindness, and mutation of Lys296 results in the degenerative disease retinitis pigmentosa. We show here that the mutants not only constitutively activate transducin but are also constitutively activated for phosphorylation by rhodopsin kinase. In addition, the phosphorylated mutants are shown to bind tightly to the inhibitory protein arrestin in a reaction that quenches the activity toward transducin. Thus the same mutations that result in constitutive activation of transducin also result in constitutive phosphorylation by rhodopsin kinase and binding of arrestin to inhibit the activity. This implies that the same conformational change may be responsible for activation of transducin and rhodopsin kinase. It also suggests that degeneration of photoreceptor cells in retinitis pigmentosa results indirectly from the activated state of the receptor, perhaps as a consequence of phosphorylation and persistent binding of arrestin.

In the retinal degeneration (rd) mouse the absence of rod cells and the progressive loss of cones does not result in a decrease in circadian phase shifting responses to light. By contrast, rd/rd mice are unable to perform simple visual tasks. In addition, rodless transgenic mice, and mice homozygous for the retinal degeneration slow (rds) mutation, show unattenuated circadian responses to light. Collectively these data suggest that cone cells lacking outer segments are sufficient to maintain normal circadian responses to light, or some unidentified photoreceptor within the retina. An action spectrum for circadian responses to light in rd/rd mice, and molecular analysis of retinally degenerate mice and blind mole rat eyes, suggests the involvement of a mid-to-long wavelength sensitive cone opsin in photoentrainment. Extraocular photoreceptors of non-mammalian vertebrates are currently being analyzed in order to identify functional and evolutionary similarities between visual and non-visual photoreceptor systems.

The hydrogen out-of-plane bending (HOOP) vibrations were studied in the difference Fourier transform infrared spectra of lumirhodopsin and metarhodopsin I by use of a series of specifically deuterated retinal derivatives of bovine rod outer segments. The 947 cm-1 band of lumirhodopsin and the 950 cm-1 band of metarhodopsin I were assigned to the mode composed of both 11-HOOP and 12-HOOP vibrations. This result suggests that the perturbation near C12-H of the retinal in the earlier intermediate, bathorhodopsin (Palings, van den Berg, Lugtenburg and Mathies, Biochemistry, 28 (1989) 1498-1507), is extinguished in lumirhodopsin and metarhodopsin I. Unphotolyzed rhodopsin and metarhodopsin I exhibited the 14-HOOP bands in the 12-D derivatives at 901 and 886 cm-1, respectively. Lumirhodopsin, however, did not show the 14-HOOP in the 12-D derivatives. The result suggests a change in geometrical alignment of the C14-H bond in lumirhodopsin with respect to the N-H bond of the Schiff base.

Recent molecular genetic studies show how changes in the protein component of a visual pigment alters its absorbance; they also explain the abnormal colour vision of one of the great pioneers of visual science.

For rapid single-step purification of recombinant rhodopsin, a baculovirus expression vector was constructed containing the bovine opsin coding sequence extended at the 3'-end by a short sequence encoding six histidine residues. Recombinant baculovirus-infected Spodoptera frugiperda cells produce bovine opsin carrying a C-terminal histidine tag (v-opshis6x). The presence of this tag was confirmed by immunoblot analysis. Incubation with 11-cis-retinal produced a photosensitive pigment (v-Rhohis6x) at a level of 15-20 pmol/10(6) cells. The histidine tag was exploited to purify v-Rhohis6x via immobilized metal affinity chromatography. Optimized immobilized metal affinity chromatography yielded a binding capacity of > or = 35 nmol of v-Rhohis6x per ml of resin and purification factors up to 500. Best samples were at least 85% pure, with an average purity of 70% (A280 nm/A500 nm = 2.5 +/- 0.4, n = 7). Remaining contamination was largely removed upon reconstitution into lipids, yielding rhodopsin proteoliposomes with a purity over 95%. Spectral analysis of v-Rhohis6x showed a small but significant red shift (501 +/- 1 nm) compared to wild type rhodopsin (498 +/- 1 nm). The pK alpha of the Meta I<==>Meta II equilibrium in v-Rhohis6x is down-shifted from 7.3 to 6.4 resulting in a significant shift at pH 6.5 toward the Meta I photointermediate. Both effects are reversed upon increasing the ionic strength. FT-IR analysis of the Rho-->Meta II transition shows that the corresponding structural changes are identical in wild type and v-Rhohis6x.

