RESUMO
The locus for autosomal dominant congenital stationary night blindness (adCSNB) has recently been assigned to distal chromosome 4p by linkage analysis in a large Danish family. Within the candidate gene encoding the beta-subunit of rod photoreceptor cGMP-specific phosphodiesterase (beta PDE), we have identified a heterozygous C to A transversion in exon 4, predicting a His258Asp change in the polypeptide. We found a perfect cosegregation (Zmax = 22.6 at theta = 0.00) of this mutation with the disease phenotype suggesting that this missense mutation is responsible for the disease in this pedigree. Homozygous nonsense mutations in the beta PDE gene have been found recently in patients with autosomal recessive retinitis pigmentosa, a common hereditary photoreceptor dystrophy.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , Genes Dominantes , Cegueira Noturna/genética , Mutação Puntual , Segmento Externo da Célula Bastonete/enzimologia , 3',5'-GMP Cíclico Fosfodiesterases/deficiência , Adulto , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos Par 4 , Dinamarca , Feminino , Heterozigoto , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Cegueira Noturna/classificação , Cegueira Noturna/enzimologia , Linhagem , Polimorfismo Genético , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
We have previously shown that postnatal expression of the viral oncoprotein SV40 T antigen in rod photoreceptors (transgene MOT1), at a time when retinal cells have withdrawn from the mitotic cycle, leads to photoreceptor cell death (Al-Ubaidi et al., 1992. Proc. Natl. Acad. Sci. USA. 89:1194-1198). To study the effect of the specificity of the promoter, we replaced the mouse opsin promoter in MOT1 by a 1.3-kb promoter fragment of the human IRBP gene which is expressed in both rod and cone photoreceptors during embryonic development. The resulting construct, termed HIT1, was injected into mouse embryos and five transgenic mice lines were established. Mice heterozygous for HIT1 exhibited early bilateral retinal and brain tumors with varying degrees of incidence. Histopathological examination of the brain and eyes of three of the families showed typical primitive neuroectodermal tumors. In some of the bilateral retinal tumors, peculiar rosettes were observed, which were different from the Flexner-Wintersteiner rosettes typically associated with human retinoblastomas. The ocular and cerebral tumors, however, contained Homer-Wright rosettes, and showed varying degrees of immunoreactivity to antibodies against the neuronal specific antigens, synaptophysin and Leu7, but not to antibodies against photoreceptor specific proteins. Taken together, the results indicate that the specificity of the promoter used for T antigen and/or the time of onset of transgene expression determines the fate of photoreceptor cells expressing T antigen.
Assuntos
Antígenos Virais de Tumores/genética , Proteínas do Olho , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas/genética , Proteínas de Ligação ao Retinol/genética , Vírus 40 dos Símios/genética , Animais , Antígenos de Diferenciação/análise , Sequência de Bases , Encéfalo/patologia , Neoplasias Encefálicas/etiologia , Neoplasias Encefálicas/patologia , Neoplasias Oculares/etiologia , Neoplasias Oculares/patologia , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos/embriologia , Dados de Sequência Molecular , Retina/patologia , Distribuição TecidualRESUMO
A bovine rhodopsin complementary DNA probe was used to detect homologous visual pigment genes in a variety of species. Under stringent DNA hybridization conditions, genomic DNA from most vertebrate species carried a single homologous fragment. Additional homologies were detected in some vertebrates by reducing the hybridization stringency. Homologous fragments were also detected in DNA isolated from invertebrate species, a unicellular alga, and an archaebacterium; many of these fragments were homologous to a Drosophila opsin probe. These results suggest that photosensory pigments in a wide variety of species arose from a common precursor.
