RESUMO
Similar to visual arrestins of other vertebrates, two subtypes of medaka visual arrestins, KfhArr-R1 and KfhArr-C, are selectively expressed in rods and cones, respectively [Hisatomi et al. (1997) FEBS Lett. 411, 12-18]. We isolated a cDNA encoding the third arrestin, KfhArr-R2, from a medaka retinal cDNA library. Phylogenetic analysis of arrestin sequences suggests that KfhArr-R2 is classified into the rod arrestin subtype. In situ hybridization indicated that KfhArr-R2 mRNA is localized in most of the rod photoreceptors, suggesting that both KfhArr-R1 and -R2 are co-expressed in rods. Antisera against KfhArr-R2 recognized outer segments, but anti-KfhArr-R1 antisera reacted with cell bodies and synaptic termini in light-adapted rods. It seems likely that KfhArr-R1 and -R2 play different roles in rod photoreceptors.
Assuntos
Arrestina/metabolismo , Proteínas de Peixes , Oryzias/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sequência de Aminoácidos , Animais , Arrestina/classificação , Arrestina/genética , Western Blotting , Clonagem Molecular , Biblioteca Gênica , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Oryzias/genética , Filogenia , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Visão OcularRESUMO
Retinal cDNAs encoding the putative opsins, dg3 and dg4, were isolated from a diurnal gecko, Phelsuma madagascariensis longinsulae. dg3 mRNA is localized in about 20% of the thin members of type C double cones, and likely encodes an opsin of the ultraviolet-sensitive pigment. Surprisingly, dg4 is very similar to chicken pinopsin, a pineal-specific photoreceptive molecule. An anti-dg4 antiserum recognized a small population of photoreceptor outer segments in the retina and a large number of pinealocytes. Our results suggest that P. m. longinsulae expresses pinopsin in its retina, which usually plays a role as a photoreceptive molecule in the pineal organ.
Assuntos
Proteínas do Tecido Nervoso/biossíntese , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Opsinas de Bastonetes/biossíntese , Sequência de Aminoácidos , Animais , Proteínas Aviárias , DNA Complementar/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Lagartos , Microscopia de Fluorescência , Dados de Sequência Molecular , Filogenia , Pigmentos Biológicos/biossíntese , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Two kinds of cDNA fragments (KfhArr-R and KfhArr-C) encoding the putative arrestins of killifish, Oryzias latipes, were isolated. The distributions of these transcripts were investigated by in situ hybridization, and it was demonstrated that KfhArr-R and KfhArr-C are expressed in, respectively, rod and all four types of cone cells. The deduced amino acid sequences of KfhArr-R and KfhArr-C are closely related to human S-antigen (rod arrestin) and X-arrestin (cone arrestin), respectively. Phylogenetic analysis of arrestin sequences suggests that vertebrate visual arrestins form a single cluster distinct from other arrestins and diverged to form rod and cone subtypes before the divergence between teleosts and tetrapods. It is speculated that the divergence pattern of vertebrate visual arrestins may prove to be reflected in the divergence of the proteins participating in the respective phototransduction cascades.
Assuntos
Arrestina/genética , Proteínas do Olho , Proteínas de Peixes , Células Fotorreceptoras/metabolismo , Sequência de Aminoácidos , Animais , Arrestina/biossíntese , Sequência de Bases , DNA Complementar , Humanos , Hibridização In Situ , Peixes Listrados , Dados de Sequência Molecular , Células Fotorreceptoras/citologia , Filogenia , Homologia de Sequência de AminoácidosRESUMO
Phosducin (PD) is a regulatory protein of vertebrate phototransduction cascades. In mammalian retina, it has been thought that only one kind of PD commonly exists in both rods and cones. However, we have found two kinds of PD (OIPD-R and OIPD-C) in the retina of a teleost, medaka (Oryzias latipes). In situ hybridization and immunohistochemical analysis demonstrated that OIPD-R and -C are selectively expressed in rods and cones, respectively. The antiserum against medaka PDs recognized two kinds of proteins in bluegill (Lepomis macrochirus) retina. These results suggest that rod- and cone-specific PDs exist in teleost retinas, probably creating differences in light adaptation between rods and cones.
