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PubMed1999

A back-propagation neural network predicts absorption maxima of chimeric human red/green visual pigments.

The absorption spectra of human red and green visual pigments have peak wavelengths, lambda max, that differ by 31 nm, yet the opsins differ in only 15 amino acids. Mutagenesis studies have demonstrated that seven of the 15 amino acids determine the spectral shift. We trained neural networks to predict the lambda max of any red/green chimeric protein. Seven mutants were excluded from the original training set. The trained networks were able to predict the lambda max for the excluded mutants. As an additional test, five new chimeric pigments were constructed and lambda max determined. The neural networks correctly predicted the lambda max of all five mutants. The use of neural networks is a novel approach to the problem of wavelength modulation in visual pigments.

Amino Acids↗
PubMed1995

Amino acid replacements and wavelength absorption of visual pigments in vertebrates.

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.

Amino Acid Sequence↗
PubMed1995

Gene conversion between red and defective green opsin gene in blue cone monochromacy.

Blue cone monochromacy is an X-linked condition in which the function of both the red pigment gene (RCP) and the green pigment gene (GCP) is impaired. Blue cone monochromacy can be due to a red/green gene array rearrangement existing of a single red/green hybrid gene and an inactivating C203R point mutation in GCP. We describe here a family with blue cone monochromacy due to the presence of the C203R mutation in both RCP and GCP. The flanking sequences of the C203R mutation in exon 4 of RCP were characteristic for GCP, indicating that this mutation was transferred from GCP into RCP by gene conversion.

Base Sequence↗
PubMed1994

Phylogeny and physiology of Drosophila opsins.

Phylogenetic and physiological methods were used to study the evolution of the opsin gene family in Drosophila. A phylogeny based on DNA sequences from 13 opsin genes including representatives from the two major subgenera of Drosophila shows six major, well-supported clades: The "blue opsin" clade includes all of the Rh1 and Rh2 genes and is separated into two distinct subclades of Rh1 sequences and Rh2 sequences; the ultraviolet opsin clade includes all Rh3 and Rh4 genes and bifurcates into separate Rh3 and Rh4 clades. The duplications that generated this gene family most likely took place before the evolution of the subgenera Drosophila and Sophophora and their component species groups. Numerous changes have occurred in these genes since the duplications, including the loss and/or gain of introns in the different genes and even within the Rh1 and Rh4 clades. Despite these changes, the spectral sensitivity of each of the opsins has remained remarkably fixed in a sample of four species representing two species groups in each of the two subgenera. All of the strains that were investigated had R1-6 (Rh1) spectral sensitivity curves that peaked at or near 480 nm, R7 (Rh3 and Rh4) peaks in the ultraviolet range, and ocellar (Rh2) peaks near 420 nm. Each of the four gene clades on the phylogeny exhibits very conservative patterns of amino acid replacement in domains of the protein thought to influence spectral sensitivity, reflecting strong constraints on the spectrum of light visible to Drosophila.

Amino Acid Sequence↗
PubMed1987

Multiple topogenic sequences in bovine opsin.

Bovine opsin, a polytopic integral membrane protein, contains seven transmembrane segments connecting eight hydrophilic domains alternating on each side of the membrane. To localize topogenic sequences that might specify the distinct topology of opsin in the membrane, we constructed various opsin mutants, each containing only one transmembrane segment. Messenger RNAs transcribed from these mutants were translated in a cell-free system supplemented with microsomal membranes. Among six of the seven transmembrane segments of opsin that were analyzed, five were able to function as signal sequences and also expressed stop-transfer sequences of variable strength. By the criteria of extractability at pH 11 and protease sensitivity, the presence of a signal sequence in combination with a "strong" stop-transfer sequence yielded integration into the lipid bilayer of the majority of chains. However, in combination with a "weak" stop-transfer sequence, we observed integration into the lipid bilayer of only some chains, with the others either completely translocated across the membrane or retained in a water-accessible space in the membrane.

Amino Acid Sequence↗
PubMed1990

Analysis of cis-acting requirements of the Rh3 and Rh4 genes reveals a bipartite organization to rhodopsin promoters in Drosophila melanogaster.

