RESUMO
We sought to determine whether differentiation agents such as retinoids and butyrate regulate transcript levels of interphotoreceptor retinoid binding protein (IRBP) and cone rod homeobox (CRX), a homeodomain transcription factor that regulates IRBP promoter activity. WERI-Rb1 retinoblastoma cells were treated with all-trans retinol, all-trans retinoic acid, or butyrate. IRBP and CRX mRNA levels were determined by quantitative RT-PCR. Butyrate at low concentrations increased both mRNA levels but suppressed them at higher concentrations. Retinoic acid had minimal effects. Retinol increased CRX mRNA over four fold. IRBP and CRX transcript levels are sensitive to butyrate and CRX expression is sensitive to retinol.
Assuntos
Butiratos/farmacologia , Proteínas do Olho , Proteínas de Homeodomínio/efeitos dos fármacos , Retinoides/farmacologia , Proteínas de Ligação ao Retinol/efeitos dos fármacos , Transativadores/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , RNA Mensageiro/genética , RNA Neoplásico/genética , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Proteínas de Ligação ao Retinol/genética , Proteínas de Ligação ao Retinol/metabolismo , Transativadores/genética , Transativadores/metabolismo , Tretinoína/farmacologia , Células Tumorais Cultivadas , Vitamina A/farmacologiaRESUMO
PURPOSE: To determine whether antisense oligonucleotides (AODNs) targeted against CRX, a photoreceptor-specific trans-acting factor, suppress CRX expression and interphotoreceptor retinoid binding protein (IRBP) promoter activity. METHODS: Cultures of human retinoblastoma cells were transfected with chloramphenicol acetyltransferase (CAT) reporter plasmids containing a mouse IRBP promoter and AODNs directed against CRX. RT-PCR using primers specific to CRX, OTX2, GAPDH, or RNase H was conducted on total RNA isolated from retinoblastoma cells at various times following transfection with AODNs. RESULTS: Transfection of retinoblastoma cells with IRBP promoter CAT constructs alone produced high activity. Co-transfection with AODNs suppressed IRBP promoter activity in a concentration-dependent manner, with half-maximal effect produced at about 2 nM AODN concentration. Transfection with CAT constructs containing an SV40 promoter produced high activity that was unaffected by co-transfection with AODNs. RT-PCR products were obtained for all target sequences. CRX RT-PCR product from cells transfected with AODNs was greatly diminished following transfection with an AODN whereas control transcripts, including that of OTX2, were relatively unaffected. CONCLUSIONS: The CRX-specific AODNs specifically and potently suppressed CRX expression and IRBP promoter activity, as measured by RT-PCR and transient transfection assays, respectively. Little or no effect was seen on controls. These data suggest that endogenous CRX is required for IRBP promoter activity in retinoblastoma cells.
Assuntos
Proteínas do Olho , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Retina/efeitos dos fármacos , Proteínas de Ligação ao Retinol/efeitos dos fármacos , Transativadores/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Sequência de Bases/genética , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Genes Reporter/fisiologia , Vetores Genéticos/fisiologia , Gliceraldeído-3-Fosfato Desidrogenases/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenases/genética , Proteínas de Homeodomínio/genética , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição Otx , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Retina/metabolismo , Retinoblastoma , Proteínas de Ligação ao Retinol/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonuclease H/efeitos dos fármacos , Ribonuclease H/genética , Transativadores/genética , Transcrição Gênica/genética , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
The expression of interphotoreceptor retinoid binding protein (IRBP) is limited to photoreceptor cells of the retina and pinealocytes of the pineal gland. We sought to define cis-elements of the mouse IRBP 5' flanking region that are required for this restricted activity. In vitro transient transfections of retinoblastoma and neuroblastoma cells and in vivo experiments with transgenic Xenopus laevis indicate that -1783/+101 and -156/+101 IRBP gene fragments directed expression predominantly to the retina and pineal, with minor neuronal expression elsewhere. In contrast, a -70/+101 fragment was less restrictive in controlling expression, exhibiting activity not only in retina, but also in forebrain, hindbrain, spinal cord, and motor neurons innervating gills.