Assuntos
Proteínas do Olho/genética , Fosfoproteínas/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas Quinases/genética , Sequência de Aminoácidos , Animais , Western Blotting , Bovinos , Clonagem Molecular , Primers do DNA/química , Proteínas do Olho/metabolismo , Reguladores de Proteínas de Ligação ao GTP , Técnicas Imunoenzimáticas , Hibridização In Situ , Dados de Sequência Molecular , Oryzias , Perciformes , Fosfoproteínas/metabolismo , Reação em Cadeia da Polimerase , Proteínas Quinases/metabolismo , Sondas RNA , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de AminoácidosRESUMO
Most geckos are nocturnal forms and possess rod retinas, but some diurnal genera have pure-cone retinas. We isolated cDNAs encoding the diurnal gecko opsins, dg1 and dg2, similar to nocturnal gecko P521 and P467, respectively. Despite the large morphological differences between the diurnal and nocturnal gecko photoreceptor types, they express phylogenetically closely related opsins. These results provide molecular evidence for the reverse transmutation, that is, rods of an ancestral nocturnal gecko have backed into cones of diurnal geckos. The amino acid substitution rates of dgl and dg2 are higher than those of P521 and P467, respectively. Changes of behavior regarding photic environment may have contributed to acceleration of amino acid substitutions in the diurnal gecko opsins.
Assuntos
Evolução Molecular , Lagartos/genética , Opsinas de Bastonetes/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Dados de Sequência Molecular , Opsinas de Bastonetes/análise , Opsinas de Bastonetes/classificaçãoRESUMO
Previously, we reported that an opsin (Rc-MS) belonging to the SWS2 group opsins is expressed in bullfrog green rods [Hisatomi, O. et al., FEBS Lett., 1999, 447, 44-48]. An anti-Rc-MS antiserum recognized the cones of the Japanese common newt, Cynops pyrrhogaster, which has no green rods. We isolated a cDNA encoding an SWS2 group opsin (Cp-SWS2) from this newt and found that Cp-SWS2 is expressed in a small population of the cones. Our results suggest that SWS2 opsins can be expressed in either green rods or cones of caudata. It seems reasonable to suppose that green rods arose before amphibia were divided into caudata and anura.
Assuntos
Células Fotorreceptoras/metabolismo , Retina/metabolismo , Opsinas de Bastonetes/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , DNA Complementar/análise , Imuno-Histoquímica , Hibridização In Situ , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/análise , Opsinas de Bastonetes/classificação , Opsinas de Bastonetes/genética , Salamandridae , Homologia de Sequência de AminoácidosRESUMO
In frog retina there are special rod photoreceptor cells ('green rods') with physiological properties similar to those of typical vertebrate rods ('red rods'). A cDNA fragment encoding the putative green rod visual pigment was isolated from a retinal cDNA library of the bullfrog, Rana catesbeiana. Its deduced amino acid sequence has more than 65% identity with those of blue-sensitive cone pigments such as chicken blue and goldfish blue. Antisera raised against its C-terminal amino acid sequence recognized green rods. It is concluded that bullfrog green rods contain a visual pigment which is closely related to the blue-sensitive cone pigments of other non-mammalian vertebrates.
Assuntos
Proteínas de Anfíbios , Proteínas do Olho/química , Rana catesbeiana , Células Fotorreceptoras Retinianas Bastonetes/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Molecular , Proteínas do Olho/genética , Dados de Sequência Molecular , Conformação Proteica , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Cones/química , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Especificidade da Espécie , Distribuição TecidualRESUMO
Two kinds of retinal cDNA fragments (OIGRK-R and -C) encoding the putative G-protein-coupled receptor kinases (GRKs) were isolated from medaka, Oryzias latipes. OIGRK-R appears to be closely related to the rhodopsin kinase (RK) found in the outer segments of mammalian photoreceptors, but the deduced amino acid sequence of OIGRK-C shows less than 50% identity to those of GRKs known to date, suggesting that OIGRK-C is a novel GRK subtype (GRK7). The mRNA of OIGRK-R is detectable in rods, and that of OIGRK-C is found in all four types of cone photoreceptor. The C-terminal of OIGRK-R has a consensus sequence for farnesylation, whereas, surprisingly, OIGRK-C has a consensus sequence for geranylgeranylation. Our result are consistent with the concept that lower vertebrates have rod- and cone-specific opsin kinases.