The rhodopsin genes of Drosophila melanogaster are expressed in nonoverlapping subsets of photoreceptor cells within the insect visual system. Two of these genes, Rh3 and Rh4, are known to display complementary expression patterns in the UV-sensitive R7 photoreceptor cell population of the compound eye. In addition, we find that Rh3 is expressed in a small group of paired R7 and R8 photoreceptor cells at the dorsal eye margin that are apparently specialized for the detection of polarized light. In this paper we present a detailed characterization of the cis-acting requirements of both Rh3 and Rh4. Promoter deletion series demonstrate that small regulatory regions (less than 300 bp) of both R7 opsin genes contain DNA sequences sufficient to generate their respective expression patterns. Individual cis-acting elements were further identified by oligonucleotide-directed mutagenesis guided by interspecific sequence comparisons. Our results suggest that the Drosophila rhodopsin genes share a simple bipartite promoter structure, whereby the proximal region constitutes a functionally equivalent promoter "core" and the distal region determines cell-type specificity. The expression patterns of several hybrid rhodopsin promoters, in which all or part of the putative core regions have been replaced with the analogous regions of different rhodopsin promoters, provide additional evidence in support of this model.

Animals↗
PubMed1992

Effects of RPE-cell factors secreted from permselective fibers on retinal cells in vitro.

This study was undertaken to determine if retinal pigment epithelial (RPE) cells encased in permselective hollow fibers survive in a tissue culture environment and secrete a diffusible trophic factor(s) that may affect retinal cell survival in vitro. In this study, RPE cells were isolated from 6- to 8-day-old Long-Evans rats, then loaded into hollow fibers. The RPE-cell fibers were then cultured for at least one week in serum-containing medium. These RPE-cell fibers were subsequently co-cultured with cells isolated from retinas of day 2 Long-Evans rats in a defined medium. For at least 6 days in culture, opsin-positive cells were observed on the surface of larger flat cells. Over 80% of the small, round cells immunostained for opsin. However, opsin-immunostained cells were seldom seen in cultures with control fibers, that lacked RPE cells. In addition, conditioned medium collected from either the RPE-cell fibers or cultured RPE cells affected survival of opsin-positive retinal cells in culture in a manner similar to that of the RPE-cell fibers. Furthermore, selected growth factors such as epidermal, nerve and fibroblast growth factors, were unable to sustain retinal cell survival and affect morphological development as seen in RPE-CM supplemented cultures. In vivo companion developmental studies demonstrated that few opsin-positive cell bodies were observed in retinas of day 2 Long-Evans rats, the age corresponding to the stage of retinal cell isolation. In retinas of day 5 Long-Evans rats, the age corresponding to the end point of the in vitro assay, a dramatic increase in the number of opsin-immunostained cell bodies was noted, which corresponds to the developmental sequence also seen in culture. Light and electron microscopic examination revealed that the RPE cells cultured in the hollow tubes maintained an RPE-like structure for several months, in that these cells contained melanosomes and extended microvilli from their apical border and formed junctional complexes with adjacent cells. Results of this study confirm our earlier findings that RPE cells secrete an apparent novel factor(s) that affects retinal cell survival in vitro and, most significantly, the described encapsulation/secretion mechanism may provide a convenient method to deliver such factors for further in vivo testing of this phenomenon.

Animals↗
PubMed2000

Molecular evolution of vertebrate visual pigments.

Dramatic improvement of our understanding of the genetic basis of vision was brought by the molecular characterization of the bovine rhodopsin gene and the human rhodopsin and color opsin genes (Nathans and Hogness, 1983; Nathans et al., 1984, 1986a,b). The availability of cDNA clones from these studies has facilitated the isolation of retinal and nonretinal opsin genes and cDNA clones from a large variety of species. Today, the number of genomic and cDNA clones of opsin genes isolated from different vertebrate species exceeds 100 and is increasing rapidly. The opsin gene sequences reveal the importance of the origin and differentiation of various opsins and visual pigments. To understand the molecular genetic basis of spectral tuning of visual pigments, it is essential to establish correlations between a series of the sequences of visual pigments and their lambda(max) values. The potentially important amino acid changes identified in this way have to be tested whether they are in fact responsible for the lambda(max)-shifts using site-directed mutagenesis and cultured cells. A major goal of molecular evolutionary genetics is to understand the molecular mechanisms involved in functional adaptations of organisms to different environments, including the mechanisms of the regulation of the spectral absorption. Therefore, both molecular evolutionary analyses of visual pigments and vision science have an important common goal.

