Article

Isolation, sequence analysis, and intron-exon arrangement of the gene encoding bovine rhodopsin

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Abstract

We have isolated cDNA clones generated from the mRNA encoding the opsin apoprotein of bovine rhodopsin and used these cDNAs to isolate genomic DNA clones containing the complete opsin gene. Nucleotide sequence analysis of the cloned DNAs has yielded a complete amino acid sequence for bovine rhodopsin and provided an intron-exon map of its gene. The mRNA homologous sequences in the 6.4 kb gene consist of a 96 bp 5' untranslated region, a 1044 bp coding region, and a surprisingly long approximately 1400 bp 3' untranslated region, and are divided into five exons by four introns that interrupt the coding region. Secondary structure analysis predicts that the bovine rhodopsin chain, like that of bacteriorhodopsin, contains seven transmembrane segments. Interestingly, three of the four introns are immediately distal to the codons for three of these segments, and one of these introns marks the boundary between the C-terminal domain and a transmembrane domain.

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... In transfection studies using primary chick retinal cell cultures, the presence of NRE showed a 3-5 fold increase in the activity of the rhodopsin promoter as compared to constructs with a mutated or deleted site(Kumar et a l, 1996), suggesting it plays a role in controlling retinal function. This important site has been identified in several rhodopsin promoter regions including those of the mouse, rat, Chinese hamster, bovine, human, and Xenopus(Zack et a l, 1991; Morabito et a l, 1991; Gale et a l, 1992;Nathans & Hogness, 1983;(Nathans & Hogness, 1984;Batni et a l, 1996).93A further group of developmentally regulated elements, termed CRS, have been identified within the promoter region of the bovine opsin gene. ...
... Without DNase I footprinting experiments, we cannot be sure which is the functional TATA box. In chicken,Xenopus, bovine(Sheshberadaran & Takahashi, 1994;Batni et a l, 1996;Nathans & Hogness, 1983) and goldfish rod, the TATA element is located between approximately -29 and -40bp upstream from the Kozak sequence, ANNATG G(Kozak, 1983). This sequence indicates the;transcription start site and suggests that the first TATA box in each green opsin gene, at -29 and -30 upstream, is the functional element. ...
... However, when the GC rich flanking regions and their location relative to the GCAAT boxes is taken into account, it is the second group of TATA elements at -51 and -52 which are more similar to the other opsin promoters.There are numerous motifs present in the goldfish opsin promoters containing the core Retl/PCEl sequence, CC/TAAT. The Retl/PCEl sites in chicken, Xenopus, bovine and human rods are centred at -125, -130, -135 and -135 respectively(Sheshberadaran & Takahashi, 1994;Batni et a l, 1996;Nathans & Hogness, 1983;Nathans & Hogness, 1984). In the goldfish rod the motif is present at -123, in green 1 at -199, and in green 2 at -185 and -198. ...
Thesis
The opsin genes of mammals have been the subject of a number of studies. As a useful comparison and to increase our understanding of gene regulation, spectral tuning and evolution, I have studied the visual pigment genes of goldfish (Carassius auratus) and a number of species of cottoid fish from Lake Baikal in Eastern Siberia. An unusual feature of the goldfish is that, due to tetraploidy, many of its genes are present as duplicate copies and both may be expressed. This is seen with the green cone opsin genes. The level of expression and localisation to particular cone cells of the two green genes was assessed. It was found that green 2 was expressed at a higher level than green 1, although it is thought that both opsins are expressed within the same subset of cone cells. Regulation of the rod and two green cone opsin genes in goldfish has also been studied by the sequencing of the promoter regions and comparing them to each other and to those of mammals, chicken and Xenopus. A number of regions of homology were identified, many of which were known cis-acting elements. Additionally, an eight base pair motif, which has not been identified before, was found to be conserved between species. The wavelength of maximum absorption (?max) of a visual pigment is determined by the structure of the opsin; changes in the amino acid composition at critical sites can significantly affect the ?max. The species flock of cottoid fish living in Lake Baikal are known to have visual pigments whose ?max is short-wave shifted with increasing depth of habitat. In order to identify amino acid substitutions responsible for these spectral shifts, the blue opsin gene of a representative subset of these fish has been sequenced. Three potential spectral tuning sites were identified at positions 118, 215 and 269. Mutations at these sites were then generated by site-directed mutagenisis and the mutant opsins expressed in 293T cells. After isolation and regeneration with 11-cis retinal, the resulting pigments were analysed spectrophotometrically. Evolution of the blue visual pigment genes in the cottoid fish has also been investigated by phylogenetic analysis of the blue opsin sequences.
... The receptor for scotopic stimulation is rhodopsin, which consists of the apoprotein rod opsin and a chromophore, W-cis retinal, which is common to all vertebrate visual pigments. Following the pubhcation of the amino acid sequence of bovine rod opsin (Ovchinnikov, 1982;Hargrave et a i, 1983;Ovchinnikov et al., 1983), the genes encoding bovine (Nathans and Hogness, 1983) and human (Nathans and Hogness, 1984) Introduction and hydrophilicity (polar), such that in vivo, the former regions are embedded in the membrane bilayer, and the latter reside at the aqueous surface of the membrane see Figure 1.4). The three dimensional structure is at present not known because of the difficulties in forming crystals. ...
... Using probes based on the nucleotide sequence of bovine rhodopsin (Nathans and Hogness, 1983), human genomic DNA was screened. At relatively high stringency a single class of clone was detected, which was identified as the human rhodopsin gene. ...
... A comparison of visual pigment genes from birds, fish, and mammals reveals that the duplication events that generated the rod opsin, SW and LW/MW photopigment genes occurred prior to the vertebrate radiation (Nathans and Hogness, 1983;Takao et a l, 1988;Kuwata et a l, 1990, Tokunaga et a l, 1990Yokoyama and Yokoyama, 1990). Construction of a phylogenetic tree using this data (Yokoyama, 1995) suggests that the cone opsins gave rise to the rod opsins. ...
Thesis
Over the 10 year period since the cone opsin genes of humans were first isolated and sequenced, a wealth of information has accumulated on the genetics of primate colour vision. However, a number of intriguing questions have remained unanswered. Do other apes and monkeys see with the same basic complement of photopigments as humans do. How are these different opsin genes temporally and spatially regulated. At present a complete understanding of human colour anomalies is lacking. Based on the analysis of exons 3 to 5 of the X-chromosome opsin genes of two members of the Hominidea (the chimpanzee, Pan troglodytes and the Gorilla, Gorilla gorilla) and five members of the Cercopithecoidea family of Old World primates (the Diana monkey, Cercopithecus diana; lesser spot-nosed guenon, Cercopithecus petaurista; African green monkey, Cercopithecus aethiops; talapoin monkey, Miopithecus talapoin; and patas monkey, Erythrocebus patas), it is predicted that the long-wave (LW) and middle-wave (MW) pigments of these primates have similar spectral properties to those of human. Multiple copies of the same opsin gene sequence were identified in the chimpanzee and talapoin. Humans and the other primates show a bunching of polymorphic sites in exon three. The ancestry of the separate LW and MW genes of Old World primates is discussed. Two anomalous trichromatic human males, each exhibiting the same phenotype, were found to present with differing genotypes. One subject, SSJ, possessed a normal LW and a MW-LW hybrid gene. The other subject, PSJ, had in addition a normal MW gene. It is suggested that the same phenotype may be possible due to selective expression of some of PSJ's opsin genes. Finally, the phenotype of John Dalton, considered the father of colour vision, has been revised to deuteranopia, following amplification, cloning, and sequencing of his opsin genes from the remains of his eyes.
... The lipophilic nature of rhodopsin was first noted by Kühne (1977), who observed that it could be solubilized in a spectrally intact form by treating rod outer segments with bile detergents. Determination of the bovine rhodopsin amino acid sequence (Hargrave et al. 1983, Nathans & Hogness 1983 has revealed seven distinct stretches of hydrophobic amino acids of appropriate length to be embedded as α-helices in a phospholipid membrane. This topology was confirmed by the projection structure of rhodopsin (Schertler et al. 1993), which provided the first detailed information concerning the configuration of the α-helical bundle of this protein. ...
... A single ROL-binding site is formed by the core, eight-stranded, β-barrel fold of RBP4 ( Figure 9a) (Newcomer et al. 1984). All-trans-ROL is oriented with its hydroxyl moiety nearest to the binding cavity entrance, where residues involved in STRA6 binding have been localized. ...
Article
Recent progress in molecular understanding of the retinoid cycle in mammalian retina stems from painstaking biochemical reconstitution studies supported by natural or engineered animal models with known genetic lesions and studies of humans with specific genetic blinding diseases. Structural and membrane biology have been used to detect critical retinal enzymes and proteins and their substrates and ligands, placing them in a cellular context. These studies have been supplemented by analytical chemistry methods that have identified small molecules by their spectral characteristics, often in conjunction with the evaluation of models of animal retinal disease. It is from this background that rational therapeutic interventions to correct genetic defects or environmental insults are identified. Thus, most presently accepted modulators of the retinoid cycle already have demonstrated promising results in animal models of retinal degeneration. These encouraging signs indicate that some human blinding diseases can be alleviated by pharmacological interventions. Expected final online publication date for the Annual Review of Vision Science Volume 2 is September 15, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
... resolved by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose specifically binds 125I-hCG (Keinanen and Rajaniemi, 1986). The other G-protein coupled receptors that have been cloned do not possess a large extracellular domain (rhodopsin - Nathans and Hogness, 1983; yeast mating factors - Nakayama et al., 1985;B-adrenergic -Dixon et al., 1986;muscarinic acetylcholine -Kubo et al., 1986;substance-K -Masu et al., 1987;a-adrenergic -Kobilka et al., 1987;serotonin -Julius et al., 1988;dopamine -Bunzow et al., 1988;angiotensin -Jackson et al., 1988;substance P -Yokoda et al., 1989;neuromedin K -Shigemoto et al., 1989) ( Fig. 1.8). ...
... The C-terminal half of molecule which includes the seven transmembrane domains are essentially equivalent in size to the entire molecule of other G-protein coupled receptors e.g. rhodopsin and adrenergic receptors (Nathans and Hogness, 1983;Lefkowitz and Caron, 1988). In adrenergic receptors, an extended third cytoplasmic loop seems to be required for specific interactions with the G-protein (Kobilka et al., 1988;O'Dowd et al., 1989;Strader et al., 1989). ...
Thesis
Cyclic AMP (cAMP) has been established as a second messenger in the stimulation of testosterone production by luteinizing hormone (LH). The aim of this thesis was to investigate the possible involvement other regulators mediating the acute stimulation of steroidogenesis in purified testis Leydig cells. The possible involvement of calcium ions, chloride ions, pH, protein kinase C (PKC) and de novo synthesis of cholesterol were investigated. Extracellular calcium was found to be essential for both maximum steroidogenesis and cAMP production. However, LH-stimulated increase in intracellular calcium could not be reproducibly demonstrated in cells loaded with fluorescent calcium indicator Fura 2. The involvement of calmodulin in testosterone production was investigated using the calmodulin inhibitor, calmidazolium. This compound inhibited cAMP production stimulated by LH, forskolin and cholera toxin, with ID50 of 2μM. It had both a stimulatory and inhibitory effect on steroidogenesis. The stimulatory effect was an unexpected finding; it was independent of cAMP, calcium and protein synthesis, and was similar in magnitude to stimulation obtained with maximum levels of LH or dibutyryl cyclic AMP (dbcAMP). It also stimulated steroidogenesis in rat adrenocortical cells and mouse Leydig cells. Using cells loaded with the fluorescent pH indicator BCECF, the intracellular pH was found to be pH 7.2, and the presence of the Na+/H+ exchanger was demonstrated. Testosterone production was found to be relatively insensitive to fluctuations in intracellular pH. It has been established that rat Leydig cells possess outward rectifying chloride channels on their plasma membrane. The replacement of extracellular chloride with gluconate salts caused a potentiation of the sub-maximal testosterone production in response to low concentrations of LH and dbcAMP. This potentiating effect of chloride replacement was also found in dbcAMP-stimulated pregnenolone production in rat adrenocortical cells. These results suggest that the efflux of chloride ions may play an important role in steroidogenesis. The chloride channel inhibitors, SITS and DIDS, inhibited both LH-stimulated cAMP and testosterone production, but had no effect on dbcAMP stimulated steroidogenesis. The precise role of SITS- and DIDS-sensitive chloride channels remains unclear. Both basal and stimulated testosterone production were stimulated two-fold when PKC activity was downregulated through preincubation with phorbol ester, phorbol 12- myristate, 13-acetate (PMA). Preincubation with an inactive phorbol ester, phorbol 12,13-dideconoate (PDD), had no effect on testosterone production. The site of this potentiation was prior to cholesterol-side-chain-cleavage. Testosterone production in Leydig cells appears to be tonically inhibited by the action PKC. Pertussis toxin-sensitive G-protein(s) has previously been found in Leydig cells. In the present studies, pertussis toxin was found to inhibit both LH- and dbcAMP-stimulated testosterone production. This suggests the involvement of pertussis toxin- sensitive G-proteins in the regulation of testosterone production. The supply of cholesterol for testosterone production in rat Leydig cells is derived from de novo synthesis. It was found that de novo synthesis of cholesterol was not required for acute testosterone production. This indicates the existence of a large pool of steroidogenic cholesterol in rat Leydig cells.