Assuntos
Oryzias/fisiologia , Proteínas Serina-Treonina Quinases , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Células Fotorreceptoras Retinianas Cones/enzimologia , Sequência de Aminoácidos , Animais , DNA Complementar/isolamento & purificação , Hibridização In Situ/métodos , Lagartos/fisiologia , Dados de Sequência Molecular , Filogenia , Retina/enzimologia , Células Fotorreceptoras Retinianas Bastonetes , Salamandridae/fisiologia , Homologia de Sequência de AminoácidosRESUMO
PURPOSE: Cone photoreceptors in teleost fish retina are organized into a precise, crystalline mosaic in which the four spectral subtypes have a consistent position relative to each other. The objective of the current study was to describe the spatial and temporal progression of photoreceptor differentiation in the embryonic goldfish retina to understand how the retinal cone mosaic might be produced. METHODS: To identify developing photoreceptors when they first begin to express a specific opsin, the authors used in situ hybridization with cRNA probes generated from cDNA for rod opsin and red, green, blue, and ultraviolet cone opsins from goldfish (Carassius auratus). RESULTS: In the retina, rod opsin was expressed first, and it was restricted to a small patch of regularly spaced, precocious rods located near the ventronasal edge of the retina, close to the choroid fissure. The patch enlarged by recruitment of additional rods in a circular path, moving from ventral to nasal to dorsal to temporal retina. Expression of cone opsins began approximately 10 hours after rod opsin was first expressed, and differentiation of cone photoreceptors followed the spatial pattern laid down by the early rods. The temporal order of onset of cone opsin expression was red, then green, then blue, then ultraviolet. When rod and red cone opsin probes were combined, the number of labeled cells was additive, suggesting that these two opsins are expressed in separate populations of photoreceptors. CONCLUSIONS: The onset of opsin expression in goldfish retina follows a highly ordered spatio-temporal pattern. Early differentiation and regular spacing of the precocious rods was unexpected and suggested that they may play a role in cone mosaic patterning. The order of subsequent cone opsin expression was related to the relative positions of cone subtype in the mosaic, suggesting the possibility that inductive interactions among developing photoreceptors may be responsible for patterning the cone mosaic array.
Assuntos
Carpa Dourada/embriologia , Células Fotorreceptoras/metabolismo , Retina/embriologia , Células Fotorreceptoras Retinianas Cones/citologia , Opsinas de Bastonetes/biossíntese , Animais , Diferenciação Celular , Feminino , Hibridização In Situ , Masculino , Células Fotorreceptoras/citologia , Células Fotorreceptoras/embriologia , Sondas RNA , Retina/citologiaRESUMO
The molecular phylogenetic tree of vertebrate visual pigments, constructed on the basis of amino acid sequence identity, suggests that the visual pigments can be classified into five groups (L, ML, MS, S and Rh) and that their genes have evolved along these five gene lines. Goldfish has a UV-sensitive visual pigment (S group) localized in miniature single cone cells. Medaka has one type of rod cell containing rhodopsin (Rh group) and four types of cone cells, each of which contains a specific visual pigment with an absorption maximum that differs from those of the others. Frogs have a violet-sensitive visual pigment (S group) in small single cone cells and a blue-sensitive visual pigment (MS group) in green rod cells. Although nocturnal and diurnal geckos have rod- and cone-based retinas, respectively, they have phylogenetically closely related visual pigments. The pigments in each line may have restricted absorption maxima. We have cloned cDNAs encoding molecules involved in the phototransduction system of visual cells, such as phosphodiesterase, opsin kinase and arrestin. We then constructed phylogenetic trees of these molecules with the deduced amino acid sequences. The resulting phylogenetic trees show that these molecules are classified into two groups; one is expressed in cones and another in rods, suggesting that rods and cones contain homologous molecules with different amino acid sequences. These differences may result in the different light responses of rods and cones.