Assuntos
Sequência de Bases , Proteínas do Olho/genética , Pigmentos da Retina/genética , Homologia de Sequência do Ácido Nucleico , Animais , Bovinos , Galinhas , DNA , Enzimas de Restrição do DNA , Drosophila , Camundongos , Hibridização de Ácido Nucleico , Plantas , Rodopsina/genética , Opsinas de Bastonetes , OvinosRESUMO
Detailed biochemical, structural and physiological studies of the role of Ca2(+)-binding proteins in mammalian retinal neurons have yielded new insights into the function of these proteins in normal and pathological states. In phototransduction, a biochemical process that is responsible for the conversion of light into an electrical impulse, guanylate cyclases (GCs) are regulated by GC-activating proteins (GCAPs). These regulatory proteins respond to changes in cytoplasmic Ca2+ concentrations. Disruption of Ca2+ homeostasis in photoreceptor cells by genetic and environmental factors can result ultimately in degeneration of these cells. Pathogenic mutations in GC1 and GCAP1 cause autosomal recessive Leber congenital amaurosis and autosomal dominant cone dystrophy, respectively. This report provides a recent account of the advances, challenges, and possible future prospects of studying this important step in visual transduction that transcends to other neuronal Ca2+ homeostasis processes.
Assuntos
Proteínas Ativadoras de Guanilato Ciclase/fisiologia , Guanilato Ciclase/metabolismo , Doenças Retinianas/fisiopatologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sinalização do Cálcio/fisiologia , Motivos EF Hand , Ativação Enzimática , Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/genética , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Células Fotorreceptoras/fisiologia , Alinhamento de Sequência , Visão Ocular/fisiologiaRESUMO
Vertebrate photoreceptor cells can signal the absorption of a single photon and then modulate their response as the intensity of the light and the intensity of the background illumination vary, and it has long been recognized that Ca2+ ions contribute to the underlying processes. Recently, several Ca(2+)-binding proteins of the EF-hand family were identified that mediate the actions of Ca2+ during the response to light. Molecular interactions between these Ca(2+)-binding proteins and their cellular targets are amenable to study owing in part to the unique features of phototransduction. In addition, two of the proteins, recoverin and guanylate cyclase activating protein (GCAP), appear to be involved in separate degenerative diseases of the retina that arise in humans and in animal models of human disease. Information obtained from these studies should also be relevant to the growing number of homologous proteins found in other neural tissues.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Cálcio/fisiologia , Retina/patologia , Retina/fisiologia , Animais , Humanos , Retina/químicaRESUMO
Examination of the role of Ca(2+)-binding proteins (CaBPs) in mammalian retinal neurons has yielded new insights into the function of these proteins in normal and pathological states. In the last 8 years, studies on guanylate cyclase (GC) regulation by three GC-activating proteins (GCAP1-3) led to several breakthroughs, among them the recent biochemical analysis of GCAP1(Y99) mutants associated with autosomal dominant cone dystrophy. Perturbation of Ca(2+) homeostasis controlled by mutant GCAP1 in photoreceptor cells may result ultimately in degeneration of these cells. Here, detailed analysis of biochemical properties of GCAP1(P50L), which causes a milder form of autosomal dominant cone dystrophy than constitutive active Y99C mutation, showed that the P50L mutation resulted in a decrease of Ca(2+)-binding, without changes in the GC activity profile of the mutant GCAP1. In contrast to this biochemically well-defined regulatory mechanism that involves GCAPs, understanding of other processes in the retina that are regulated by Ca(2+) is at a rudimentary stage. Recently, we have identified five homologous genes encoding CaBPs that are expressed in the mammalian retina. Several members of this subfamily are also present in other tissues. In contrast to GCAPs, the function of this subfamily of calmodulin (CaM)-like CaBPs is poorly understood. CaBPs are closely related to CaM and in biochemical assays CaBPs substitute for CaM in stimulation of CaM-dependent kinase II, and calcineurin, a protein phosphatase. These results suggest that CaM-like CaBPs have evolved into diverse subfamilies that control fundamental processes in cells where they are expressed.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Oftalmopatias/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/biossíntese , Calmodulina/biossíntese , Bovinos , Mapeamento Cromossômico , Clonagem Molecular , Ativação Enzimática , Escherichia coli/metabolismo , Guanilato Ciclase/biossíntese , Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Células Fotorreceptoras de Vertebrados/enzimologia , Retina/enzimologia , Alinhamento de SequênciaRESUMO