... In 1983 the first rod opsin gene was identified when Nathans and Hogness published the cDNA sequence of bovine rhodopsin (Nathans and Hogness, 1983). Subsequently, Nathans and colleagues identified the human long-wave, middle-wave and short-wave sensitive cone opsins (Nathans, et a l, 1986). ...
... The mechanism has been reported previously for the NADH dehydrogenase subunit 4 gene of lettuce, Lactuca sativa (Geiss et a l, 1994). With the exception of teleost rod opsin and the LWS/MWS opsin class all visual pigment genes contain five exons (Nathans and Hogness., 1983;Nathans et al., 1986a;Yokoyama and Yokoyama, 1993). The LWS/MWS opsin genes contain an extra exon (Nathans et al., 1986a;Kawamura and Yokoyama, 1993) at the 3' end of the gene. ...
Thesis
Over evolutionary time the planet has become populated by a multitude of organisms highly adapted to their own ecological niche. Each individual species relies on a range of modalities that have become fine-tuned to provide accurate and reliable information. Vision is one such sense and shows interspecific variation in both acuity and chromatic sensitivity. One of the prime examples in recent years has been the short-wave shift in sensitivity found in the visual pigments of deep-sea fish (Lythgoe, 1972; Douglas et al., 1998, 2003). In the majority of species a single rod opsin is expressed, which is maximally sensitive to the wavelengths of bioluminescence and down-welling sunlight (λmax = 470 - 490 nm). This study examines two deep-sea species that have further adapted their visual sense to aid prey detection, and investigates the notothenioid family of ice fish that live beneath pack ice and inhabit a variety of depths. The three stomiid genera, Malacosteus, Pachystomias and Aristostomias are unique in emitting far-red light as well as the typical blue bioluminescence. All three utilize a rhodopsin/porphyropsin pigment pair with λmax values around 520/550 nm, approximately 40 and 70 nm long-wave shifted when compared to other deep-sea pigments, to increase sensitivity to long wavelengths (O'Day and Fernandez, 1974; Dartnall, 1975; Bowmaker et al., 1988; Partridge and Douglas, 1995). There is also evidence that Pachystomias and Aristostomias express a second visual opsin, which has a rhodopsin λmax at around 580 nm (Partridge and Douglas, 1995; Douglas et al., 1998). The aim of this study was to identify these two opsins. A single rod opsin has been identified from both Pachystomias and Aristostomias, and there has been no evidence for a second opsin. Histology shows a single class of rod photoreceptor in the Pachystomias retina. Another deep-sea species, Scopelarchus analis, has highly adapted eyes with seven different retinal specializations. Previously MSP identified three rhodopsins with λmax values at 444, 479 and 505 nm, which show different expression patterns in the main and accessory retinae. It was also demonstrated that there might be a switch in expression from the 505 nm to the 444 nm pigment during maturation (Partridge et al., 1992). This study has identified four retinally expressed opsin genes, an RH2, an SWS2 and two RH1 opsins. The two rod opsins regenerate in vitro with λmax at 480 and 490 nm. Both are expressed in adult mRNA, but the 480 nm rod is absent from juvenile mRNA. Further experiments linking the identified genes to the MSP values and examining expression patterns are also shown. Work has also been undertaken on members of the notothenioid family of ice fish. These live at various depths under thick pack ice in the Antarctic. The light that reaches the water below has been filtered, with long and short wavelengths restricted. This study shows that a number of species express SWS1, SWS2 and RH2 opsins, though the level of SWS1 expression may be low in the majority of species. In situ hybridization and histology have helped to localize expression of these genes to different classes of photoreceptor. MSP and in vitro regeneration have identified the λmax of these pigments.
... From the fragments o f amino acid sequence determined by labeling experiments, Nathans and Hogness (1983) were able to design nucleotide probes for use on bovine retinal RNA, enabling them to isolate the nucleotide sequence and deduce the amino acid sequence for bovine rod opsin They then went on to identify the sequences for human rod opsin from a human genomic library, using bovine sequences for probes (Nathans and Hogness (1984)). Both human and bovine rod opsin are 348m amino acids long and highly homologous (93.4% identity). ...
... An example o f this is the plV primer pair (Fig 4 .1b), which as well as The duck and budgerigar sequences both cover all seven of the transmembrane regions, and all the expected amino acid sites (section 1.4.1) are present lysine 296 the 11-cA-retinal binding site (Nathans and Hogness (1983)), glutamate 113, the counter-ion (Sakmar, et a i (1989)), the pair of palmitoylated cysteines 322 and 323 (Ovchinnikov, et a i (1988)), the cysteines which form the disulphide bridge that helps maintain the tertiary structure (C l 10 and C l 87 Karnik and Khorana (1990)), and tryptophan 265 which is involved in the binding o f the 13-ionine ring o f retinal (Nakayama and Khorana (1991)). These are indicated on the sequence alignment, numbering as for bovine rod opsin. ...
Thesis
Avian vision is more complex than mammalian vision. The most complex mammalian systems, for example in old world primates, have three classes of cone, which mediate daylight vision and a single class of rod, which mediates low light vision Birds, however, often have four cone pigments alongside the rod system and sensitivity which extends into the ultraviolet. The makes avian vision very interesting, both to study the spectral tuning - looking at which amino acids cause variations in the spectral sensitivity of pigments - and the evolution of visual pigments. Previously it has been shown that the rod and green cone pigments in birds are very closely related - about 70% sequence identity - and it was suggested that rod pigments have evolved from green cone pigments (Okano et at. (1992)). I looked at the rod and green cone pigments from two othe avian species. Mallard duck (Anas platyrhynchos) and budgerigar (Mellopscittacus undulatus), to see if this pattern was repeated. The polymerase chain reaction was used to specifically amplify the central parts of the genes for the rod and green cone pigments from Mallard duck and budgerigar. These fragments were cloned and sequenced and the amino acid sequence deduced. The green cone and rod pigments are highly homologous in these avian species as well as chicken. The green pigemnt sequences were like the rod ones in almost all the major positions postulated to be important in rod function. The major difference between the rod and green cone pigments seemed to be the isoelectric point. The green cones share a basic isoelectric point with the other cone pigments, despite the overall similarity to rods, which have an acidic or neutral pl. This work extends and confirms earlier work on opsin evolution as it reinforces the theory that the rod opsins have evolved from cone opsins and adapted to work at relatively low light levels, possibly by changing their ioninc properties.
... Therefore, it is not surprising that studies on opsins and the visual system have evolved in parallel. In 1983, the bovine rhodopsin gene was first cloned and sequenced as an opsin gene (46). Subsequently, the bovine rhodopsin crystal structure was solved in 2000, providing the first GPCR structure to the scientific community (29). ...
Article
Full-text available
Driven by the energy of a photon, the visual pigments in rod and cone photoreceptor cells isomerize 11-cis-retinal to the all-trans configuration. This photochemical reaction initiates the signal transduction pathway that eventually leads to the transmission of a visual signal to the brain and leaves the opsins insensitive to further light stimulation. For the eye to restore light sensitivity, opsins require recharging with 11-cis-retinal. This trans–cis back conversion is achieved through a series of enzymatic reactions composing the retinoid (visual) cycle. Although it is evident that the classical retinoid cycle is critical for vision, the existence of an adjunct pathway for 11-cis-retinal regeneration has been debated for many years. Retinal pigment epithelium (RPE)–retinal G protein-coupled receptor (RGR) has been identified previously as a mammalian retinaldehyde photoisomerase homologous to retinochrome found in invertebrates. Using pharmacological, genetic, and biochemical approaches, researchers have now established the physiological relevance of the RGR in 11-cis-retinal regeneration. The photoisomerase activity of RGR in the RPE and Müller glia explains how the eye can remain responsive in daylight. In this review, we will focus on retinoid metabolism in the eye and visual chromophore regeneration mediated by RGR.
... Among these receptors, opsins have the oldest study history of biochemistry and biophysics (Wald 1968) and molecular genetics (Nathans and Hogness 1983). Gene identification of ORs (Buck and Axel 1991), TAS1Rs (Hoon et al. 1999;Li et al. 2002), and TAS2Rs (Adler et al. 2000) utilized the knowledge generated from study of opsins and was enabled more recently. ...
Chapter
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Primates are generally regarded as visually oriented mammals, trading a sense of smell for good sight. However, recent studies have questioned this simplistic view, and it is not well understood the extent to which senses have evolved interactively or in concert with each other in primates including humans. For example, the number of olfactory receptor genes is not as clearly differentiated between species with different color vision as once asserted. Among senses, receptors of stimuli for vision, olfaction, and bitter/sweet/umami tastes all belong to the G-protein-coupled receptor (GPCR) family, for which the genetic mechanism of signal perception is well understood. Thus, it is now possible to explore the evolutionary correlation among different senses in primates by studying these receptor groups for interspecies divergence, intraspecies diversity, and functional differences among variants. In this chapter, we review recent findings on these receptors and senses in humans and other primates and discuss the future directions of studies on their sensory evolution.
... Opsin proteins catalyze light-dependent processes in all three domains of life, including vision and circadian cycling in animals (Koyanagi & Terakita, 2013), as well as chlorophyll-independent phototrophy, osmoregulation and phototaxis in bacteria, archaea, microbial eukaryotes, and multi-cellular fungi (Inoue et al, 2014). The evolutionary relationship between microbial (type 1) opsins and metazoan (type 2) opsins is unresolved (Larusso et al, 2008;Mackin et al, 2014;Shen et al, 2013), however, in both groups, photosensing depends upon a covalent interaction between a conserved lysine residue in the seventh transmembrane helix (bovine visual rhodopsin K296 / bacteriorhodopsin K216) and a retinal chromophore (Nathans & Hogness, 1983;Mullen et al, 1981). Recent studies have reported discovery of genes encoding opsin homologs lacking this residue in fungal, haloarchaeal and placozoan genomes (Spudich et al, 2000;Siddaramappa et al, 2012;Feuda et al, 2012). ...
Preprint
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Opsins are photosensitive proteins catalyzing light-dependent processes across the tree of life. For both microbial (type 1) and metazoan (type 2) opsins, photosensing depends upon covalent interaction between a retinal chromophore and a conserved lysine residue. Despite recent discoveries of potential opsin homologs lacking this residue, phylogenetic dispersal and functional significance of these abnormal sequences have not yet been investigated. We report discovery of a large group of putatively non-retinal binding opsins, present in a number of fungal and microbial genomes and comprising nearly 30% of opsins in the Halobacteriacea, a model clade for opsin photobiology. Based on phylogenetic analyses, structural modeling, genomic context and biochemistry, we propose that these abnormal opsin homologs represent a novel family of sensory opsins which may be involved in taxis response to one or more non-light stimuli. This finding challenges current understanding of microbial opsins as a light-specific sensory family, and provides a potential analogy with the highly diverse signaling capabilities of the eukaryotic G-protein coupled receptors (GPCRs), of which metazoan type 2 opsins are a light-specific sub-clade.