Assuntos
Evolução Molecular , Pigmentos da Retina/classificação , Pigmentos da Retina/genética , Animais , Humanos , Filogenia , Visão Ocular/fisiologiaRESUMO
The gene encoding photoactive yellow protein (PYP) was isolated from Ectothiorhodospira halophila, and a high-level expression system for PYP was constructed in Escherichia coli. The molecular weight and the absorption spectrum of PYP expressed in E. coli were identical with those of the native PYP isolated from E. halophila. The amino acid residues which might interact with the chromophore (Tyr42, Glu46, Thr50, Arg52, and Cys69) were mutated by site-directed mutagenesis and the absorption spectra of these mutants were examined to study the chromophore/protein interaction in PYP. The former three substitutions (Y42F, E46Q, and T50V) brought about red-shifts of the absorption spectra, but the substitution of Arg52 (R52Q) brought about no change and that of Cys69 (C69S) led to no formation of pigments. These results suggest that Tyr42, Glu46, and Thr50 strongly interact with the chromophore, while Arg52 does not contribute the color tuning of PYP.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Mutagênese Sítio-Dirigida/genética , Fotorreceptores Microbianos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sítios de Ligação/genética , Chromatiaceae/química , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Solubilidade , EspectrofotometriaRESUMO
Structural changes during the thermal phase transition of purple membrane were observed by freeze-fracture electron microscopy. Native Halobacterium halobium cells contain broad purple membrane areas about 1 micron in diameter. The boundary separating purple and red membranes is obvious. On warming at 80 degrees C, particles of red membrane spread out beyond the boundary. Then a purple membrane area is eventually divided into small areolae of similar size, about 0.1 micron in diameter. By cooling down slowly, the purple membrane area is reformed and the crystalline arrangement also restored.
Assuntos
Bacteriorodopsinas/metabolismo , Carotenoides/metabolismo , Halobacterium/metabolismo , Técnica de Fratura por Congelamento , Cinética , Microscopia Eletrônica , TemperaturaRESUMO
The primary structures of two opsins from the brachyuran crab Hemigrapsus sanguineus were deduced from the cDNA nucleotide sequences. Both deduced proteins were composed of 377 amino acid residues and included residues highly conserved in visual pigments of other species, and the proteins were 75 % identical to each other. The distribution of opsin transcripts in the compound eye, determined by in situ hybridization, suggested that the mRNAs of the two opsins were expressed simultaneously in all of the seven retinular cells (R1-R7) forming the main rhabdom in each ommatidium. Two different visual pigments may be present in one photoreceptor cell in this brachyuran crab. The spectral sensitivity of the compound eye was also determined by recording the electroretinogram. The compound eye was maximally sensitive at about 480 nm. These and previous findings suggest that both opsins of this brachyuran crab produce visual pigments with maximal absorption in the blue-green region of the spectrum. Evidence is presented that crustaceans possess multiple pigment systems for vision.
RESUMO
A visual pigment was extracted from lamprey retina and was expressed in cultured mammalian cells (293S) using a cDNA fragment isolated from lamprey retina. The extracted pigment, a putative lamprey rhodopsin, had an absorption maximum at 503 nm. The recombinant lamprey rhodopsin, reconstituted with 11-cis-retinal, showed an absorption maximum at about 500 nm. Both pigments reacted with an anti-bovine rhodopsin antibody (Rh29), which recognizes the short photoreceptor cells in lamprey retina. Unlike rhodopsins of higher vertebrates, the lamprey rhodopsin bleached gradually in the presence of 100 mM hydroxylamine even in the dark. Our results suggest that, despite its high similarities with other vertebrate rhodopsins, lamprey rhodopsin has a character different from those of higher vertebrates.
Assuntos
Retina/metabolismo , Rodopsina/química , Animais , Bovinos , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Lampreias , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Rodopsina/genética , Rodopsina/isolamento & purificação , Análise EspectralRESUMO
Five cDNA fragments (KFH-R, -V, -G, -B and -Rh) encoding the putative visual pigments of killifish were isolated and sequenced. Judging from the deduced amino acid sequences, each cDNA falls into a different group of the five major families of vertebrate visual pigment genes. In situ hybridization localized the mRNA of KFH-R and -G to the principle and accessory members, respectively, of double cones. Visual pigment genes KFH-Rh, -B and -V were expressed in the rods, and the long and short single cones, respectively. It is suggested that the relationships between the cell types and their respective visual pigment gene groups may be a common pattern among teleost fishes.
