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1.
Adv Exp Med Biol ; 664: 79-87, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20238005

RESUMO

To identify genes expressed in cone photoreceptors, we previously carried out subtractive hybridization and microarrays of retinal mRNAs from normal and cd (cone degeneration) dogs. One of the isolated genes encoded ZBED4, a novel protein that in human retina is localized to cone photoreceptors and glial Müller cells. ZBED4 is distributed between nuclear and cytoplasmic fractions of the retina and it readily forms homodimers, probably as a consequence of its hATC dimerization domain. In addition, the ZBED4 sequence has several domains that suggest it may function as part of a co-activator complex facilitating the activation of nuclear receptors and other factors (BED finger domains) or as a co-activator/co-repressor of nuclear hormone receptors (LXXLL motifs). We have identified several putative ZBED4-interacting proteins and one of them is precisely a co-repressor of the estrogen receptor alpha.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas do Olho/metabolismo , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas do Olho/química , Proteínas do Olho/genética , Citometria de Fluxo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Frações Subcelulares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transfecção
2.
Mamm Genome ; 11(6): 422-7, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10818205

RESUMO

C57BL/6J-c(2J) (c2J) albino mice showed much less damage to their photoreceptors after exposure to prolonged light than BALB/c mice and seven other albino strains tested. There were no gender differences, and preliminary studies suggested that the c2J relative protective effect was a complex trait. A genome-wide scan using dinucleotide repeat markers was carried out for the analysis of 194 progeny of the backcross (c2J x BALB/c)F(1) x c2J and the thickness of the outer nuclear layer (ONL) of the retina was the quantitative trait reflecting retinal damage. Our results revealed a strong and highly significant quantitative trait locus (QTL) on mouse Chromosome (Chr) 3 that contributes almost 50% of the c2J protective effect, and three other very weak but significant QTLs on Chrs 9, 12, and 14. Interestingly, the Chrs 9 and 12 QTLs corresponded to relative susceptibility alleles in c2J (or relative protection alleles in BALB/c), the opposite of the relative protective effect of the QTLs on Chrs 3 and 14. We mapped the Rpe65 gene to the apex of the Chr 3 QTL (LOD score = 19.3). Northern analysis showed no difference in retinal expression of Rpe65 message between c2J and BALB/c mice. However, sequencing of the Rpe65 message revealed a single base change in codon 450, predicting a methionine in c2J and a leucine in BALB/c. When the retinas of aging BALB/c and c2J mice reared in normal cyclic light were compared, the BALB/c retinas showed a small but significant loss of photoreceptor cells, while the c2J retinas did not. Finding light damage-modifying genes in the mouse may open avenues of study for understanding age-related macular degeneration and other retinal degenerations, since light exposures may contribute to the course of these diseases.


Assuntos
Cromossomos/genética , Células Fotorreceptoras/efeitos da radiação , Característica Quantitativa Herdável , Envelhecimento , Animais , Sequência de Bases , Feminino , Genótipo , Luz , Escore Lod , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Repetições de Microssatélites , Células Fotorreceptoras/patologia , Retina/patologia , Retina/efeitos da radiação
3.
Proc Natl Acad Sci U S A ; 97(10): 5551-6, 2000 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-10805811

RESUMO

The rd7 mouse, an animal model for hereditary retinal degeneration, has some characteristics similar to human flecked retinal disorders. Here we report the identification of a deletion in a photoreceptor-specific nuclear receptor (mPNR) mRNA that is responsible for hereditary retinal dysplasia and degeneration in the rd7 mouse. mPNR was isolated from a pool of photoreceptor-specific cDNAs originally created by subtractive hybridization of mRNAs from normal and photoreceptorless rd mouse retinas. Localization of the gene corresponding to mPNR to mouse Chr 9 near the rd7 locus made it a candidate for the site of the rd7 mutation. Northern analysis of total RNA isolated from rd7 mouse retinas revealed no detectable signal after hybridization with the mPNR cDNA probe. However, with reverse transcription-PCR, we were able to amplify different fragments of mPNR from rd7 retinal RNA and to sequence them directly. We found a 380-nt deletion in the coding region of the rd7 mPNR message that creates a frame shift and produces a premature stop codon. This deletion accounts for more than 32% of the normal protein and eliminates a portion of the DNA-binding domain. In addition, it may result in the rapid degradation of the rd7 mPNR message by the nonsense-mediated decay pathway, preventing the synthesis of the corresponding protein. Our findings demonstrate that mPNR expression is critical for the normal development and function of the photoreceptor cells.