Light-dependent changes in the binding of G-protein were analyzed in outer segment disk membranes obtained from photoreceptors of the toad (Bufo marinus) retina. Isolated, intact retinas, incubated in oxygenated Ringer's solution at 23 +/- 1 degree C, were subjected to various conditions of illumination and then incubated in darkness for specified periods. The retinas were then chilled (0-4 degrees C) and the receptor outer segments (ROS) were isolated. Binding of the alpha- and beta-subunits of G-protein to the ROS membranes was analyzed by quantitating G alpha and G beta extracted from the membranes with hypotonic medium lacking GTP vs. hypotonic medium containing GTP (H and HG extracts, respectively). For retinas illuminated and then immediately chilled for analysis, the extent of G binding (relative abundance of G alpha, beta in the HG extract) increased with the extent of bleaching of the visual pigment. Near-maximal binding was observed after bleaches of greater than or equal to 30%. With an increasing period of incubation in darkness after approximately 70% bleaching, the extent of binding declined gradually to low levels characteristic of unbleached retinas. The period required for half-completion of the decline was approximately 10(3) s. A gradual decline in G binding, from a rapidly developing peak value, was also observed with an increasing period of exposure to intense light. Viewed in the context of previous electrophysiological data, our results indicate that sustained bleaching desensitization of the rods does not depend upon a persisting state of "tight binding" (immobilization) of G-protein by bleached visual pigment.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Luz , Células Fotorreceptoras/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Adaptação Fisiológica , Animais , Bufo marinus , Bovinos , Centrifugação com Gradiente de Concentração , Adaptação à Escuridão , Membranas/metabolismo , Peptídeos/isolamento & purificaçãoRESUMO
Absorption of light by rhodopsin or cone pigments in photoreceptors triggers photoisomerization of their universal chromophore, 11-cis-retinal, to all-trans-retinal. This photoreaction is the initial step in phototransduction that ultimately leads to the sensation of vision. Currently, a great deal of effort is directed toward elucidating mechanisms that return photoreceptors to the dark-adapted state, and processes that restore rhodopsin and counterbalance the bleaching of rhodopsin. Most notably, enzymatic isomerization of all-trans-retinal to 11-cis-retinal, called the visual cycle (or more properly the retinoid cycle), is required for regeneration of these visual pigments. Regeneration begins in rods and cones when all-trans-retinal is reduced to all-trans-retinol. The process continues in adjacent retinal pigment epithelial cells (RPE), where a complex set of reactions converts all-trans-retinol to 11-cis-retinal. Although remarkable progress has been made over the past decade in understanding the phototransduction cascade, our understanding of the retinoid cycle remains rudimentary. The aim of this review is to summarize recent developments in our current understanding of the retinoid cycle at the molecular level, and to examine the relevance of these reactions to phototransduction.
Assuntos
Retina/metabolismo , Retinoides/metabolismo , Visão Ocular/fisiologia , Animais , Adaptação à Escuridão/fisiologia , Humanos , Luz , Retina/efeitos da radiação , Rodopsina/fisiologia , Rodopsina/efeitos da radiaçãoRESUMO
An isoform of RGS9 was recently identified as the GTPase activating protein in bovine and mouse rod and cone photoreceptors. To explore the potential role of the RGS9 gene in human retinal disease, we determined its exon/intron arrangement, and investigated its expression in human retina. The results show that the gene, located on 17q24, consists of 19 exons and spans more than 75kb of genomic DNA. The entire gene was found to be contained on a single BAC clone with an insert size of 170kb. The major transcripts of the gene are alternatively spliced into a 9.5kb retina-specific transcript (RGS9-1) and a brain specific 2.5kb transcript (RGS9-2). Exons 1-16 are constitutive and present in both variants. Exon 17 contains the 3' end of the open reading frame and the 3'-UTR of the RGS9-1 variant. In RGS9-2, exon 17 is alternatively spliced and joined to exons 18 and 19 that are not present in the retina variant. Immunolocalization with a monoclonal antibody recognizing the retina and brain variants shows abundant expression in photoreceptors and possibly very low levels in cell types of the inner retina. Owing to the specific expression of RGS9-1 in photoreceptors the RGS9 gene is a candidate gene for RP17, a form of autosomal retinitis pigmentosa, located on the long arm of chromosome 17.