... The first steps toward the understanding of the GPCR structure were made in 1980s with the purification of the β2-adrenergic receptor (Shorr and Heald, 1982), sequencing of the gene encoding the bovine rhodopsin receptor (Nathans and Hogness, 1983) and cloning of the β2adrenergic hamster receptor (Dixon et al., 1986). Already at that time it was suggested that the β2-adrenergic and the rhodopsin receptors possessed multiple membrane-spanning regions. ...
Thesis
In order to better understand the role of OTR and AVPR in ASD, to reveal new features in its pharmacology and signaling and to establish high-throughput screening method on wild-type G protein-coupled receptors, we developed imaging probes for the oxytocin-vasopressin receptors family, namely radiotracers for positron emission tomography and optical probes for fluorescence detection and imaging. The fluorescent ligands have been used to establish TR-FRET binding assay for OTR and to initiate the development the screening assay for the wild-type oxytocin receptor. The PET radiotracers will be shortly tested in mice and monkeys to evaluate their potency in detecting the central oxytocin receptors.
... In all neuralians studied so far, all opsins are linked via a highly conserved lysine (Schiff base) to a chromophore to form a visual pigment [25]. A covalent interaction between the Schiff base in the seventh transmembrane helix and the retinal chromophore leads to photosensation [36,37]. In the scaphopod Antalis entalis, the predicted Go-opsin amino acid sequence does not contain this lysine (K296, named after the position of the residue in bovine Rhodopsin) (Fig. 2). ...
Article
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Background: Eyes have evolved and been lost multiple times during animal evolution, however, the process of eye loss has only been reconstructed in a few cases. Mollusks exhibit eyes as varied as the octopod camera eye or the gastropod cup eye and are ideal systems for studying the evolution of eyes, photoreceptors, and opsins. Results: Here, we identify genes related to photoreceptor formation and function in an eyeless conchiferan mollusk, the scaphopod Antalis entalis, and investigate their spatial and temporal expression patterns during development. Our study reveals that the scaphopod early mid-stage trochophore larva has putative photoreceptors in a similar location and with a similar gene expression profile as the trochophore of polyplacophoran mollusks. The apical and post-trochal putative photoreceptors appear to co-express go-opsin, six1/2, myoV, and eya, while expression domains in the posterior foot and pavilion (posterior mantle opening) show co-expression of several other candidate genes but not go-opsin. Sequence analysis reveals that the scaphopod Go-opsin amino acid sequence lacks the functionally important lysine (K296; Schiff base) in the retinal-binding domain, but has not accumulated nonsense mutations and still exhibits the canonical G-protein activation domain. Conclusions: The scaphopod Go-opsin sequence reported here is the only known example of a bilaterian opsin that lacks lysine K296 in the retinal-binding domain. Although this may render the Go-opsin unable to detect light, the protein may still perform sensory functions. The location, innervation, development, and gene expression profiles of the scaphopod and polyplacophoran apical and post-trochal photoreceptors suggest that they are homologous, even though the scaphopod post-trochal photoreceptors have degenerated. This indicates that post-trochal eyes are not a polyplacophoran apomorphy but likely a molluscan synapomorphy lost in other mollusks. Scaphopod eye degeneration is probably a result of the transition to an infaunal life history and is reflected in the likely functional degeneration of Go-opsin, the loss of photoreceptor shielding pigments, and the scarce expression of genes involved in phototransduction and eye development. Our results emphasize the importance of studying a phylogenetically broad range of taxa to infer the mechanisms and direction of body plan evolution.
... G-protein-coupled receptors (GPCRs) constitute one of the largest gene families in the human genome, and provide the target for about 20-30% of all drugs currently on the market. Since the discovery of rhodopsin and the beta-adrenergic receptor as first GPCRs (1,2), currently more than 800 different genes are classified as members of the GPCR family. The pharmacologic and biologic importance of this huge receptor family led to enormous efforts world-wide to delineate activation and signaling mechanisms of GPCRs. ...
Article
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The family of G-protein-coupled receptors (GPCRs) is one of the most important drug targets. Mechanisms underlying GPCR activation and signaling are therefore of great pharmacologic interest. It was long thought that GPCRs exist and function as monomers. This feature was considered to distinguish GPCRs from other membrane receptors such as receptor tyrosine kinases or cytokine receptors, which signal from dimeric receptor complexes. But during the last two decades it was increasingly recognized that GPCRs can undergo aggregation to form dimers and higher order oligomers, resulting in homomeric and/or heteromeric protein complexes with different stoichiometries. Moreover, this protein complex formation could modify GPCR signaling and function. We contributed to this paradigm shift in GPCR pharmacology by the discovery of the first pathologic GPCR aggregation, which is the protein complex formation between the angiotensin II AT1 receptor and the bradykinin B2 receptor. Increased AT1-B2 heteromerization accounts for the angiotensin II hypersensitivity of pregnant women with preeclampsia hypertension. Since the discovery of AT1-B2, other pathologic GPCR aggregates were found, which contribute to atherosclerosis, neurodegeneration and Alzheimer's disease. As a result of our findings, pathologic GPCR aggregation appears as an independent and disease-specific process, which is increasingly considered as a novel target for pharmacologic intervention.
... receptor which displays a short cytoplasmic segment of only 50 residues . Another characteristic of the protein-tyrosine kinase family of receptors is that they possess only a single transmembrane domain whereas the group of receptors, including the rhodopsin (Nathans and Hogness, 1983) and muscarinic (Kubo et al., 1986) receptors, contain seven m em brane-spanning segm ents. ...
Thesis
Peptide growth factors are important in a wide variety of physiological and pathological processes as a consequence of their ability to generate a mitogenic signal via their interaction with and activation of a specific membrane-associated receptor. The subversion of the normal growth factor signalling pathway, albeit by the enhanced expression of the growth factor or receptor, the unregulated activity of the receptor protein-tyrosine kinase or the uncoupling of intracellular pathways can lead to uncontrolled cellular proliferation resulting in cellular transformation and the formation of tumours in vivo. The major part of this thesis was devoted to the development of baculoviral expression systems producing large amounts of biologically active EGF and TGFa for the assessment of the structural and functional basis of ligand-receptor interactions. Insect cells infected with the recombinant baculoviruses expressed growth factors with the correct putative molecular weights which were immunologically indistinguishable from their authentic counterparts. The recombinant growth factors were able to bind to the EGF receptor expressed in insect cells and NR6+ fibroblasts and activate the receptor protein-tyrosine kinase activity, Furthermore, they induced a mitogenic response in NR6+ and Swiss 3T3 fibroblasts. It is anticipated that milligram quantities of these biologically important growth factors will be available for crystallisation. The elucidation of the three-dimensional structures of the growth factors by diffractional analysis of the crystals and structural mutational analysis will establish the nature of the receptor binding face. This information will lead to the design of clinically important EGF and TGFa antagonists. The recombinant ligands will allow the continued biophysical characterisation of the conformation changes occurring in the extracellular domain of the EGF receptor in response to ligand binding. Cocrystallisation will ultimately lead to the determination of the ligand binding site of the receptor. The biosynthetic precursors for EGF and TGFa are integral membrane glycoproteins suggesting that they may function to mediate physiological cell-cell recognition events. The TGFa precursor expressed in insect cells was able to stimulate the EGF receptor protein-tyrosine kinase activity. Characterisation of the precursor in insect cells will determine the relevance of this mechanism for the production of secreted growth factors and investigate further potential roles for their membrane- associated precursors. The expression of c-erbB-2 in insect cells has allowed the evaluation of a putative growth factor receptor which is highly homologous to the EGF receptor. Furthermore, an in vivo association of the EGF receptor and p185c-erbB-2 been demonstrated. This system will provide a valuable tool for the determination of the mechanism responsible for the transmembrane translocation of the mitogenic signal. It is anticipated that baculoviral coexpression of growth factor receptors and their cognate ligands, in conjunction with intracellular substrates will play an important role in the identification of their intracellular signalling pathways. The elucidation of the growth factor signalling pathway is crucial to the understanding of how oncogenic activation of anyone of the components can result in cellular transformation.
... The cone class of pigments include short wavelength sensitive type 1 opsin (SWS1/OPN1SW/S) sensitive in the ultraviolet/violet range of the spectrum at 355?440 nm, SWS type 2 opsin (SWS2) sensitive in the blue range from about 410?490 nm, RH2 opsin (close homolog of RH1) sensitive in the green range from about 480? 535 nm, long wavelength opsin (LWS/OPN1LW/L) and middle wavelength-sensitive opsin (MWS/OPN1MW/M) sensitive in the red range from about 490?570 nm (Yokoyama, 2002;Bowmaker, 2008). The cloning of bovine rhodopsin gene byNathans & Hogness (1983)inspired efforts to understand variations in molecular genetic mechanisms underlying vision in mammals and vertebrates in general. Comparison of different classes of opsin genes among vertebrates reveals gene loss, gene duplication and nucleotide substitutions to play a fundamental role in the evolution of color vision in vertebrates (Yokoyama & Radlwimmer, 1998;Yokoyama, 2002;Horth, 2007;Jacobs, 2009). ...
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Background The capacity of visually oriented species to perceive and respond to visual signal is integral to their evolutionary success. Giraffes are closely related to okapi, but the two species have broad range of phenotypic differences including their visual capacities. Vision studies rank giraffe’s visual acuity higher than all other artiodactyls despite sharing similar vision ecological determinants with many of them. The extent to which the giraffe’s unique visual capacity and its difference with okapi is reflected by changes in their vision genes is not understood. Methods The recent availability of giraffe and okapi genomes provided opportunity to identify giraffe and okapi vision genes. Multiple strategies were employed to identify thirty-six candidate mammalian vision genes in giraffe and okapi genomes. Quantification of selection pressure was performed by a combination of branch-site tests of positive selection and clade models of selection divergence through comparing giraffe and okapi vision genes and orthologous sequences from other mammals. Results Signatures of selection were identified in key genes that could potentially underlie giraffe and okapi visual adaptations. Importantly, some genes that contribute to optical transparency of the eye and those that are critical in light signaling pathway were found to show signatures of adaptive evolution or selection divergence. Comparison between giraffe and other ruminants identifies significant selection divergence in CRYAA and OPN1LW . Significant selection divergence was identified in SAG while positive selection was detected in LUM when okapi is compared with ruminants and other mammals. Sequence analysis of OPN1LW showed that at least one of the sites known to affect spectral sensitivity of the red pigment is uniquely divergent between giraffe and other ruminants. Discussion By taking a systemic approach to gene function in vision, the results provide the first molecular clues associated with giraffe and okapi vision adaptations. At least some of the genes that exhibit signature of selection may reflect adaptive response to differences in giraffe and okapi habitat. We hypothesize that requirement for long distance vision associated with predation and communication with conspecifics likely played an important role in the adaptive pressure on giraffe vision genes.
... The C-terminal half of the molecule which includes the seven transmembrane domains is essentially equivalent in size to the entire molecule o f other G-protein coupled receptors (e.g. rhodopsin and adrenergic receptors (Nathans and Hogness, 1983; see review Lefkowitz and Caron, 1987)). In adrenergic receptors, an extended third cytoplasmic loop seems to be required for specific interactions with the G-protein (Kobilka et a l, 1988;see review O'Dowd et a l, 1989;Strader et a l, 1989;Cheung et a l, 1991). ...