Assuntos
Peixes Listrados/genética , Células Fotorreceptoras/metabolismo , Pigmentos da Retina/genética , Sequência de Aminoácidos , Animais , DNA Complementar/genética , Expressão Gênica , Hibridização In Situ , Peixes Listrados/anatomia & histologia , Peixes Listrados/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/genética , Retina/metabolismo , Pigmentos da Retina/química , Pigmentos da Retina/metabolismoRESUMO
Genomic DNA fragments in exon 4 of chicken, goldfish and salmon visual pigments were amplified by polymerase chain reaction, using oligonucleotide mixtures as primers, and hypothetical phylogenetic trees were drawn up from the deduced amino acid sequences. The results suggest that vertebrate visual pigments have evolved along at least five lines, and that these lines diverged from an ancestral gene before the bony fishes diverged from the rest of the higher vertebrates.
Assuntos
Filogenia , Pigmentos da Retina/classificação , Sequência de Aminoácidos , Animais , Galinhas , Éxons , Amplificação de Genes , Carpa Dourada , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Pigmentos da Retina/análise , Pigmentos da Retina/genética , SalmãoRESUMO
A cDNA full length encoding a putative ultraviolet (UV)-sensitive visual pigment of goldfish was isolated. The deduced amino acid sequence shows 64% identity to those of human blue and chicken violet, and less identity (40-49%) to those of other vertebrate visual pigment. The mRNA is localized in the miniature short single cone cells, which are known to have a sensitivity maximum in the near UV-region.
Assuntos
DNA Circular/genética , Carpa Dourada/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Pigmentos da Retina/genética , Raios Ultravioleta , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas , Clonagem Molecular , Primers do DNA/genética , Humanos , Hibridização In Situ , Lampreias , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/análise , Células Fotorreceptoras Retinianas Cones/química , Peixe-ZebraRESUMO
Seven types of ferritinized lectin were used to examine the distribution of glycoconjugates on the outer segment membranes of lamprey photoreceptor cells. Ultrastructural pre-embedding labeling revealed that peanut agglutinin, soybean agglutinin and Ricinus communis agglutinin I were preferentially bound to the proximal, lateral and luminal surfaces of the long cell outer segments, whereas Griffonia simplicifolia agglutinin II and concanavalin A agglutinin were bound to the corresponding surfaces of the short cell outer segments. The results indicate that there is marked difference in the composition of glycoconjugates over the outer segment membranes between long and short photoreceptors.
Assuntos
Glicoconjugados/análise , Lampreias/metabolismo , Células Fotorreceptoras/química , Animais , Sítios de Ligação , Lectinas , Microscopia Eletrônica , Células Fotorreceptoras/ultraestruturaRESUMO
The cDNA encoding the putative rhodopsin of frog (Rana catesbeiana) was cloned and expressed in cultured cells. The deduced amino acid sequence (354 residues) has more than 90% identity with the rhodopsins of two other frogs (Rana pipiens and Xenopus laevis) and 80% identity with other vertebrate rhodopsins. The isoelectric point calculated from the sequence was about 8.2, which is intermediate between rhodopsins and the cone visual pigments of higher vertebrates. The cloned cDNA was expressed in cultured mammalian cells. The difference absorbance maximum before and after photobleaching was about 500 nm, the same as that observed in the retina, demonstrating that the cloned cDNA does indeed encode functional rhodopsin.
Assuntos
DNA Complementar/genética , Rana catesbeiana/genética , Rodopsina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Expressão Gênica , Dados de Sequência Molecular , Filogenia , Rana pipiens/genética , Especificidade da Espécie , Xenopus laevis/genéticaRESUMO
A cDNA fragment encoding a putative visual pigment (FCV pigment) was isolated from the bullfrog, Rana catesbeiana. Its deduced amino acid sequence shows high similarities to those of short wavelength-sensitive pigments such as human blue-, chicken violet- and goldfish ultraviolet-sensitive pigments. An antiserum against its C-terminal amino acid sequence recognized the outer segments of small cone photoreceptor cells without oil droplets. It is suggested that the FCV pigment is a short wavelength-sensitive pigment contained in small single cones which have not been characterized previously.