Assuntos
Processamento Alternativo , Genes/genética , Proteínas RGS/genética , Idoso , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Western Blotting , Encéfalo/metabolismo , Pré-Escolar , Mapeamento Cromossômico , Cromossomos Humanos Par 17/genética , Corpo Estriado/química , Cricetinae , DNA/análise , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Variação Genética , Humanos , Células Híbridas , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Íntrons , Mamíferos/genética , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Proteínas RGS/análise , Retina/química , Retina/metabolismo , Homologia de Sequência de AminoácidosRESUMO
We expressed the gamma subunit of mouse rod photoreceptor cGMP phosphodiesterase (PDE) in the bacterial pGFX-2TK expression vector which produces a cleavable 40 kDa fusion protein. The fusion protein can be isolated in a one step procedure by affinity chromatography on glutathione beads. The yield of purified fusion protein is approximately 10 mg from 1 liter of bacterial culture, or about 3 mg of PDE gamma equivalent to the PDE gamma content of approximately 200,000 mouse retinas. Both the fusion protein and the cleaved PDE gamma, to which a short kinase domain remains attached, are biologically active, inhibiting activated PDE in a manner comparable to native PDE gamma. Immobilized PDE gamma binds transducin alpha subunit charged with GTP, PDE alpha and beta subunits, and, unexpectedly, arrestin (S-antigen).
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , Sequência de Aminoácidos , Animais , Antígenos/metabolismo , Arrestina , Sequência de Bases , Clonagem Molecular , Proteínas do Olho/metabolismo , Guanosina Trifosfato/metabolismo , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Proteínas Recombinantes/metabolismo , Transducina/metabolismoRESUMO
Cyclic GMP phosphodiesterase (PDE) is rod photoreceptor disk membrane-associated via C-terminal lipid tails. PDEdelta, a recently identified subunit, was shown to disrupt PDE/membrane interaction under physiological conditions, without affecting PDE catalytic activity. We found that a PDEdelta ortholog from the eyeless nematode Caenorhabditis elegans (termed CEdelta) solubilizes bovine PDE in vitro with an EC50 very similar to PDEdelta. Immobilized PDEdelta and CEdelta both bind, in addition to bovine PDE, an N-terminal fragment of human retinitis pigmentosa GTPase regulator, but not rhodopsin kinase and Ran binding protein 1. The results suggest that PDEdelta and CEdelta may regulate membrane binding of a variety of proteins in photoreceptors and other tissues.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Helminto/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/biossíntese , 3',5'-GMP Cíclico Fosfodiesterases/genética , Animais , Caenorhabditis elegans/genética , Bovinos , Membrana Celular/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Glutationa Transferase/metabolismo , Proteínas de Helminto/biossíntese , Proteínas de Helminto/genética , Humanos , Diester Fosfórico Hidrolases/metabolismo , Ligação Proteica , Dobramento de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
Amphibians have been employed extensively to study the anatomy, physiology, biochemistry, and cell biology of the visual system for decades, yet there have been no reports concerning the primary structure of amphibian visual transduction components. Thus, we have determined the entire nucleotide sequence of frog (Rana pipiens) rhodopsin cDNA, including a putative transcription start point and poly A tail, by sequence analysis of PCR products and mRNA. The open reading frame predicts an opsin of 354 residues, six residues longer than the mammalian rod opsins, containing 11 potential phosphorylation sites in the C-terminal domain. RNA blot analysis revealed two transcripts of ca. 1.7 and 3.1 kb. Frog rhodopsin exhibits approximately 85% identity to mammalian rhodopsin at the amino acid level. Sequence analysis of additional components will produce the framework from which a more detailed understanding of amphibian phototransduction can emerge.
Assuntos
Rodopsina/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Rana pipiensRESUMO
We have characterized overlapping cDNA clones encoding cGMP phosphodiesterase (PDE) alpha- and beta-subunits of mouse retinal rod photoreceptors. The open reading frames predict an alpha-subunit of 100 kDa (856 residues), and a beta-subunit of 99 kDa (853 residues). Sequence analysis of two of twelve beta-subunit clones predicts the presence in the retina of an additional PDE, termed beta', which is generated by alternative splicing of the beta-subunit gene. beta' differs from beta only at the C-terminus being 55 residues shorter and lacking the Caax motif found at the C-termini of both the alpha- and beta-subunits. A 300 residue segment thought to contain the active site is present in the C-terminal half of alpha, beta and beta'.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , DNA/genética , Isoenzimas/genética , Células Fotorreceptoras/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Íntrons , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Splicing de RNARESUMO
Glucagon and beta-adrenergic agents increase cAMP levels and stimulate Ca2+ influx in liver cells. There is no consensus as to the mechanism by which these hormones stimulate the influx of Ca2+. Using mouse retinal rod CNGCalpha cDNA probes, we cloned rat liver and skeletal muscle, and human hepatic CNGCalpha subunit sequences showing 97-100% identity with the human rod channel. In order to assess channel activity, the effect of cyclic nucleotides on free intracellular Ca2+ levels of isolated hepatocytes was measured. Dibutyryl-cAMP was more effective in increasing free Ca2+ levels than dibutyryl-cGMP. These data indicate that the CNGCalpha subunit is expressed in both the liver and skeletal muscle possibly mediating hormonal effects on ion fluxes.
Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/genética , Fígado/química , Músculo Esquelético/química , Nucleotídeos Cíclicos/farmacologia , Sequência de Aminoácidos , Animais , Bucladesina/farmacologia , Cálcio/metabolismo , Membrana Celular/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Dibutiril GMP Cíclico/farmacologia , Expressão Gênica , Humanos , Canais Iônicos/análise , Canais Iônicos/química , Fígado/fisiologia , Masculino , Camundongos , Dados de Sequência Molecular , Peso Molecular , Músculo Esquelético/fisiologia , Nitroprussiato/farmacologia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Homologia de Sequência de AminoácidosRESUMO
We have identified three overlapping 5'-truncated mouse opsin cDNA clones by immunologically screening a lambda gt11 retina expression library. Using one of the cDNA clones as a probe, we isolated a 5 kb genomic fragment that encompassed the complete coding sequence for mouse opsin. The coding region for opsin was interrupted by four introns positioned precisely as those previously described for other mammalian opsins. In contrast to the single major opsin mRNA in the bovine and human retina, Northern analysis of mouse retina RNA demonstrated the presence of at least five distinct species of polyadenylated opsin mRNAs. Their sizes ranged from 1.7 kb to 5.1 kb.
Assuntos
DNA/isolamento & purificação , Proteínas do Olho/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/genética , Enzimas de Restrição do DNA , DNA Recombinante , Éxons , Íntrons , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Células Fotorreceptoras , Retina/análise , Pigmentos da Retina , Opsinas de Bastonetes , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
The 23 kDa protein was localized by immunocytochemistry to photoreceptor cells of the mouse retina, and bovine and mouse cDNA clones were isolated and sequenced. The deduced amino acid sequences showed that the mouse 23 kDa protein is 91% identical to the bovine protein, and is the same as S-modulin, the CAR (cancer-associated retinopathy) protein and recoverin, the Ca(2+)-dependent activator of photoreceptor guanylate cyclase. The amino acid sequence reveals two Ca2+ binding sites, no internal repeats, 59% homology to the chicken visinin protein and 40% homology to calmodulin while Northern analysis demonstrated a single 1.0 kb mRNA species in bovine and mouse retina.