Thesis
Site-directed antibodies were used to investigate the structure / activity relationships of the LH/CG receptor. Polyclonal antibodies were raised against synthetic peptides corresponding to regions within the extracellular N-terminal domain (antibody 1: Arg48-Glu65; antibody 2: Tyr187-Asp206) and the cytoplasmic C-terminal domain (antibody 3: Cys622-Ala636) of the receptor. Affinity purified antibodies proved to be peptide-specific by both ELISA and dot-blotting assays. On Western blots of membrane proteins prepared from superovulated rat ovaries, mouse Leydig tumour (MA10) cells and rat testes, all three antibodies recognised a single broad band (95- 100 kDa) corresponding to the putative LH/CG receptor. The 95-100 kDa protein also bound 125I-hCG on ligand blots and this binding was inhibited by the two N-terminal antibodies. Antibody 1 significantly inhibited 125I-hCG binding to intact MA10 cells to a greater extent than did antibody 2, whereas antibody 3 and pre-immune IgG were without effect. Similarly both N-terminal antibodies significantly inhibited the cAMP and progesterone response of MA10 cells to LH (antibody 1 being more potent than antibody 2), whereas antibody 3 and pre-immune IgG had no significant effect. Both the cAMP and progesterone responses of MA10 cells to dbcAMP, cholera-toxin and forskolin were unaffected by any of the antibodies, as was the stimulation of membrane adenylyl cyclase activity by NaF and a GTP analogue. These results indicate that the antibodies inhibit LH action at the level of the LH/CG receptor and that residues Arg48-Glu65 (and to a lesser extent Tyr187-Asp206) are close to the binding site for LH/CG. Data obtained with antibody 3 suggest that antibody binding to the C-terminal residues Cys622-Ala636 does not interfere with hormone binding or actions. The differential effects of these antibodies on LH-induced signal transduction provide important information on the structure / activity relationships of the LH receptor.
... It is possible that different mechanisms were responsible for the two distinct cell lines. We observed that "A-H-specific gains" were enriched in signal transduction through G-protein coupled receptors, which involve in many biological functions, such as the sensing of taste, light and odor, chemotaxis, and inflammatory and immune responses [38,39]. In addition, the loss of genes involved in cell-cell adhesion in A-H cells might have contributed to their high level of invasion/migration. ...
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Ovarian cancer has the worst prognosis of any gynecological malignancy, and generally presents with metastasis at advanced stages. Copy number variation (CNV) frequently contributes to the alteration of oncogenic drivers. In this study, we sought to identify genetic targets in heterogeneous clones from human ovarian cancers cells. We used array-based technology to systematically assess all the genes with CNVs in cell models clonally expanded from A2780 and SKOV3 ovarian cancer cell lines with distinct highly and minimally invasive/migratory capacities. We found that copy number alterations differed between matched highly and minimally invasive/migratory subclones, differentially affecting specific functional processes including immune response processes, DNA damage repair, cell cycle and cell proliferation. We also identified seven genes as strong candidates, including DDB1, ERCC1, ERCC2, PRPF19, BCAT1, CDKN1B and MARK4, by integrating the above data with gene expression and clinical outcome data. Thus, by determining the molecular signatures of heterogeneous invasive/migratory ovarian cancer cells, we identified genes that could be specifically targeted for the treatment and prognosis of advanced ovarian cancers.
... When the chromophore absorbs a photon, conformational changes in the chromophore and opsin protein activate Gprotein signal transduction (Terakita 2005). Despite their widespread importance in animal photosensitivity, most work on the function and evolution of opsins focused initially on those expressed in the eyes of vertebrates and arthropods (Nathans and Hogness 1983;O'Tousa et al. 1985). Only more recently has work on opsins included those expressed outside eyes or from other animal phyla (Velarde et al. 2005;Radu et al. 2008;Hering et al. 2012;Hering and Mayer 2014;D'Aniello et al. 2015). ...
Article
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The opsin gene family encodes key proteins animals use to sense light and has expanded dramatically since it originated early in animal evolution. Understanding the origins of opsin diversity can offer clues to how separate lineages of animals have repurposed different opsin paralogs for different light-detecting functions. However, the more we look for opsins outside of eyes and from additional animal phyla, the more opsins we uncover, suggesting we still do not know the true extent of opsin diversity, nor the ancestry of opsin diversity in animals. To estimate the number of opsin paralogs present in both the last common ancestor of the Nephrozoa (bilaterians excluding Xenoacoelomorpha), and the ancestor of Cnidaria + Bilateria, we reconstructed a reconciled opsin phylogeny using sequences from 14 animal phyla, especially the traditionally poorly-sampled echinoderms and molluscs. Our analysis strongly supports a repertoire of at least nine opsin paralogs in the bilaterian ancestor and at least four opsin paralogs in the last common ancestor of Cnidaria + Bilateria. Thus, the kernels of extant opsin diversity arose much earlier in animal history than previously known. Further, opsins likely duplicated and were lost many times, with different lineages of animals maintaining different repertoires of opsin paralogs. This phylogenetic information can inform hypotheses about the functions of different opsin paralogs and be used to understand how and when opsins were incorporated into complex traits like eyes and extraocular sensors.
... Type 2 opsins by contrast are found in metazoan species and serve primarily as light dependent photoreceptors in animal eyes and various other tissues of higher eukaryotes [3]. The evolutionary relationships between these two classes of opsins remains unresolved [4][5][6], however, in both groups, photosensing depends upon a covalent interaction between a conserved lysine residue in the seventh transmembrane helix (bovine visual rhodopsin K296 / bacteriorhodopsin K216) and a retinal chromophore [7,8]. Recent studies have reported discovery of genes encoding opsin homologs lacking this residue in fungal, haloarchaeal and placozoan genomes [3,9,10]. ...
Article
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Opsins are photosensitive proteins catalyzing light-dependent processes across the tree of life. For both microbial (type 1) and metazoan (type 2) opsins, photosensing depends upon covalent interaction between a retinal chromophore and a conserved lysine residue. Despite recent discoveries of potential opsin homologs lacking this residue, phylogenetic dispersal and functional significance of these abnormal sequences have not yet been investigated. We report discovery of a large group of putatively non-retinal binding opsins, present in a number of fungal and microbial genomes and comprising nearly 30% of opsins in the Halobacteriacea, a model clade for opsin photobiology. We report phylogenetic analyses, structural modeling, genomic context analysis and biochemistry, to describe the evolutionary relationship of these recently described proteins with other opsins, show that they are expressed and do not bind retinal in a canonical manner. Given these data, we propose a hypothesis that these abnormal opsin homologs may represent a novel family of sensory opsins which may be involved in taxis response to one or more non-light stimuli. If true, this finding would challenge our current understanding of microbial opsins as a light-specific sensory family, and provides a potential analogy with the highly diverse signaling capabilities of the eukaryotic G-protein coupled receptors (GPCRs), of which metazoan type 2 opsins are a light-specific sub-clade.
... When the chromophore absorbs a photon, conformational changes in the chromophore and opsin protein result in the activation of a G-protein based signal transduction cascade (Terakita 2005 ). Despite their widespread importance in animal photosensitivity , most work on the function and evolution of opsins focused initially on those expressed in the eyes of vertebrates and arthropods (O'Tousa et al. 1985); (Nathans & Hogness 1983). Only recently . ...
Article
Full-text available
The opsin gene family encodes key proteins animals use to sense light and has expanded dramatically since it originated early in animal evolution. Understanding the origins of opsin diversity can offer clues to how separate lineages of animals have repurposed different opsin paralogs for different light-detecting functions. However, the more we look for opsins outside of eyes and from additional animal phyla, the more opsins we uncover, suggesting we still do not know the true extent of opsin diversity, nor the ancestry of opsin diversity in animals. To estimate the number of opsin paralogs present in both the last common ancestor of the Nephrozoa (bilaterians excluding Xenoacoelomorpha), and the ancestor of Cnidaria + Bilateria, we reconstructed a reconciled opsin phylogeny using sequences from 14 animal phyla, especially the traditionally poorly-sampled echinoderms and molluscs. Our analysis strongly supports a repertoire of at least nine opsin paralogs in the bilaterian ancestor and at least four opsin paralogs in the last common ancestor of Cnidaria + Bilateria. Thus, the kernels of extant opsin diversity arose much earlier in animal history than previously known. Further, opsins likely duplicated and were lost many times, with different lineages of animals maintaining different repertoires of opsin paralogs. This phylogenetic information can inform hypotheses about the functions of different opsin paralogs and be used to understand how and when opsins were incorporated into complex traits like eyes and extraocular sensors.
Thesis
Les acteurs neuroendocriniens régulant la physiologie des Lophotrochozoaires, groupe frère des Ecdysozoaires parmi les Protostomiens, demeurent peu connus. Grâce à l’émergence récente de ressources génomiques, transcriptomiques et peptidomiques chez l’huître creuse Crassostrea gigas, l’étude des couples ligand(s)/récepteur(s) régulant les fonctions physiologiques est désormais facilitée. Ainsi, par une approche d’endocrinologie inverse consistant à éprouver l’activité d’un panel de ligands potentiels, plusieurs récepteurs couplés aux protéines G (RCPGs), jusqu’alors orphelins ont pu être caractérisés sur le plan fonctionnel. Trois voies de signalisation ont été étudiées : la voie de type cholécystokinine/sulfakinine (CCK/SK) la voie de type calcitonine (CT) et la voie de type dopamine (DA). Grâce à des tests fonctionnels, deux neuropeptides et deux récepteurs de type CCK/SK ont pu être caractérisés. Des tests d’activité biologique in vitro et des expériences de conditionnement alimentaire ont montré la potentielle implication de ces couples dans la régulation de la digestion et de la satiété chez l’huître. Par ailleurs, deux couples neuropeptide/récepteur de type CT ont été caractérisés montrant, à l’image de leurs homologues chez les vertébrés, leur possible rôle dans la régulation hydrique ou ionique. D’autre part, un récepteur activé de manière spécifique par la dopamine et la tyramine a été caractérisé. Ce système de signalisation semble être impliqué dans la médiation du stress et intervenir dans les processus régulateurs de la reproduction au niveau de la gonade. Ainsi, les différents résultats obtenus au cours de ces travaux ont permis d’identifier des couples ligands/récepteurs d’huître homologues de systèmes de signalisation présents chez les Ecdysozoaires et les vertébrés confirmant l’origine de ces systèmes neuroendocriniens depuis l’ancêtre commun des Bilatériens. Les résultats obtenus ont également permis de mieux comprendre comment l’huître intègre les paramètres du milieu et donc s’acclimate aux différentes contraintes environnementales.
Article
Non-visual opsins were discovered in the early 1990s. These genes play roles in circadian rhythm in mammals, seasonal reproduction in birds, light avoidance in amphibian larvae, and neural development in fish. However, the interpretation of such studies and the success of future work are compromised by the fact that non-visual opsin repertoires have not been properly characterized in any of these lineages. Here, we show that non-visual opsins from tetrapods and ray-finned fish are distributed among 18 monophyletic subfamilies. An amphibian sequence occurs in every subfamily, whereas mammalian orthologs occur in only seven. Species in the major ray-finned fish lineages, Holostei, Osteoglossomorpha, Otomorpha, Protacanthopterygii, and Neoteleostei, have large numbers of non-visual opsins (22–32 genes) as a result of gene duplication events including, but not limited to, the teleost genome duplication (TGD). In contrast to visual opsins, where lineage-specific duplication is common, the ray-finned fish non-visual opsin repertoire appears to have stabilized shortly after the TGD event and consequently even distantly related species have repertoires of similar size and composition. Most non-visual opsins have been named without the benefit of a phylogenetic perspective and, accordingly, major revisions are proposed.
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We conduct a cartography of rhodopsin-like non-olfactory G protein-coupled receptors in the Ensembl database. The most recent genomic data (releases 90–92, 90 vertebrate genomes) are analyzed through the online interface and receptors mapped on phylogenetic guide trees that were constructed based on a set of ~14.000 amino acid sequences. This snapshot of genomic data suggest vertebrate genomes to harbour 142 clades of GPCRs without human orthologues. Among those, 69 have not to our knowledge been mentioned or studied previously in the literature, of which 28 are distant from existing receptors and likely new orphans. These newly identified receptors are candidates for more focused evolutionary studies such as chromosomal mapping as well for in-depth pharmacological characterization. Interestingly, we also show that 37 of the 72 human orphan (or recently deorphanized) receptors included in this study cluster into nineteen closely related groups, which implies that there are less ligands to be identified than previously anticipated. Altogether, this work has significant implications when discussing nomenclature issues for GPCRs.