Assuntos
Mapeamento Cromossômico , Células Fotorreceptoras de Vertebrados/fisiologia , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/genética , Degeneração Retiniana/genética , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Sequência de Bases , Códon de Terminação , Primers do DNA , Eletrorretinografia , Marcadores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Mutantes , Dados de Sequência Molecular , Receptores Nucleares Órfãos , Células Fotorreceptoras de Vertebrados/patologia , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência
4.
Gene ; 227(2): 257-66, 1999 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-10023077

RESUMO

Retinal photoreceptor cells are particularly vulnerable to degenerations that can eventually lead to blindness. Our purpose is to identify and characterize genes expressed specifically in photoreceptors in order to increase our understanding of the biochemistry and function of these cells, and then to use these genes as candidates for the sites of mutations responsible for degenerative retinal diseases. We have characterized a cDNA, a fragment of which (SR3.1) was originally isolated by subtractive hybridization of adult, photoreceptorless rd mouse retinal cDNAs from the cDNAs of normal mouse retina. The full-length sequence of this cDNA was determined from clones obtained by screening mouse retinal and eye cDNA libraries and by using the 5'- and 3'-RACE methods. Both Northern blot analysis and in situ hybridization showed that the corresponding mRNA is expressed in rod and cone photoreceptors. The gene encoding this cDNA was mapped to the X chromosome using an interspecific cross. Based on the nucleotide and amino acid sequences, as well as chromosome mapping, we determined that this gene is the mouse ortholog (Xlrs1) of the human X-linked juvenile retinoschisis gene (XLRS1). Analysis of the predicted amino acid sequence indicates that the Xlrs1 mRNA may encode a secretable, adhesion protein. Therefore, our data suggest that X-linked juvenile retinoschisis originates from abnormalities in a photoreceptor-derived adhesion protein.


Assuntos
Proteínas do Olho/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Doenças Retinianas/genética , Cromossomo X/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Proteínas do Olho/química , Humanos , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Células Fotorreceptoras de Vertebrados/citologia , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Homologia de Sequência
5.
Mol Vis ; 4: 16, 1998 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-9743540

RESUMO

PURPOSE: To screen the exons of the genes encoding the beta3-subunit (GNB3) and gammac-subunit (GNGT2) of cone transducin for mutations in a large number of unrelated patients with various forms of inherited retinal disease including cone dystrophy, cone-rod dystrophy and macular dystrophy. METHODS: Exons of the two genes were screened for mutations by denaturing gradient gel electrophoresis (DGGE) and/or single strand conformation polymorphism electrophoresis (SSCP); any variants were sequenced directly. RESULTS: Although many sequence variants were found in both genes, none could be associated with disease. Additionally, the gene structure and sequence of the coding exons of GNB3 were determined and compared with those of the dog homolog. Both human and canine GNB3 have nine coding exons and their two predicted amino acid sequences have 97% identity. CONCLUSIONS: The results indicate that GNB3 and GNGT2 are unlikely sites of mutations responsible for inherited retinal degenerations that predominantly effect cone-mediated function (cone and cone-rod dystrophies) or have a predilection for disease in the macula (macular dystrophies).


Assuntos
Doenças Retinianas/genética , Transducina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cães , Testes Genéticos , Humanos , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , Homologia de Sequência
6.
Invest Ophthalmol Vis Sci ; 39(10): 1775-81, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9727399

RESUMO

PURPOSE: To characterize the cDNA and the organization of the gene encoding the cone-specific gamma subunit of transducin (Tgamma c) and to examine this gene as a candidate for the recessively inherited cone photoreceptor degeneration in the cd dog. METHODS: Canine Tgamma c cDNA was cloned and sequenced. Polymerase chain reaction (PCR) was used to define the Tgamma c gene structure, northern blot analysis to examine the level of expression of Tgamma c mRNA in control and cd-affected retinas, and immunocytochemistry to determine the presence and localization of Tgamma c in normal and cd retinas. RESULTS: Immunocytochemical results showed Tgamma c localized to cone photoreceptor outer segments in the normal retina, whereas no Tgamma c immunoreactivity was observed in the cd retinas. However, the level of transcription and the primary structure of the cloned cDNA coding for the 69-amino acid protein were identical in retinas from wild-type and affected dogs. CONCLUSIONS: Although Tgamma c immunoreactivity was specifically absent in the cd dog retina, no differences were detected between normal and cd retinas in the nucleotide sequence of Tgamma c mRNA or in its synthesis. These results indicate that a mutation in the Tgamma c gene may not be causally associated with the cd dog disease. These findings suggest that possible abnormalities in posttranslational modification of Tgamma c or defective assembly of the transducin alphabetagamma complex could lead to rapid degradation of Tgamma c.