Animals↗
PubMed1996

Structure and function in rhodopsin: expression of functional mammalian opsin in Saccharomyces cerevisiae.

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.

Animals↗
PubMed1989

Morphological defects in oraJK84 photoreceptors caused by mutation in R1-6 opsin gene of Drosophila.

The Drosophila mutant, oraJK84, lacks rhabdomeres in the major (R1-6) class of photoreceptors because these rhabdomeres rapidly degenerate in young flies. Genetic analysis reveals that oraJK84 actually contains two mutations (a ninaE and an ort allele) that affect the visual process. The mutation in ort appears to have no effect on photoreceptor structure. The other mutation occurs within the ninaE gene, which encodes the species of rhodopsin found in the R1-6 class of photoreceptors. Our analysis shows that this mutation is responsible for R1-6 rhabdomere degeneration in oraJK84 mutants. We also examined a ninaE mutant, denoted ninaEo117, that produces no ninaE transcript. The morphological phenotype observed in ninaEo117 is similar to that seen in oraJK84 mutants. We conclude that rhodopsin plays a vital role in maintaining photoreceptor structure in Drosophila.

Animals↗
PubMed2000

Green cone opsin and rhodopsin regulation by CNTF and staurosporine in cultured chick photoreceptors.

PURPOSE: To investigate the regulation of visual pigment expression in chick embryo photoreceptor cells by ciliary neurotrophic factor (CNTF), and by the protein kinase inhibitor staurosporine. METHODS: Embryonic day (ED) 8 chick embryo retinal cells were dissociated and cultured at low densities for 3 days, either in control medium or in medium supplemented with CNTF or staurosporine. The cultures were analyzed by immunocytochemistry with the monoclonal antibody Rho4D2, which recognizes chicken rhodopsin and green cone pigment, and by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis to investigate visual pigment expression at the mRNA level. RESULTS: CNTF increased the number of Rho4D2-immunoreactive photoreceptors in retinal cell cultures, in agreement with previous reports. RT-PCR and Northern blot analysis, however, showed that rhodopsin mRNA was undetectable in both control and CNTF-treated cultures but that CNTF induced significant increases in mRNA levels for the green cone pigment. Staurosporine-treated cultures also had more Rho4D2-immunoreactive cells than control cultures, but this increase was accompanied by induction of rhodopsin expression, with concomitant decreases in levels of green cone pigment mRNA. No significant differences were found between CNTF- or staurosporine-treated cultures and the corresponding control cultures regarding the red cone pigment, which was expressed in all cases, and the blue and violet pigments, which were not detected in any of the samples. CONCLUSIONS: The results suggest that multiple regulatory systems control visual pigment expression during differentiation of chick embryo photoreceptor cells. CNTF appears to stimulate specifically the differentiation of green cones, without the previously suggested effects on the differentiation of rod photoreceptors in ED 8 chick retinal cultures.

Animals↗
PubMedDate not supplied

Cone visual pigments of the Australian marsupials, the stripe-faced and fat-tailed dunnarts: sequence and inferred spectral properties.