Article
Inherited mutations in the rod visual pigment, rhodopsin, cause the degenerative blinding condition, retinitis pigmentosa (RP). Over 150 different mutations in rhodopsin have been identified and, collectively, they are the most common cause of autosomal dominant RP (adRP). Mutations in rhodopsin are also associated with dominant congenital stationary night blindness (adCSNB) and, less frequently, recessive RP (arRP). Recessive RP is usually associated with loss of rhodopsin function, whereas the dominant conditions are a consequence of gain of function and/or dominant negative activity. The in-depth characterisation of many rhodopsin mutations has revealed that there are distinct consequences on the protein structure and function associated with different mutations. Here we categorise rhodopsin mutations into seven discrete classes; with defects ranging from misfolding and disruption of proteostasis, through mislocalisation and disrupted intracellular traffic to instability and altered function. Rhodopsin adRP offers a unique paradigm to understand how disturbances in photoreceptor homeostasis can lead to neuronal cell death. Furthermore, a wide range of therapies have been tested in rhodopsin RP, from gene therapy and gene editing to pharmacological interventions. The understanding of the disease mechanisms associated with rhodopsin RP and the development of targeted therapies offer the potential of treatment for this currently untreatable neurodegeneration.
Article
Rhodopsin is the classical light sensor. Although rhodopsin has long been known to be important for image formation in the eye, the requirements for opsins in non-image formation and in extraocular light sensation were revealed much later. Most recent is the demonstration that an opsin in the fruit fly, Drosophila melanogaster, is expressed in pacemaker neurons in the brain and functions in light entrainment of circadian rhythms. However, the biggest surprise is that opsins have light-independent roles, countering more than a century of dogma that they function exclusively as light sensors. Through studies in Drosophila, light-independent roles of opsins have emerged in temperature sensation and hearing. Although these findings have been uncovered in the fruit fly, there are hints that opsins have light-independent roles in a wide array of animals, including mammals. Thus, despite the decades of focus on opsins as light detectors, they represent an important new class of polymodal sensory receptor. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 33 is October 6, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Chapter
Structurally and functionally, the retina consists of two components: (1) the nonneural retinal pigment epithelium, a single layer of hexagonally shaped cells resting on Bruch’s membrane, and (2) the tightly apposed, but not anatomically joined, neural retina (Fig. 7.1). The neural retina is a peripheral extension of the forebrain, with which it has common embryological origins. It consists of six distinct neuronal cell types: photoreceptors (rods and cones), bipolar cells, horizontal cells, amacrine cells, interplexiform cells, and ganglion cells. Its principal glial cell is the Müller cell, whose fibers extend from the vitreous surface (or inner limiting “membrane” of the retina) to beyond the outer limiting “membrane,” where they surround the photoreceptor inner segments. In histological cross section, the neural retina is organized into ten distinct layers, including the pigment epithelium, as described in the legend to Fig. 7.1.
Chapter
The very large G protein-coupled receptor 1 (VLGR1), also known as MASS1 or GPR98, is most notable among the family of adhesion GPCR for its size. Encoded by an 18.9 kb open reading frame, the ~700 kDa primary translation product is by far the largest GPCR and, additionally, the largest cell surface protein known to date. The large ectodomain of the protein contains several repeated motifs, including some 35 calcium binding, Calx-β repeats and seven copies of an epitempin repeat thought to be associated with the development of epilepsy. The extreme carboxyl-terminus contains a consensus PDZ ligand sequence, suggesting interactions with other cytosolic or cytoskeletal proteins. At least two spontaneous and two targeted mutant mouse lines are currently known. The mutant mice present with sensitivity to audiogenic seizures but also have cochlear defects and significant progressive hearing impairment. VLGR1 is one of the few adhesion GPCR family members in which mutations have been shown to be responsible for a human malady. Mutations in VLGR1 in humans result in one form (2C) of Usher syndrome, the most common genetic cause of combined blindness and deafness. Consistent with this phenotype, VLGR1 is a major component of the ankle link of stereocilia in cochlear hair cells and the preciliary complex in retinal photoreceptor cells.
Chapter
G protein-coupled receptors (GPCRs) are the largest family of integral membrane proteins. With more than 800 members, they play an important role in mediating cell signaling across the cytoplasmic membrane to activate downstream proteins such as G proteins and β-arrestins. The first GPCR crystal structure determined was bovine rhodopsin in 2000. Since 2007, there have been more than 300 crystal structures and 12 cryo-EM structures published, while those numbers keep growing. This article is aimed at providing some historical perspectives of this so-called golden age of GPCR structural biology, as well as some new insights from the structures into the mechanism of GPCR signaling at molecular and atomic levels.
Chapter
G protein‐coupled receptors (GPCRs) continue to be popular drug targets and recent advances in the elucidation of their structures are likely to refine and accelerate further drug design. A subset of GPCRs, the orphan GPCRs, are not yet targeted by drugs, mainly because their endogenous ligands are unknown and thus information on their putative ligand binding pocket is missing. Recent advances in identification of natural and surrogate ligands for orphan GPCRs, combined with new formats of functional assays and large‐scale bioinformatics analysis have started to change this picture. In the future, important progress can be anticipated in the field of orphan GPCRs as summarized in the current chapter.
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Opsins play important roles in the image‐forming visual pathways and numerous biological systems such as the biological clock and circadian rhythm. However, the nonvisual opsins involved in nonimage forming process are not clear to date. The aim of this study was to characterize nonvisual opsins in Paralichthys olivaceus. A total of 24 nonvisual opsin genes were identified. Expressions of these genes in eye, brain, heart, testis, and fin were investigated by quantitative real‐time polymerase chain reaction (qRT‐PCR). Testis contained a surprisingly large number of nonvisual opsins including Opn4m2, Tmt2a, Tmt3b, Opn3, RRH, Opn7a, and Opn7b. Syntenic and phylogenetic analyses confirmed that the RGRa and RGRb originated from the teleost‐specific genome duplication (TSGD). qRT‐PCR results demonstrated high RGRa and RGRb expression in the eye, while the expression levels in the brain, heart, testis, and fin were relatively weak. In situ hybridization results presented here revealed the presence of both RGRa and RGRb mRNA‐positive signals in the ganglion cell layer but absence in the intracellular compartment of retinal pigment epithelium (RPE) and Müller glial cells. Therefore, we hypothesized that RGRa and RGRb had undergone subfunctionalization in P. olivaceus after TSGD. In conclusion, this study provides novel insights into the evolutionary fates of the RGR genes, still, further studies need to be done to explore the mechanism about the lack of RGR genes' expression in RPE. Research Highlights Non‐visual opsins involve in non‐image forming are studied in flounder. Testis contained large number of non‐visual opsins. RGRa/b originated from TSGD have functionally diversified. The results provided novel insights into the evolution RGRs.
Article
Loss of function of GPA1, which encodes a guanine-nucleotide-binding protein, arrests the cell at the G1 phase and allows it to mate, suggesting that the gpa1 mutation spontaneously exerts an intracellular signal that mimics the action of mating factor. We have cloned the SGP2 gene, which was first identified as a secondary mutation that allowed a gpa1::HIS3 mutant to grow and to show a non-cell-type-specific sterile phenotype. Disruption of SGP2 confers temperature-sensitive growth and a-specific sterile phenotypes, characteristics similar to those conferred by the dpr1 (ram) mutation, a suppressor of RAS2Val-19. The following observations indicate that SGP2 and DPR1 are in fact identical. (i) The cloned SGP2 complements both the temperature-sensitive growth and the a-specific sterility of the dpr1 mutant and can be integrated into the chromosomal DPR1 locus. (ii) The cloned DPR1, in turn, complements the ability of sgp2 to suppress the lethality of gpa1::HIS3. (iii) The dpr1 mutation suppresses the growth defect of gpa1::HIS3, and the dpr1 gpa1::HIS3 strain shows a non-cell-type-specific sterile phenotype. (iv) sgp2 is closely linked to the dpr1 locus. The DPR1 product has been shown to be responsible for processing and fatty acid acylation of a-factor and RAS proteins at their carboxyl termini. Therefore, the SGP2 (DPR1) product may be involved in membrane localization of an essential component in the mating-factor signaling pathway.
Chapter
Snakes comprise nearly 4,000 extant species found on all major continents except Antarctica. Morphologically and ecologically diverse, they include burrowing, arboreal, and marine forms, feeding on prey ranging from insects to large mammals. Snakes are strikingly different from their closest lizard relatives, and their origins and early diversification have long challenged and enthused evolutionary biologists. The origin and early evolution of snakes is a broad, interdisciplinary topic for which experts in palaeontology, ecology, physiology, embryology, phylogenetics, and molecular biology have made important contributions. The last 25 years has seen a surge of interest, resulting partly from new fossil material, but also from new techniques in molecular and systematic biology. This volume summarises and discusses the state of our knowledge, approaches, data, and ongoing debates. It provides reviews, syntheses, new data and perspectives on a wide range of topics relevant to students and researchers in evolutionary biology, neontology, and palaeontology.
Chapter
Research on type 1 rhodopsins spans now a history of 50 years. Originally, just archaeal ion pumps and sensors have been discovered. However, with modern genetic techniques and gene sequencing tools, more and more proteins were identified in all kingdoms of life. Spectroscopic and other biophysical studies revealed quite diverse functions. Ion pumps, sensors, and channels are imprinted in the same seven-helix transmembrane protein scaffold carrying a retinal prosthetic group. In this review, molecular biology methods are described, which enabled the elucidation of their function and structure leading to optogenetic applications.
Article
The International Narcotics Research Conference (INRC), founded in 1969, has been a successful forum for research into the actions of opiates, with an annual conference since 1971. Every year, scientists from around the world have congregated to present the latest data on novel opiates, opiate receptors and endogenous ligands, mechanisms of analgesic activity and unwanted side effects, etc. All the important discoveries in the opiate field were discussed, often first, at the annual INRC meeting. With an apology to important events and participants not discussed, this review presents a short history of INRC with a discussion of groundbreaking discoveries in the opiate field and the researchers who presented from the first meeting up to the present.
Article
We identified a bovine papilloma virus function encoded by the E6/E7 gene, which is required for both BPV high-copy-number replication and maintenance of transformation of cultured cells. A cDNA copy of this gene was isolated and expressed from a retrovirus vector. We found that complete complementation of a BPV low-copy-number mutant (dl576) by the cDNA encoding the E6/E7 gene was temporally dependent. When both the E6/E7 cDNA and dl576 were introduced together into cells, wild-type replication and stable transformation resulted. In contrast, introduction of the complementing cDNA into cells already carrying dl576 led to only partial amplification of the resident mutant DNA accompanied by a restoration of the transformed phenotype. These results, along with other findings, suggest that the establishment of BPV plasmid replication occurs in two stages: an initial amplification of the incoming DNA followed by stable homeostatic replication which maintains the existing copy number.
Article
The Saccharomyces cerevisiae GPA1, STE4, and STE18 genes encode products homologous to mammalian G-protein alpha, beta, and gamma subunits, respectively. All three genes function in the transduction of the signal generated by mating pheromone in haploid cells. To characterize more completely the role of these genes in mating, we have conditionally overexpressed GPA1, STE4, and STE18, using the galactose-inducible GAL1 promoter. Overexpression of STE4 alone, or STE4 together with STE18, generated a response in haploid cells suggestive of pheromone signal transduction: arrest in G1 of the cell cycle, formation of cellular projections, and induction of the pheromone-inducible transcript FUS1 25- to 70-fold. High-level STE18 expression alone had none of these effects, nor did overexpression of STE4 in a MATa/alpha diploid. However, STE18 was essential for the response, since overexpression of STE4 was unable to activate a response in a ste18 null strain. GPA1 hyperexpression suppressed the phenotype of STE4 overexpression. In addition, cells that overexpressed GPA1 were more resistant to pheromone and recovered more quickly from pheromone than did wild-type cells, which suggests that GPA1 may function in an adaptation response to pheromone.
Article
Both 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase isozymes of the yeast Saccharomyces cerevisiae are predicted to contain seven membrane-spanning domains. Previous work had established the utility of the histidinol dehydrogenase protein domain, encoded by HIS4C, as a topologically sensitive monitor that can be used to distinguish between the lumen of the endoplasmic reticulum and the cytoplasm. This study directly tested the structural predictions for HMG-CoA reductase by fusing the HIS4C domain to specific sites in the HMG-CoA reductase isozymes. Yeast cells containing the HMG-CoA reductase-histidinol dehydrogenase fusion proteins grew on histidinol-containing medium if the HIS4C domain was present on the cytoplasmic side of the endoplasmic reticulum membrane but not if the HIS4C domain was targeted to the endoplasmic reticulum lumen. Systematic exchanges of transmembrane domains between the isozymes confirmed that both isozymes had equivalent membrane topologies. In general, deletion of an even number of putative transmembrane domains did not interfere with the topology of the protein, but deletion or duplication of an odd number of transmembrane domains inverted the orientation of the protein. The data confirmed the earlier proposed topology for yeast HMG-CoA reductase, demonstrated that the yeast enzymes are core glycosylated, and provided in vivo evidence that the properties of transmembrane domains were, in part, dependent upon their context within the protein.