Assuntos
Proteínas do Olho/genética , Regiões Promotoras Genéticas , Proteínas de Ligação ao Retinol/genética , Animais , Animais Geneticamente Modificados , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Expressão Gênica , Humanos , Camundongos , Xenopus laevisRESUMO
PURPOSE: To determine the sequences of the mouse and bovine interphotoreceptor retinoid-binding protein (IRBP) 5' flanking regions and whether these 5' flanking regions contain functional IRBP promoter activity in multiple cell types using both quantitative and statistical analyses. METHODS: We sequenced the bovine and mouse 5' flanking regions of the IRBP gene and compared these sequences to the human gene sequence. To test for functional activity of this region, we used the same DNA construct, p1783, in four different cell types. Mobility shift, DNase footprints, and southwestern blots were used to determine where nuclear protein complexes bind the IRBP 5' flanking region. RESULTS: The 5' flanking regions of the bovine, human, and mouse IRBP genes exhibit sequence similarity in regions immediately adjacent to the start of transcription (roughly 350 bases in length) and also over a 220 base sequence about 1.25 to 1.50 kb upstream of the transcription start site. Two different statistical approaches showed that the IRBP 5' flanking region possesses promoter activity in four different cell types. By using mobility shift, DNase I-protection experiments, and southwestern blotting, a region of about 45 bases at position -300 was identified that specifically binds a protein from the nuclei of bovine retina and Y79 cells. CONCLUSIONS: Specific DNA binding events are an essential part of IRBP promoter activity. The conservation of sequences far upstream of the transcription start suggest that unknown physiological processes remain to be understood in IRBP transcriptional regulation.
Assuntos
Região 5'-Flanqueadora/genética , Proteínas do Olho/genética , Regiões Promotoras Genéticas/genética , Proteínas de Ligação ao Retinol/genética , Animais , Sequência de Bases , Southern Blotting , Bovinos , Pegada de DNA , Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Retina/citologia , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Transfecção , Células Tumorais CultivadasRESUMO
Interphotoreceptor retinoid binding protein (IRBP), a putative component of the visual cycle, is expressed selectively in the retina and pineal gland. This study examined whether site-specific DNA hypomethylation plays a role in this expression regulation. Southern blotting of HpaII and MspI digests of DNA from various bovine and murine tissues (whole brain, retina, pineal gland, superior colliculus, cortex, thymus, habenular nucleus, cornea, liver, tail, and kidney) revealed that specific CpG dinucleotides in the IRBP gene promoter are hypomethylated in DNA from retinal photoreceptor cells and pineal gland compared to DNA from other tissues. These sites are methylated in DNA from non-photoreceptor retinal cells. Exogenous methylation of these sites diminished DNA:protein binding in electrophoretic mobility shift assays. HpaII methylation of chloramphenicol acetyltransferase reporter constructs suppressed IRBP but not SV40 promoter activity in transiently transfected primary cultures of embryonic chick retinal cells. These data indicate that specific cytosines in the bovine and murine IRBP promoters are unmethylated in photoreceptive cells but methylated in other tissues. This differential DNA methylation may modulate IRBP gene expression since exogenous methylation of the murine sites suppresses reporter gene transcription, apparently by inhibiting DNA:protein binding events.
Assuntos
Metilação de DNA , Proteínas do Olho , Regulação da Expressão Gênica/fisiologia , Células Fotorreceptoras/metabolismo , Glândula Pineal/metabolismo , Regiões Promotoras Genéticas/fisiologia , Células Ganglionares da Retina/metabolismo , Proteínas de Ligação ao Retinol/metabolismo , Animais , Bovinos , Células Cultivadas , Embrião de Galinha , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Retina/metabolismoRESUMO
The human mitochondrial genome has not been previously known to differentially express specific mRNA transcripts. Results of northern analysis, using total RNA from two different retinal regions, demonstrate that there is differential expression of five mitochondrial genes. There is a correlation of regional expression of one of these differentially expressed genes with the gene responsible for the majority of cases of foveo-macular mitochondropathy. These findings suggest that there is selective control over specific mitochondrial messenger steady state levels.
Assuntos
DNA Mitocondrial/genética , Células Fotorreceptoras Retinianas Cones/fisiologia , Northern Blotting , Sondas de DNA , Proteínas de Ligação a DNA/genética , Expressão Gênica/fisiologia , Humanos , Isoenzimas/genética , Mitocôndrias/química , Mitocôndrias/enzimologia , Dados de Sequência Molecular , NAD/genética , RNA Mensageiro/análise , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Proteínas Virais/genéticaRESUMO
PURPOSE: Interphotoreceptor retinoid binding protein (IRBP) is expressed exclusively and to high levels in photoreceptive cells. This study was an attempt to delineate the minimal regulated control region of the murine IRBP promoter involved in this expression pattern. METHODS: Fragments of the mouse IRBP 5' flanking region were tested for promoter activity in transient transfections of embryonic chick retina cells in primary culture. Electrophoretic mobility shift assays were used to identify specific cis-acting DNA elements within these fragments. RESULTS: Nested deletion analysis of a 1783 bp fragment of the murine IRBP 5' flanking region shows that high promoter activity is maintained with truncated fragments as short as 70 bp 5' to transcription start, but is lost with truncation to 45 bases. The 1783 bp promoter is active in cultures of retina cells but not brain cells or fibroblasts. The 70 bp fragment is active in retina and brain cells but not fibroblasts. Within retina cell cultures, the 1783 bp fragment is active in photoreceptor-like and amorphous or unidentifiable cells whereas the 70 bp is additionally active in multipolar neuron-like cells. The -70 to -45 interval contains Ret-1/PCE-I (AATTAG in the IRBP gene), a proposed retina-specific consensus sequence cis element, and a same-strand reversed copy of this sequence, GATTAA, the consensus binding element of the photoreceptor-specific trans-acting factor CRX. Mutation of either element suppresses promoter activity. Paralleling promoter tissue-specificity, the -70 to -45 fragment binds a sequence-specific protein complex found in retina and brain extracts but not fibroblasts. Mutation of both or either element inhibits this binding. CONCLUSIONS: These data suggest that a trans-acting complex binds a cis-element in the -70 to -45 sequence. This binding fully activates transcription but confers only partial tissue-specificity to IRBP gene expression.