Studies of color vision in marsupial mammals have been very limited. Two photoreceptor genes have been characterized from the tammar wallaby, but a third cone pigment was suggested by microspectrophotometric measurements on cone photoreceptors in two other species, including the fat-tailed dunnart, Sminthopsis crassicaudata. To determine the sequence and infer absorption maxima of the cone photoreceptor pigments of S. crassicaudata and the related stripe-faced dunnart (Sminthopsis macroura), we have used evolutionarily conserved sequences of the cone pigments of other species, including the tammar wallaby, to design primers to amplify the S. macroura and S. crassicaudata pigment sequences by the polymerase chain reaction (PCR) using genomic DNA or retinal cDNA as a template. These primers will be useful for amplifying cone opsin coding sequences from a variety of vertebrates. Amplified products were directly sequenced to determine gene structure and coding sequences. The inferred amino acid sequences of the cone visual pigments indicated that both species have middle-wave-sensitive (MWS) pigments with a predicted absorption maximum (lambda(max)) at 530 nm, and ultraviolet-sensitive (UVS) pigments with a predicted lambda(max) at 360 nm. The MWS pigments of the two species differ by two, and UVS by three amino acid positions. No evidence was obtained for a third cone pigment in either species.

Amino Acid Sequence↗
PubMed2004

Evolving eyes.

Despite the incredible diversity among extant eyes, laws of physics constrain how light can be collected resulting in only eight known optical systems in animal eyes. Surprisingly, all animal eyes share a common molecular strategy using opsin for catching photons, but there are a diverse collection of mechanisms with proteins unrelated to each other used to focus light for vision. However, opsin is expressed in either one of two types of photoreceptor that differ fundamentally in their structure and tissue of origin. Taken together, this collection of observations strongly suggests that eyes have had multiple origins with remarkable convergence due to physics and molecular conservation of the opsin protein. Yet recent work has shown that a family of conserved genes are involved in eye formation despite substantial differences in their structure and origin, leading to a controversy over whether eyes evolved once or repeatedly. A likely resolution of this discussion is that particular genes and genetic programs have become associated with specific features needed for eyes and such suites of genes have been recruited as new eyes evolve. Since specific genes and their products are used repeatedly, it is somewhat difficult to conceptualize their causal relationships relative to evolutionary processes. However, detailed comparison of developmental programs may offer clues about multiple origins.

Animals↗
PubMed2002

Variations in long- and middle-wavelength-sensitive opsin gene loci in crab-eating monkeys.

We analyzed variations in long (L)- and middle (M)-wavelength-sensitive opsin gene loci in crab-eating monkeys. Unlike humans, most monkeys have a single L and a single M gene. Two variant genotypes, one with only one opsin gene (dichromatic) and one with tandemly arrayed multiple genes, were also found in the monkeys. However, the frequency of the former was 0.47%, and that of the latter was 5% in the monkeys, while 2% and 66%, respectively, in Caucasian males. The two variants were found only in Java Island, Indonesia, and South Thailand, respectively. The data suggest that the frequency of each genotype is different among Old World primates.

Animals↗
PubMed2004

Detection of pairwise residue proximity by covariation analysis for 3D-structure prediction of G-protein-coupled receptors.

G-protein-coupled receptor (GPCR) is one of the most important targets for medicines. Homology modeling based on the crystal structure of bovine rhodopsin is currently the most frequently used method for GPCR targeted drug design. Information about residue-residue contacts and the structural specificity in the subfamily is essential for constructing more precise 3D structures, to distinguish the structural differences between the template and targets. In this study, we adopted the covariation analysis to extract information about residue-residue interactions from the amino acid sequence. In the opsin family, a large number of adjacent covarying residue pairs were detected. The detected residue pairs have a strong tendency to gather in some regions important for the structure and function. These results suggest that the covariation analysis is practically utilized to detect adjacent residue pairs and also to apply for predicting functional sites. Analyses of other GPCR subfamilies, olfactory receptor and chemokine receptor families, demonstrated that some adjacent covarying residue pairs were common. Thus, the covariation analysis has possibilities in the substantial improvement of the 3D-structure modeling of GPCRs and in the detection of functional sites such as the ligand-binding sites.

Animals↗
PubMed2004

Classical photoreceptors regulate melanopsin mRNA levels in the rat retina.