Article
We constructed and screened a cDNA library made from simian virus 40 (SV40)-transformed NIH 3T3 cells, and we isolated cDNAs representing genes that are differentially expressed between the parental cell and its SV40-transformed derivative. We found only a small number of cDNAs representing such genes. Two isolated cDNA clones represented RNAs expressed at elevated levels in the transformed cell line in a manner relatively independent of growth conditions. The expression of two other cDNAs was growth specific because transformed cells and nonconfluent parental cells contained higher levels of the homologous RNAs than did confluent, contact-inhibited parental cells. Another cDNA was well expressed in confluent parental and confluent transformed cells, but not in nonconfluent cells. The expression of some of these cDNAs varied strikingly in different mouse cell lines. Thus the genotype or histories of different cell lines can also affect the expression of certain genes. Interestingly, the only cDNA isolated that was expressed exclusively in the transformed cell was from an SV40 message. We focused on a growth-specific cDNA which we show is derived from a mouse endogenous retrovirus-like family called VL30. We sequenced the 3' long terminal repeat (LTR) of this transcriptionally active VL30 gene. This LTR has good homology with other VL30 LTR sequences, but differences occur, particularly upstream of the VL30 promoter. We found that VL30 gene expression varied in different mouse cell lines such that C3H cell lines had very low levels of VL30 transcripts relative to NIH 3T3 cell lines. However, Southern analysis showed that both cell lines had about the same number of VL30 genes homologous to our probe and that the position of the majority of these genes was conserved. We discuss possible explanations for this difference in VL30 expression.
Article
The gene for rhodopsin, the primary light sensor of the visual system, is specifically expressed in the rod photoreceptor cells of the retina. We show here that in the rat, opsin RNA first accumulates to detectable levels at postnatal day 2 (PN2) and that nascent transcripts can be detected at PN1; this is the time when peak numbers of photoreceptor cells are generated by the final division of their neuroepithelial precursors. Accumulated opsin RNA then increases to reach the adult level, 0.06% of total retinal RNA, at about PN10. The transcription rate of the opsin gene increases to a similar extent over the same time course between PN3 and adulthood, suggesting that transcriptional activation is responsible for the increase in opsin expression. We used the antibody RET-P1 to show that rhodopsin protein is also detectable at PN2 and that the number of cells expressing the protein increases with time in a central-to-peripheral gradient in the retina. This increase in the number of differentiating photoreceptors in the tissue appears to account for much of the increase in opsin gene transcription and RNA accumulation. In situ hybridization to opsin RNA shows that it is restricted to the photoreceptor layer from the time it can first be detected, at PN7. Later in development, when RET-P1 staining shifts to the photoreceptor outer segments, opsin RNA becomes localized to the inner segments, suggesting that the distributions of opsin protein and RNA are related.
Thesis
Generalised Progressive Retinal Atrophy (GPRA) is a collective term describing a number of phenotypically and genotypically heterogeneous, autosomal recessive retinal dystrophies and degenerations segregating in specific breeds of dog. All result in blindness and collectively, show much similarity to Retinitis Pigmentosa, a group of important human inherited retinal degenerations. GPRA in the Irish setter is given the locus designation rcd-1 (Rod-Cone Dysplasia Type 1) and presents as an early onset photoreceptor dystrophy with rapid, progressive loss of cells complete by approximately one year. Biochemically, the earliest detected abnormality is an accumulation of retinal cGMP, due to a defective phosphodiesterase activity. Therefore, the possibility that a mutation in the rod cGMP phosphodiesterase beta subunit (pdeb) gene causes rcd-1 in Irish setters was investigated. The normal canine pdeb cDNA, was cloned and sequenced, spanning the entire coding region to the poly-A tail. Additionally, partial intron/exon characterisation and nucleotide substitution analysis was undertaken to investigate the relationship of canine pdeb to its homologue in other species and to other members of the photoreceptor PDE family. A recently reported G to A transition in the pdeb gene described in rcd-1 setters in the USA was confirmed in affected dogs in the UK. In addition, GPRA in two other breeds, miniature longhaired dachshunds and Tibetan terriers was shown not to be due to this mutation, which creates a premature stop codon at residue 807. A rapid, non-isotopic diagnostic test was developed and used to show cosegregation of the mutation with rcd-1 phenotype in an Irish setter pedigree. Anchored PCR has been utilised to generate upstream sequence information from genomic DNA, and in tandem with mapping of the 5' end of the transcript has revealed sequence motifs in the proximal promoter of the canine pdeb gene which are conserved with the human gene. The tissue distribution of pdeb transcripts was investigated by Reverse Transcriptase-PCR and Northern analysis and the results show that transcription is not confined to the retina, but can be detected in a variety of different tissues.
Article
The ste mutants (ste2, ste4, ste5, ste7, ste11, and ste12) are insensitive to mating factors and are, therefore, sterile. Roles of the STE gene products in the GPA1-mediated mating factor signaling pathway were studied by using ste gpa1 double mutants. Mating efficiency of a ste2 mutant defective in the alpha-factor receptor increased 1,000-fold in a gpa1 background, while G1 arrest and aberrant morphology (shmoo) caused by gpa1 were not suppressed by ste2. Furthermore, the steady-state level of the FUS1 transcript, which normally increases in response to mating factors, was also elevated when the GPA1 function was impaired. These results suggest that the GPA1 protein functions downstream of the STE2 receptor. Conversely, the sterility of ste4, ste5, ste7, ste11, and ste12 mutants was not suppressed by gpa1, but the lethal phenotype of gpa1 was suppressed by these ste mutations. Northern (RNA) blotting analysis revealed that the ste7, ste11, and ste12 mutations caused reductions of 50 to 70% in the steady-state levels of the GPA1 transcript, while ste4 had a slight effect and ste5 had no effect. This implies that the suppression by ste7, ste11, and ste12 could be due to reduced syntheses of additional components, including an effector, and that suppression by ste4 and ste5 may result from direct effects on the signaling pathway. The STE4, STE5, STE7, STE11, and STE12 products, therefore, appear to specify components of the signal transduction machinery, directly or indirectly, which function together with or downstream of GPA1.
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Neurokinin A (substance K) is a peptide neurotransmitter of the tachykinin family with potential as a major mediator in human airway and gastrointestinal tissues. Neurokinin A acts via a receptor (the NK-2 receptor) believed to be localized on smooth muscle cells and pharmacologically coupled to a GTP-binding protein. To characterize the human NK-2 receptor, we prepared a partial cDNA from human tracheal RNA using the polymerase chain reaction with oligonucleotide primers derived from the bovine NK-2 receptor cDNA sequence (Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M., Nakanishi, S. (1987) Nature 329, 836-838). This partial human NK-2 receptor cDNA was used to screen a human genomic DNA library and yielded a clone, NGNK-2, of approximately 25 kilobases. Analysis of NGNK-2 indicates that it contains the entire coding sequence of the NK-2 receptor as well as 5'- and 3'-flanking sequences. The gene is organized with five exons interrupted by four introns. The complete sequence of the exons and the intron-exon junctions was determined, as were the transcription initiation site and the 3'-polyadenylation signal. Analysis of EcoRI digests of genomic DNA from human-mouse cell hybrids indicates a single gene for the human NK-2 receptor localized to chromosome 10. Sequence analysis of exons 1 and 5, where major differences occur between the human and animal species, provided information for polymerase chain reaction primers which allowed us to prepare full-length cDNA for the human NK-2 receptor. The protein predicted from the gene sequence is extended by 14 amino acids at the COOH terminus compared to the bovine and 9 residues compared to the rat molecules. The seven membrane-spanning regions are encoded by exons 1-4 and none is interrupted by introns. These regions are highly conserved among the species studied, suggesting stringent evolutionary control over these molecules.
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The complete amino acid sequence of human retina and muscle aldose reductase was determined by nucleotide analysis of cDNA clones isolated using synthetic oligonucleotide probes based on partial amino acid sequences of purified human psoas muscle aldose reductase. The cDNA sequence differs substantially in the noncoding and coding regions of recently published sequences of this enzyme. The mRNA for aldose reductase was abundantly expressed in HeLa cells, but only scarcely in a neuroblastoma cell line. Recombinant baculovirus containing one of the muscle cDNA clones was constructed and used to infect Spodoptera frugiperda (SF9) cells. A prominent protein with an apparent molecular size of 36 kDa was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the culture medium as well as in the homogenate of SF9 cells after 2 days of infection. Culture medium or the supernatant fraction of cell homogenates containing this protein had high aldose reductase activity which showed characteristics of the reported human enzyme. These findings indicate that the amino acid sequence reported in this paper represents human retina and muscle aldose reductase and that functional human aldose reductase can be expressed in large amounts in a baculovirus expression system. The result should facilitate refined structural analysis and the development of new specific aldose reductase inhibitors for the treatment of diabetic complications.
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The deletion of residues 239-272 from the hamster beta-adrenergic receptor resulted in a loss of the ability of the receptor, expressed in mouse L cells, to stimulate adenylate cyclase (Dixon, R. A. F., Sigal, I. S., Rands, E., Register, R. B., Candelore, M. R., Blake, A. D., and Strader, C. D. (1987) Nature 326, 73-77). This mutant receptor (D(239-272)beta AR) bound the agonist isoproterenol with a single class of binding sites, in contrast to the wild-type beta-adrenergic receptor, which exhibited two classes of agonist affinity sites. We now report that the affinity of D(239-272)beta AR for isoproterenol is relatively insensitive to detergent solubilization or to treatment with either GTP or NaF, indicating the absence of a receptor-Gs interaction. Whereas deletions within the region of amino acids 229-258 did not reduce the ability of the receptor to couple to Gs or to stimulate adenylate cyclase, the deletion of either of the regions 222-229 or 258-270 resulted in receptors which were unable to couple to Gs. The affinities of D(222-229)beta AR, D(239-272)beta AR, and D(258-270)beta AR toward isoproterenol were greater than that observed for the low affinity, uncoupled form of the wild-type receptor. These results suggest a role for the regions of the beta-adrenergic receptor encompassing amino acids 222-229 and 258-270, which are predicted to form amphiphilic helices, in the agonist-promoted activation of Gs.
Article
G protein-coupled receptors (GPCRs) have seven transmembrane spanning domains and comprise the largest superfamily with ~800 receptors in humans. GPCRs are attractive targets for drug discovery because they transduce intracellular signaling in response to endogenous ligands via heterotrimeric G proteins or arrestins, resulting in a wide variety of physiological and pathophysiological responses. The endogenous ligands for GPCRs are highly chemically diverse and include ions, biogenic amines, nucleotides, peptides, and lipids. In this review, we follow the KonMari method to better understand druggable lipid GPCRs. First, we have a comprehensive tidying up of lipid GPCRs including receptors for prostanoids, leukotrienes, specialized pro-resolving mediators (SPMs), lysophospholipids, sphingosine 1-phosphate (S1P), cannabinoids, platelet-activating factor (PAF), free fatty acids (FFAs), and sterols. This tidying up consolidates 46 lipid GPCRs and declutters several perplexing lipid GPCRs. Then, we further tidy up the lipid GPCR-directed drugs from the literature and databases, which identified 24 clinical drugs targeting 16 unique lipid GPCRs available in the market and 44 drugs under evaluation in more than 100 clinical trials as of 2019. Finally, we introduce drug designs for GPCRs that spark joy, such as positive or negative allosteric modulators (PAM or NAM), biased agonism, functional antagonism like fingolimod, and monoclonal antibodies (MAbs). These strategic drug designs may increase the efficacy and specificity of drugs and reduce side effects. Technological advances will help to discover more endogenous lipid ligands from the vast number of remaining orphan GPCRs and will also lead to the development novel lipid GPCR drugs to treat various diseases.