Assuntos
DNA Complementar/análise , Doenças do Cão/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/veterinária , Transducina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting/veterinária , Clonagem Molecular , Doenças do Cão/metabolismo , Doenças do Cão/patologia , Cães , Técnicas Imunoenzimáticas/veterinária , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/veterinária , RNA/isolamento & purificação , RNA Mensageiro/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Análise de Sequência de DNA , Transducina/metabolismo
7.
Gene ; 194(1): 47-56, 1997 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-9266671

RESUMO

The cDNA for the beta3-subunit of cone-specific transducin (Tbeta3) was cloned and characterized from wild type dogs, and used in linkage studies as a candidate gene for cone degeneration. Sequence analysis of the Tbeta3 cDNA revealed an open reading frame of 1020 bp, potentially coding for a protein of 340 amino acids (aa). The deduced aa sequence of canine Tbeta3 shares 97% identity with the previously identified human Tbeta3, and 82% identity with bovine rod-specific transducin (Tbeta1). RT-PCR and sequencing of the amplified products demonstrated that the retinal canine Tbeta3 gene is expressed in two different transcripts which can be generated by alternative splicing of the intron in the 3'-untranslated region (UTR). The short and the long mRNAs differ in the length of their 3'-UTR by 456 nt. We have also determined the genomic organization of the canine Tbeta3 gene; it consists of ten exons and the first exon is in the 5'-UTR. The cDNA encoding Tbeta3 from cd-affected dogs was also cloned and sequenced. We found no differences at the nucleotide level between the cDNAs isolated from normal and diseased retinas. The level of transcription of Tbeta3 mRNA in the cd dog retina appeared to be normal. Linkage analysis of a crossbred informative pedigree showed five obligate recombinants out of nine informative offspring. These results suggest that Tbeta3 is not a candidate gene for the cone degeneration of the cd mutant.


Assuntos
Doenças do Cão/genética , Cães/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/veterinária , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transducina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Primers do DNA , Éxons , Humanos , Íntrons , Escore Lod , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutação Puntual , Reação em Cadeia da Polimerase , Degeneração Retiniana/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transducina/química
8.
Nucleic Acids Res ; 20(23): 6189-94, 1992 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-1282235

RESUMO

A comparison of the nucleotide sequences from genomic DNA and cDNA of the ndhB gene from maize chloroplasts shows that the ndhB transcript is edited by C-to-U transitions at six positions which appear to exist as editing sites also in the chloroplast ndhB genes from rice and tobacco but not from liverwort. In order to identify possible sequence determinants necessary for editing, the sequences surrounding the newly identified ndhB and previously identified ndhA editing sites were compared with each other and with editing sites observed in plant mitochondrial transcripts. Among the chloroplast editing sites two closely positioned ndhB sites show similarity by sharing a common octanucleotide. The existence of the identical octanucleotide in the ndhJ gene whose transcript is not edited at the respective position, shows, however, that this octanucleotide is not sufficient to elicit the editing process. On the other hand, several of the chloroplast editing sites show sequence similarities with certain sets of consensus sequences reported earlier for editing sites of plant mitochondria. This supports the view that the editing processes of both plant organelles share common components and/or mechanistic steps and that the consensus sequences are part of the determinants necessary for editing.


Assuntos
Cloroplastos/metabolismo , Mitocôndrias/metabolismo , Proteínas de Plantas/genética , Edição de RNA , Zea mays/genética , Sequência de Aminoácidos , Sequência de Bases , Códon , DNA , Genes de Plantas , Dados de Sequência Molecular , Plantas/genética , RNA , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica
10.
FEBS Lett ; 204(2): 288-92, 1986 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-3015681

RESUMO

The primary structure of the gamma-subunit of cyclic GMP phosphodiesterase was determined by parallel analysis of the amino acid sequence of the protein and nucleotide sequence of the corresponding cDNA. The enzyme gamma-subunit contains 87 amino acid residues, its N-terminal amino group being acetylated.


Assuntos
3',5'-GMP Cíclico Fosfodiesterases , DNA/isolamento & purificação , Retina/enzimologia , 3',5'-GMP Cíclico Fosfodiesterases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , Brometo de Cianogênio , Enzimas de Restrição do DNA , Fragmentos de Peptídeos/isolamento & purificação , Escatol/análogos & derivados , Tripsina
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