Pineal opsin (P-opsin), an opsin from chick that is highly expressed in pineal but is not detectable in retina, was cloned by the polymerase chain reaction. It is likely that the P-opsin lineage diverged from the retinal opsins early in opsin evolution. The amino acid sequence of P-opsin is 42 to 46 percent identical to that of the retinal opsins. P-opsin is a seven-membrane spanning, G protein-linked receptor with a Schiff-base lysine in the seventh membrane span and a Schiff-base counterion in the third membrane span. The primary sequence of P-opsin suggests that it will be maximally sensitive to approximately 500-nanometer light and produce a slow and prolonged phototransduction response consistent with the nonvisual function of pineal photoreception.

The vast diversity in spectral sensitivities in the vision of many organisms is mediated mostly (although not entirely) through variation in the photosensitive visual pigments (opsins) of the eye. Specifically, shifts in absorption maxima of visual pigments are thought to be a result of interactions within the binding pocket of the opsin, between amino acid side chains and the retinal chromophore, However, it has proven difficult to identify specific amino acid residues important in determining wavelength absorption maxima, especially for some of the short wavelength (blue) opsins. In this paper, a comparative phylogenetic approach was applied to opsin protein sequence data to identify residues important in opsin wavelength regulation. In essence, this approach consisted of interpreting evolutionary history as a series of experiments in which natural selection has repeatedly favored amino acid replacements of certain residues to shift the opsin absorption spectra to either shorter or longer wavelengths. Opsin protein sequences were obtained from GenBank, aligned, and used to reconstruct a phylogenetic tree. Amino acid replacements were traced along the branches of this opsin tree, focusing only on residues likely to reside within the chromophore-binding pocket. A number of functionally convergent, nonconservative amino acid replacements in independently evolved opsins with similar shifts in spectral properties were identified. In short, reconstruction of the phylogeny of the opsin molecule allowed us to track amino acid substitutions in specific sites within the opsin and to target those particular substitutions that are repeatedly associated with marked changes in peak absorbance, shifting the spectral sensitivity of the opsin toward shorter or longer wavelengths. Based on these results, we propose a model for blue shifts of opsin absorption spectra. Amino acid replacements of four polar and charged residues near the protonated Schiff base (SBH+) end of the chromophore are proposed to result in blue shifts of the opsin absorption spectra. This model may explain some of the diversity of blue opsins apparent in both vertebrates and invertebrates.

An important unanswered question in phototransduction is how visual pigments (VPs) regulate their wavelength of maximal absorption (lambda max). By constructing the evolutionary tree for 28 opsins with known lambda max values, we can identify the times and directions of lambda max shift of different VPs. A total of 55 amino acid changes are shown to correlate with the directions of lambda max shift and might have been important in determining lambda max of a VP. Among these, three amino acid changes are already proven to be responsible in modifying the green-sensitive VP to the red-sensitive VP. The present evolutionary analysis opens a new direction in understanding the mechanism for the regulation of wavelength absorption by a VP and, more generally, in studying molecular mechanism involved in adaptive evolution.

In avian pinealocytes, an environmental light signal resets the phase of the endogenous circadian pacemaker that controls the rhythmic production of melatonin. Investigation of the pineal phototransduction pathway should therefore reveal the molecular mechanism of the biological clock. The presence of rhodopsin-like photoreceptive pigment, transducin-like immunoreaction, and cyclic GMP-dependent cation-channel activity in the avian pinealocytes suggests that there is a similarity between retinal rod cells and pinealocytes in the phototransduction pathway. We have now cloned chicken pineal cDNA encoding the photoreceptive molecule, which is 43-48% identical in amino-acid sequence to vertebrate retinal opsins. Pineal opsin, produced by transfection of complementary DNA into cultured cells, was reconstituted with 11-cis-retinal, resulting in formation of a blue-sensitive pigment (lambda max approximately 470 nm). In the light of this functional evidence and because the gene is specifically expressed only in the pineal gland, we conclude that it is a pineal photosensor and name it pinopsin.