Thesis
Numerous studies have addressed the issue of the photopigment complement of the retinae of different vertebrate species. However, in most non-mammalian vertebrates, extraretinal photoreceptive sites also exist, of which the pineal complex is the most evident. The pineal organ of lower vertebrates contains photoreceptive cells, similar to the cones of the retina, and is thought to play an important role in the temporal organization of the animal, through both a neural output to the brain and the synthesis of the photoperiod-mediating hormone melatonin. The visual system of teleost fishes has been studied extensively, particularly in regard to its adaptations to different photic environments. Although several studies have addressed extraretinal photoreception in teleosts, little is known as to the exact nature of the photopigments found in the teleost pineal organ, in terms of the number of photopigments present, their spectral sensitivity and their relationship to the visual pigments of the retina. Microspectrophotometry (MSP) was used to determine the absorbance characteristics of the pineal photoreceptors of the goldfish (Carassius auratus), revealing a single photoreceptor population with a λmax of 511 nm. Because of the variable A₁/A₂ chromophore content in these photoreceptors, the pineal photopigment was also bleached and regenerated with a single artificial chromophore, 9-cis retinal. Comparison with 9-cis data from goldfish retina suggested the opsin present in the pineal was most similar to the retinal rod opsin, although slightly blue-shifted. MSP was also conducted on the native pineal photoreceptors of another cyprinid, the golden orfe (Leuciscus idus) and a characid, the Mexican tetra (Astyanax fasciatis) in which similar pineal-specific photopigments were identified. The goldfish pineal was screened for retinal opsins, and during this process a novel rod-like opsin, exo-rod opsin, was identified, which has since been demonstrated to occur uniquely within the teleost pineal organ. The full coding sequence of the goldfish exorod opsin was obtained, including gene structure.
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In optogenetics, not only channelrhodopsins but also animal opsins have been used as efficient optical control tools. Animal opsins have been extensively investigated using various spectroscopic, biochemical, electrophysiological, and biophysical research techniques for more than 150 years. Based on the accumulated insights, I introduce functional characteristics of animal opsins, and discuss their potentials as optical control tools to expand our understanding of biological phenomena. In particular, I summarize advantages of “invertebrate-type” opsins as optical control tools, because they can function without supply of “cis-” retinal isomers and they can be activated and inactivated by stimulation of different color of light.
Chapter
The vertebrate retina is a tissue that has become adapted through evolution to act as an efficient collector of photons of light. The retina acts as a two-dimensional array detector that samples light from different points in space within set time intervals, integrating the signal and counting the frequency of photon arrival. Cells in the neural retina perform some initial processing and enhancement on the captured image before it is relayed to higher order processing areas in the brain. One of the most astonishing features of the retina is that, in performing this function of converting a complex visual scene into a neural representation, all of the information initially passes via one type of cellular membrane ‘receptor’, the so-called ‘visual pigments’. When visual pigments were first extracted from the retina two important characteristics were noted. One formed the basis of the now largely abandoned visual pigment naming system and concerned the fact that the visual pigments were colored (i.e. rhodopsin, porphyropsin, cyanopsin etc.).
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Sequential reverse transcriptase, DNA polymerase, and S1 nuclease reactions can be employed to synthesize double-stranded DNA representing messenger RNA. Using reverse transcriptase products made from partially purified lysozyme, ovomucoid, and ovalbumin messengers from hen oviduct, we have characterized the Escherichia coli DNA polymerase I reaction. We have optimized for a high yield of full length second strands under conditions which require only a small amount of mRNA. The effects of several parameters (time, enzyme levels, salt concentration, monovalent cation, and temperature) on the length of products synthesized by DNA polymerase I have been investigated. Each has a significant influence on the proportion of products which are full length. Under our conditions the three reactions are efficient in synthesizing full length duplex DNA from partially purified mRNA fractions or from total poly(A)-containing RNA.
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Total poly(A)-containing RNA prepared from hen oviduct and centrifuged on an isokinetic sucrose gradient displays four peaks of optical absorbance. These have been identified by translation in vitro as lysozyme, ovomucoid, ovalbumin, and conalbumin mRNAs. Isolation and recentrifugation of the peaks results in partial purification of each mRNA. Molecular weights have been determined for the mRNAs on agarose gels containing 20 mM methylmercury hydroxide. Each mRNA possesses a number of apparently untranslated nucleotides ranging from approximately 900 bases for ovalbumin and conalbumin mRNAs to 200 bases for ovomucoid and lysozyme mRNAs. The mRNAs have been copied with avian myeloblastosis virus reverse transcriptase. Each mRNA with the exception of conalbumin gives rise to a high proportion of full length cDNA. Several parameters previously reported to influence the size distribution of cDNA had no effect on the length of cDNA made from any mRNA fraction. The proportion of full length copy does depend on the reverse transcriptase lot.
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A 7-A resolution map of the purple membrane has been obtained by electron microscopy of tilted, unstained specimens. The protein in the membrane contains seven, closely packed, alpha-helical segments which extend roughly perpendicular to the plane of the membrane for most of its width. Lipid bilayer regions fill the spaces between the protein molecules.
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A sensitive and general technique has been devised for the dual purposes of cloning genes by using antibodies as probes and isolating unknown proteins encoded by cloned DNA. The method uses an expression vector, lambda gt11 (lac5 nin5 cI857 S100), that permits insertion of foreign DNA into the beta-galactosidase structural gene lacZ and promotes synthesis of hybrid proteins. Efficient screening of antigen-producing clones in lambda gt11 recombinant cDNA libraries is achieved through lysogeny of the phage library in hflA (high-frequency lysogeny) mutant cells of Escherichia coli; lysogens produce detectable quantities of antigen on induction, even when plated at high cell densities. The vector is also designed to facilitate the isolation of proteins specified by previously cloned gene sequences. Hybrid proteins encoded by recombinant phage accumulate in strains defective in protein degradation (lon mutants) in amounts amenable to large-scale purification. Antibodies produced against the portion of the hybrid encoded by foreign DNA could in turn be used to isolate the native polypeptide from eukaryotic cells.
Article
This chapter summarizes the information that can be used to identify the helical regions and attempt to improve the assignments based on several approaches. An important source of information is provided by the action of proteolytic enzymes on the bacteriorhodopsin structure. The underlying assumption in such studies is that the soluble enzymes will act to cleave only polypeptide links that are exposed to the aqueous environment. Consequently, one expects that such cleavage sites will be in connecting loops between helices and can, therefore, be used to identify the spacer regions between helices. In addition to proteolysis, information from other kinds of modification may be useful. One important result comes from the application of lactoperoxidase-catalyzed iodination. Researchers have used an immobilized lactoperoxidase molecule to modify purple membrane fragments. Subsequent analysis showed 75% of the radioactive iodine to be located in the cyanogen bromide fragment from amino acid 118 to 145. In this region only two tyrosines are present, tyrosine 131 and 133. Consequently, the expectation is that one or both of these tyrosines must be exposed at the aqueous surface of the bactcriorhodopsin molecule. Another modification of interest is the derivitization of the membrane by a biotinyl reagent that reacts with lysine amino groups. In this study it is thought that a single lysine was modified by the reagent in such a way as to permit binding of an avidin-fcrritin complex. The use of electron microscopy resulted in a determination that only one side of the membrane, that part oriented toward the outside of the plasma membrane of the Halobacterium, is labeled.
Article
We find prompt, high stoichiometry phosphorylation of rhodopsin in response to low fractional rhodopsin bleaching in bovine rod outer segments (ROS). For example, 4 ± 1 phosphates are incorporated per bleached rhodopsin (Rho*) in 30 sec and 6 ± 2 phosphates are incorporated in 75 sec in response to bleaching 1% of the rhodopsin in the presence of 1 mM [γ-32P]ATP and 0.1 mM nonradioactive GTP. Omission of GTP leads to ca 70% inhibition of rhodopsin phosphorylation, presumably due to the GTP-binding protein blocking access of rhodopsin kinase to rhodopsin. Light induced phosphodiesterase (PDE) activation is rapidly quenched in the presence of ATP as first reported by Liebman and Pugh [Nature287, 734–736 (1980)]. The kinetics of rhodopsin phosphorylation vary with conditions and from preparation to preparation, however, they are always at least as fast as the ATP dependent quenching of PDE activation. The maximum extent of rhodopsin phosphorylation was limited by specific proteolytic trimming of the carboxyl-terminal phosphorylation sites in washed ROS membranes “stripped” of extrinsic proteins. Membrane preparations with 6,4,2, or 1 phosphate(s) incorporated/Rho* (after a 75 sec post bleach incubation) were produced by treatment with: no protease, carboxypeptidase Y (C), trypsin (T), or both T and C (TC), respectively, followed by reassociation with extrinsic membrane proteins and phosphorylation. Low fractional bleaching was required for maximum phosphorylation/Rho* in membranes which were stripped and reassociated with extrinsic proteins and in isolated ROS. Removal of C-terminal rhodopsin phosphorylation sites has little or no effect on light activation of PDE in the absence of ATP. However, in the presence of ATP the extent of the removal of C-terminal rhodopsin residues has large effects on the light activation and the shut-off of PDE. The single phosphate/Rho* that was added to TC digested membranes reduced the lifetime of Rho* but apparently was not incorporated rapidly enough under our conditions to inhibit the Vmax of PDE. The two phosphates/Rho* which were incorporated after T digestion cause a large decrease in the lifetime of Rho* as well as a decrease in the Vmax of PDE (at low bleaching levels). The four phosphates/Rho* that were added after C digestion further reduce the lifetime of Rho* and the Vmax of PDE. The 6 phosphates/Rho* which were incorporated into the unproteolyzed membranes have little additional effect on Rho* lifetime compared to 4 phosphates/Rho*. However, increasing the phosphorylation observed from 4 to 6 phosphates greatly inhibits the Vmax of PDE at intermediate bleaching levels. The extent of rhodopsin phosphorylation appears to control both the maximum velocity of the light activated PDE and the lifetime of the excited rhodopsin.
Article
We have developed a simple and rapid system for the denaturation of nucleic acids and their subsequent analysis by gel electrophoresis. RNA and DNA are denatured in 1 M glyoxal (ethanedial) and 50% (vol/vol) dimethyl sulfoxide, at 50 degrees. The glyoxalated nucleic acids are then subjected to electrophoresis through either acrylamide or agarose gels in a 10 mM sodium phosphate buffer at pH 7.0. When glyoxalated DNA molecules of known molecular weights are used as standards, accurate molecular weights for RNA are obtained. Furthermore, we have employed the metachromatic stain acridine orange for visualization of nucleic acids in gels. This dye interacts differently with double- and single-stranded polynucleotides, fluorescing green and red, respectively. By using these techniques, native and denatured DNA and RNA molecules can be analyzed on the same slab gel.
Article
A modified procedure for immunoprecipitation of specific polysomes (Mueller-Lantzsch, N., & Fan, H. (1976) Cell 9, 579) has been examined as a method for purification of specific mRNAs. Antibody-polysome complexes were recovered by adsorption to inactivated Staphylococcus aureus which has a cell wall component (protein A) with a high affinity for immunoglobulins. The technique was evaluated by precipitation of polysomes synthesizing immunoglobulin K chains from mouse myelomas. Under the immunoprecipitation conditions used neither affinity purified antibody nor formaldehyde-fixed staphylococci degraded polysomes. Using purified sheep anti-mouse immunoglobulin, 10-15% of the polysomes from the myeloma MOPC41A and about 5% from M PC11 (66.2) could be precipitated, values compatible with the amount of K chain mRNA present in these tumors. Experiments to assess specificity, in which antibody or bacteria were omitted, or in which non-immune sheep immunoglobulin or liver polysomes were substituted, indicated that nonspecific precipitation was about 5-10% of specific precipitation. Kinetic hybridization analysis showed that κ chain sequences were specifically and efficiently enriched during immunoprecipitation. The level of nonspecific precipitation was higher when the scale of the reaction was increased to allow purification of mRNA. By a combination of immunoprecipitation of specific polysomes and size fractionation of polyadenylated RNA, it was possible to isolate intact κ mRNA from MPC11 (66.2); hybridization analysis indicated that this mRNA was about 90% pure.