Assuntos
Antígenos de Neoplasias/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas do Olho , Lipoproteínas , Células Fotorreceptoras/química , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/química , Sequência de Bases , Sítios de Ligação , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Calmodulina/química , Bovinos , Clonagem Molecular , DNA/química , DNA/isolamento & purificação , Hipocalcina , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Peso Molecular , Proteínas do Tecido Nervoso/química , Reação em Cadeia da Polimerase , Recoverina , Retina/química , Homologia de Sequência do Ácido NucleicoRESUMO
We cloned the guanylate cyclase activating proteins, GCAP1 and GCAP2, from chicken retina and examined their expression in normal and predegenerate rdlrd chicken retina. Northern analyses show that the amounts of the single transcripts encoding GCAP1 and GCAP2 are reduced to about 70% of normal levels in rdlrd retina. Western analyses reveal that GCAP2 levels appear normal in this retina, while GCAP1 levels are reduced by more than 90%. The specific downregulation of GCAP1 in rdlrd retina is consistent with a model for this disease in which activation of guanylate cyclase in the photoreceptors is abnormal, resulting in low levels of cGMP and an absence of phototransduction.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Regulação da Expressão Gênica/fisiologia , Retina/química , Degeneração Retiniana/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/análise , Galinhas , Clonagem Molecular , DNA Complementar/genética , Proteínas Ativadoras de Guanilato Ciclase , Dados de Sequência Molecular , Fenótipo , RNA Mensageiro/análise , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
PURPOSE: To study mechanisms leading to photoreceptor degeneration in mouse models for autosomal dominant retinitis pigmentosa (adRP) based on the rhodopsin P23H mutation. METHODS: Mice of a transgenic line expressing a rhodopsin triple mutant, V20G, P23H, and P27L (GHL), were mated with rhodopsin (rho) knockout mice. Littermates of various ages and genotypes (GHL+rho+/+, GHL+rho+/-, and GHL+rho-/-) were examined for outer nuclear layer thickness and outer segment formation (histology), fate of mutant rhodopsin (immunocytochemistry), and photoreceptor function (electroretinogram; ERG). RESULTS: Mice expressing GHL-rhodopsin in the absence of wild-type rhodopsin had severe retinopathy, which was nearly complete by postnatal day (P)30. GHL-rhodopsin formed homodimers nearly exclusively on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, whereas wild-type rhodopsin predominantly formed monomers. Expression level of mutant rhodopsin in predegenerate (P10) GHL+rho-/- retinas was low, approximately 10% to 25% of normal levels. No elaboration of disc membrane or outer segment formation was observed at any time point examined. The mutant rhodopsin was found mostly in perinuclear locales (endoplasmic reticulum; ER) as evidenced by colocalization using the antibodies Rho1D4 and calnexin-NT. CONCLUSIONS: GHL-rhodopsin dimerizes, localizes to the ER, and fails to transport and support outer segment formation. Additionally, the mutant protein does not support a scotopic ERG a-wave and accelerates photoreceptor degeneration over that occurring with the rhodopsin knockout alone. These findings indicate a cytotoxic effect of the mutant protein, probably elicited by an unfolded protein response.
Assuntos
Mutação , Células Fotorreceptoras de Vertebrados/ultraestrutura , Degeneração Retiniana/genética , Rodopsina/genética , Transgenes/genética , Animais , Primers do DNA/química , Eletroforese em Gel de Poliacrilamida , Eletrorretinografia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Genótipo , Immunoblotting , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Imunoeletrônica , Células Fotorreceptoras de Vertebrados/metabolismo , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Rodopsina/metabolismoRESUMO
PURPOSE: A line of transgenic mice containing the simian virus (SV) 40 T-antigen (T-ag) gene driven by the beta-luteinizing hormone (BLH) promoter developed bilateral retinoblastoma and primitive neuroectodermal tumors (PNET) of the midbrain. Midbrain tumors arose from the subependymal layer of the cerebral aqueduct. Bilateral ocular and brain tumors ("trilateral") were found in three other SV40 T-ag transgenic murine lines containing different promoters (murine interphotoreceptor retinoid-binding protein (IRBP), human IRBP, and alpha A-crystallin). To gain insight into the regulatory mechanisms involved in central nervous system tumorigenesis, the authors examined brain tumors from four lines of SV40 T-ag mice with different promoters. METHODS: Formalin-fixed brain tumors were examined from four lines of transgenic mice containing different promoters linked to the protein coding region of the enhancerless SV40 T-ag oncogene. Transgenes contained the following promoters: BLH, mouse 1.8-kb IRBP, human 1.3-kb IRBP, and alpha A-crystallin. RESULTS: Mice with a 1.8-kb IRBP promoter develop retinal photoreceptor and pineal tumors. Intracranial tumors arising from the subependymal layer of the third ventricle also were observed. Mice with a 1.3-kb IRBP promoter exhibit bilateral retinal PNET and PNET originating from the subependymal layer of the third ventricle. Mice with the alpha A-crystallin promoter exhibit bilateral lens tumors and PNET of the midbrain. CONCLUSIONS: Ocular tumors in these mice may be ascribed to the promoter-driven, tissue-specific expression of SV40 T-ag. The common finding of PNET arising from the subependymal layer of the diencephalon is unlikely to be promoter related. These findings indicate that a regulatory region specific to the subependymal layer of the cerebral aqueduct and third ventricle resides in the structural region of the SV40 T-ag gene.