Recent studies have demonstrated that melanopsin is a key photopigment in the mammalian circadian system. This novel opsin is exclusively expressed in retinal ganglion cells that are intrinsically sensitive to light, perhaps responding via a melanopsin-based signaling pathway. Previous investigations using transgenic mice have also demonstrated that ablation of the classical photoreceptors and of melanopsin prevents entrainment of several circadian rhythms, thus demonstrating that these photoreceptors are necessary and sufficient for circadian photoreception. In this study, we investigated the effect of photoreceptor degeneration on melanopsin mRNA regulation in RCS/N-rdy rats (Royal College of Surgeons rats with a defect in the retinal dystrophy gene). We used animals at postnatal day 21 (P21), P33, P45, and P60. At P60 degeneration of the retina in RCS/N-rdy has advanced to the point where the majority of the photoreceptors have degenerated. Our data indicate that melanopsin mRNA levels were rhythmic in light/dark cycle and in constant darkness in congenic controls (RCS/N-rdy+) and in RCS/N-rdy at P21 (i.e., before the degeneration of the photoreceptors). On the other hand, in RCS/N-rdy at P60, melanopsin mRNA levels were greatly reduced (<90%) and not rhythmic. Photoreceptor degeneration did not affect the expression of pituitary adenylate cyclase-activating polypeptide mRNA (a marker for melanopsin-containing ganglion cells). Our results suggest that classical photoreceptors (rods and cones) regulate the expression of melanopsin mRNA in the rat. Because RCS/N-rdy rats are a model for studies on retinitis pigmentosa in human, our data may provide an important insight on melanopsin function in patients affected by retinitis pigmentosa.

Animals↗
PubMed1998

Expression of ocular autoantigens in the mouse thymus.

UNLABELLED: PURPOSE. The occurrence of eye diseases of autoimmune nature, as well as experimental models of these diseases, has been attributed to the sequestration of ocular antigens from the immune system, that prevents the development of tolerance against these antigens. Here, we tested this assertion by examining whether transcripts of certain ocular antigens are constitutively expressed in the thymus, the site of central tolerance induction. METHOD: RNA was isolated from the eyes and thymi of two mouse strains and analyzed for the expression of genes encoding four retinal and three lens proteins by reverse transcribed-polymerase chain reaction. Southern blot and DNA sequence analyses. RESULTS: We detected gene transcripts of S-Antigen (S-Ag), interphotoreceptor retinoid-binding protein, opsin, recoverin, lens major intrinsic protein (MIP), alphaA-, alphaA(-ins)- and gamma-crystallins in the thymi of BALB/c and FVB/N mouse strains. DNA sequence analysis of the thymic MIP and S-Ag transcripts confirmed their identity to the lens and retinal proteins, respectively. CONCLUSIONS: Our results reveal that transcripts of several ocular-specific proteins are expressed in the thymus and suggest that the commonly held view that ocular-specific antigens are sequestered from the immune system should be modified.

Animals↗
PubMed1992

Maintenance of opsin and S-antigen gene expression in RCS dystrophic rats following RPE transplantation.

The effects of retinal pigment epithelium (RPE) transplantation in rescuing photoreceptor cells (PRC) were studied in RCS rats bearing the rdy mutation. RPE preparations from Long-Evans rats were transplanted into the subretinal space of post-natal (P) 17-19-day-old RCS rats. Age-matched RCS animals in the control group received injections of the transplantation vehicle buffer alone. The animals were killed at various post-natal ages after RPE transplantation. Immunocytochemical studies using antibodies to opsin and S-antigen indicated intact photoreceptor cells in the transplanted retinas. In contrast age-matched sham-injected animals did not show any rescue of photoreceptor cells at P90. Single RPE transplantation of 60,000 cells in each eye resulted in restoration of near normal mRNA levels for opsin, and S-antigen proteins. Comparisons of mRNA levels of two visual proteins using cDNA probes demonstrated a therapeutic effect of RPE transplantation in preventing a progressive decline in mRNA levels due to retinal degeneration. In contrast, P109 sham-injected controls showed no detectable mRNA levels for these proteins. In vitro protein synthesis in RPE-transplanted retinas implied further competence of these retinas. These data suggest that RPE transplantation not only rescues photoreceptors from degeneration, but more importantly, it allows normal transcription and translation in these cells rendering them capable of participating in the visual and signal transduction cascade.

Animals↗