Article
Opsin, the apoprotein of the visual pigment rhodopsin, is synthesized on membranes of the rough endoplasmic reticulum and subsequently passes through the Golgi apparatus to the rod outer segment. This pathway parallels the early stages of biosynthesis of some secretory proteins and viral membrane glycoproteins. Most of these proteins are initially synthesized as precursor molecules with a short-lived hydrophobic extra peptide segment at the NH(2) terminus. Therefore we investigated whether or not the immediate translation product of opsin mRNA contains a similar short-lived NH(2)-terminal extra peptide. The mRNA coding for opsin was isolated from bovine retina polysomes precipitated by antibodies to opsin. The mRNA directed the cell-free synthesis of a protein comparable in size to opsin that was specifically precipitated by anti-opsin antibodies. Sequence analyses of the immunoprecipitated protein labeled with six radioactive amino acids (Met, Asn, Pro, Phe, Tyr, Val) provided the following result: [Formula: see text] (X is unknown). This partial sequence of the cell-free product corresponds exactly to the published NH(2)-terminal segment of native opsin (21 residues long) and extends beyond this region. Met-1 was shown to be the initiator methionine residue, because only the initiator [(35)S]Met-tRNA(1) (Met)-not the internal [(35)S]Met-tRNA(2) (Met)-donated the NH(2)-terminal methionine. This finding essentially rules out the possibility that Met-1 was preceded by a peptide that was rapidly cleaved. Thus opsin, and not a precursor, is the immediate product of opsin mRNA translation.
Article
Derivatives of phage lambda are described for use as vectors for fragments of DNA generated with the HindIII and EcoRI restriction enzymes. With some vectors, hybrid molecules are recognised by a change from a turbid to a clear plaque morphology resulting from the insertion of a fragment of DNA into the lambda gene coding for the phage regressor. Other vectors contain a central, replaceable fragment of DNA which imparts a readily recognisable phenotype. This central fragment may include either a gene for a mutant transfer RNA (suppressor) or a part of the lacZ gene of E. coli able to complement a lacZ host. The appropriate lacZ host and indicator plates permit the ready distinction between recombinant and vector phages by the colour of the plaques.
Article
A rapid, direct method for screening single plaques of Agt recombinant phage is described. The method allows at least 10(6) clones to be screened per day and simplifies physical containment of recombinants.
Article
Recombinant phage genomes made in reactions with purified enzymes may be recovered directly by packaging into phage heads in vitro. The process is efficient and nonselective and offers containment in initial stages of handling recombinant DNA. Ligase [poly(deoxyribonucleotide):poly-(deoxyribonucleotide) ligase (AMP-forming), EC 6.5.1.1] reaction products can recombine with endogenous phage DNA during packaging, but UV-irradiation eliminates the biological activity of the endogenous DNA.
Article
I have derived a complete restriction map of pBR322 from the total nucleotide sequence of the plasmid. Most of the restriction sites also have been demonstrated empirically. The exact sizes of all restriction fragments and the relative positions of the cuts are presented. These fragments can serve as accurate DNA size markers from small pieces up to the 4362 base pair length of pBR322. Inserts cloned in this vector may be characterized easily using this data.
Article
We describe a technique for transferring electrophoretically separated bands of RNA from an agarose gel to paper strips. The RNA is coupled covalently to diazobenzyloxymethyl groups on the paper. After transfer and appropriate treatment of the paper to destroy remaining diazo groups, specific RNA bands can be detected by hybridization with 32P-labeled DNA probes followed by autoradiography. This procedure allows detection of specific RNA bands with high sensitivity and low background.
Article
Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.
Article
We present a procedure for eucaryotic structural gene isolation which involves the construction and screening of cloned libraries of genomic DNA. Large random DNA fragments are joined to phage lambda vectors by using synthetic DNA linkers. The recombinant molecules are packaged into viable phage particles in vitro and amplified to establish a permanent library. We isolated structural genes together with their associated sequences from three libraries constructed from Drosophila, silkmoth and rabbit genomic DNA. In particular, we obtained a large number of phage recombinants bearing the chorion gene sequence from the silkmoth library and several independent clones of beta-globin genes from the rabbit library. Restriction mapping and hybridization studies reveal the presence of closely linked beta-globin genes.
Article
The Charon lambda bacteriophages have been developed as vectors for cloning. Their construction incorporates mutations that make them simple to use and also greatly increases their safety for the biological containment of cloned recombinant DNA. Three of the Charon vector phages, 3A, 4A, and 16A, have been certified for use as EK2 vector-host systems, when propagated in bulk in a special bacterial host, DP50SupF. We present here some of the data on which the safety of these systems was evaluated. DNA fragments ranging in size from 0 to 2.2 X 10(4) base pairs can be cloned in these EK2 Charon phages.
Article
Circular (e.g. simian virus 40) and linear (e.g. λ phage) DNAs have been labeled to high specific radioactivities (>108 cts/min per μg) in vitro using deoxynucleoside [α-32P]triphosphates (100 to 250 Ci/mmol) as substrates and the nick translation activity of Escherichia coli DNA polymerase I. The reaction product yields single-stranded fragments about 400 nucleotides long following denaturation. Because restriction fragments derived from different regions of the nick-translated DNA have nearly the same specific radioactivity (cts/min per 10[su3] bases), we infer that nicks are introduced, and nick translation is initiated, with equal probability within all internal regions of the DNA. Such labeled DNAs (and restriction endonuclease fragments derived from them) are useful probes for detecting rare homologous sequences by in situ hybridization and reassociation kinetic analysis.
Article
An immunological procedure for the preparation of biologically pure mRNA from cells that contain a multitude of different mRNAs is described. The double antibody technique was used employing purified antibodies prepared by means of specific immunoadsorbents. The amount of mRNA obtained from immune-precipitated polysome was within the expected theoretical yield and the procedure could be run on a large scale (25,000 A260 units of polysomes can be processed in one batch). This procedure was used for the isolation from a mouse myeloma of the mRNA coding for immunoglobulin light chain (see also Schechter, I. (1973), Proc. Nat. Acad. Sci. U. S. 70, 2256). The biological purity of this mRNA (i.e., the capacity to program the synthesis only of L chain in a cell-free system) was found to be ≥95%. Because unique features of the antigen and antibodies were not required in this system, it seems that this procedure would serve as a general solution for the isolation of biologically pure mRNA molecules from fully functional eukaryotic and prokaryotic cells.
Article
In 1967 Professor Wald, together with Professors H. K. Hartline and R. Granit, received the Nobel Prize for Medicine. The article that follows consists of most of the lecture delivered by Professor Wald last December when he received the prize in Stockholm.
Article
This chapter discusses the sequencing end-labeled DNA with base-specific chemical cleavages. In the chemical DNA sequencing method, one end-labels the DNA, partially cleaves it at each of the four bases in four reactions, orders the products by size on a slab gel, and then reads the sequence from an autoradiogram by noting which base-specific agent cleaved at each successive nucleotide along the strand. This technique sequences the DNA made in and purified from cells. No enzymatic copying in vitro is required, and either single- or double-stranded DNA can be sequenced. Most chemical schemes that cleave at one or two of the four bases involve three consecutive steps: modification of a base, removal of the modified base from its sugar, and DNA strand scission at that sugar. Base-specific chemical cleavage is only one step in sequencing DNA. The chapter presents techniques for producing discrete DNA fragments, end-labeling DNA, segregating end-labeled fragments, extracting DNA from gels, and the protocols for partially cleaving it at specific bases using the chemical reactions. The chapter also discusses the electrophoresis of the chemical cleavage products on long-distance sequencing gels and a guide for troubleshooting problems in sequencing patterns.
Article
We have isolated 16 peptides from a cyanogen bromide digest of rhodopsin. These cyanogen bromide peptides account for the complete composition of the protein. Methionine-containing peptides from other chemical and enzymatic digests of rhodopsin have allowed us to place the cyanogen bromide peptides in order, yielding the sequence of the protein. We have completed the sequence of most of the cyanogen bromide peptides. This information, in conjunction with that from other laboratories, forms the basis for our prediction of the secondary structure of the protein and how it may be arranged in the disk membrane.
Article
The Drosophila melanogaster gene Sgs4 encodes one of the glue polypeptides, sgs-4, synthesized in the larval salivary gland. We have examined the structure and expression of Sgs4 in five strains that produce abundant amounts of sgs-4 and its mRNA and in four that do not. The nonproducers include three Japanese strains that accumulate trace amounts of mRNA and one strain, BER-1, that contains no detectable Sgs4 RNA. Sgs4 carries a tandem array of repeated 21 bp elements within its coding sequence. The number of elements per array varies, causing considerable differences in the lengths of Sgs4 and its mRNA among the strains. These differences in length are not correlated with differences in mRNA abundance; rather, the low or zero abundance in nonproducers correlates with the loss of DNA upstream from the gene. The Japanese nonproducers carry a 52 bp deletion 305 bp upstream from the 5' end of Sgs4, and BER-1 carries a 95 bp deletion 392 bp upstream. Curiously, each deletion encompasses one or more of the salivary-gland-specific DNAase I-hypersensitive sites which are known to flank the Sgs4 gene.
Article
Our previous work has shown that the treatment of bovine rhodopsin with the proteolytic enzyme papain gives rise to a cleaved, but fully functional, complex consisting of three fragments, H, M and L (heavy, medium and light), held together by strong non-covalent forces. By using some of the chemical and physical differences between the three fragments, a protocol for the preparative isolation of each fragment was devised. Purified M-fragment, which had been radiochemically labelled at the retinal-binding site was treated with CNBr and the mixture subjected to a multi-step separation to furnish a retinyl peptide. The sequence analysis of the latter showed that the retinal-binding lysine residue was located at position 296 from the N-terminal of rhodopsin (or residue 53 from the C-terminal). In order to ascertain the position of the cytoplasmic loop which exists between the M- and L-fragments, radiochemically labelled L-fragment was isolated from the cleaved complex. The purified L-fragment was shown to consist of two populations of peptides which were produced by the action of papain on the bonds between Lys-311 and Gln-312 and between Gln-312 and Phe-313.
Article
The covalent sequence of the carboxyl-terminal one-third of bovine rhodopsin has been determined. It is 108 amino acids in length and consists of three hydrophilic regions exposed to the aqueous environment at membrane surfaces and two hydrophobic regions which penetrate the disc membrane lipid bilayer. This carboxyl-terminal third of rhodopsin contains the retinal binding site, sites of phosphorylation, the fast-reacting sulfhydryl group and the thermolytic fragment F2. Hydrophobic helices in the sequence have been predicted by a new method developed for application to membrane proteins and a hypothetical "helical hairpin" space-filling working model has been constructed.
Article
Preparation of the three hexadecanucleotides, dGpTpApTpCpApCpGpApGpGpCpCpCpTpT, dCpGpApCpGpApGpCpGPTpGpApCpApCpC and cTpGpCpCpGpGpCpCpApCpGpApTpGpCpG, is described by a rapid and simple solid-phase method on polyacrylamide supports. The syntheses were performed by the extension of the method described in the previous paper using di and trinucleotides of defined sequences as an incoming 3′-phosphodiester unit. Although the coupling yields to form phosphotriester bonds are slightly lower than those for the homothymidylic acid series, pure polydeoxyribonucleotides of defined sequences can be synthesized without any major difficulty.
Article
Synthesis of two oligothymidylic acids, tridecamer and nonadecamer, is described by a rapid and simple solid-phase method on two kinds of polyacrylamide supports derivatized from commercially available Enzacryl Gel K-2. The syntheses were performed by the phosphotriester method using di- and trithymidylic acid blocks as the incoming 3′-phosphodiester component. High coupling yields were consistently obtained and the final product was isolated very easily by high performance liquid chromatography on Permaphase AAX.
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Synthesis of double-stranded DNA complementary to lysozyme. ovomucoid, and ovalbumin mRNAs
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Characterization of bovine rhodcpsin mRNA and cDNA Screening lambda gt recombinant clones by hybridization to single plaques in situ
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Characterization of bovine rhodopsin mRNA and cDNA
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Baehr, W.. and Applebury. M. (1983). Characterization of bovine rhodcpsin mRNA and cDNA. Biophys. J. 41, 34Oa.