Assuntos
Proteínas de Homeodomínio/genética , Retina/metabolismo , Proteínas de Ligação ao Retinol/genética , Transativadores/genética , Animais , Encéfalo/metabolismo , Células Cultivadas , Embrião de Galinha , Eletroforese , Proteínas do Olho/genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese , Especificidade de Órgãos , Células Fotorreceptoras/metabolismo , Regiões Promotoras Genéticas , TransfecçãoRESUMO
The primate fovea is the region of the retina responsible for acute vision. This region constitutes less than 5% of the total area of the retina and has not been intensely studied at the molecular level. As a first step in the molecular characterization of the fovea, we have constructed a primary human fovea cDNA library. Experiments confirm that our cDNA library reflects a nonbiased distribution of foveal expressed sequences. Single-pass sequencing was performed on 209 randomly isolated clones from this library. Analysis of the sequences generated reveals that the distributions of fovea clones with either human mitochondrial gene sequences or repetitive elements are different than those observed in cDNA libraries made from other tissues. A significant number of the fovea expressed sequence tags (ESTs) (88, 42.1%) represent novel human ESTs. This suggests that the library will be useful in identifying new human genes. Northern analysis of specific fovea ESTs defined in this study suggests that there are significant quantitative differences in gene expression that distinguish the fovea from the rest of the retina.
Assuntos
DNA Complementar/genética , Fóvea Central/fisiologia , Expressão Gênica , Biblioteca Gênica , Retina/fisiologia , Química Encefálica , DNA Mitocondrial/genética , Fóvea Central/química , Humanos , Fígado/química , Dados de Sequência Molecular , RNA Mensageiro/análise , Sequências Repetitivas de Ácido Nucleico/genética , Retina/química , Células Fotorreceptoras Retinianas Cones , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
PURPOSE: In humans, the fovea is the region of the retina responsible for acute vision. Disorders affecting the fovea are responsible for the majority of cases of untreated blindness in the developed world, yet are poorly understood at the molecular level. Our goal is to identify genes that are preferentially expressed within the human fovea as compared to the midperipheral retina (differential fovea clones). MATERIALS AND METHODS: An unamplified fovea cDNA library was differentially screened with cDNA probes derived from either human fovea or midperipheral retina. Rounds of secondary screening and northern analysis were used to verify the expression pattern of a selective number of clones isolated. RESULTS: Forty-one differential fovea clones were isolated from a screening of 10,000 phage clones (clones). Of these clones, 31.5 % correspond to known sequences present in GenBank/EMBL and 70.7% represent novel human fovea expressed sequence tags (ESTs). Northern analysis of selected clones demonstrated that they represent genes expressed at higher levels in the human fovea than in the midperipheral retina. CONCLUSIONS: Genes that are more highly expressed in the fovea as opposed to the midperipheral retina are likely to represent essential genes for fovea function. Using our fovea cDNA library, we are able to isolate differential human fovea clones at an incidence of 41/10,000 clones screened. We demonstrate that there is a high level of differential gene expression within different regions of the human retina.
Assuntos
Fóvea Central/química , Regulação da Expressão Gênica , Genes/genética , Adolescente , Adulto , Idoso , Northern Blotting , Criança , Pré-Escolar , Clonagem Molecular/métodos , Biblioteca Gênica , Humanos , Pessoa de Meia-Idade , Mitocôndrias/química , Retina/químicaRESUMO
Retinitis pigmentosa (RP) is a genetically and clinically heterogeneous group of human disorders that is characterized by diminished retinal function, visual cell loss, and blindness. Elevated levels of TRPM-2/clusterin mRNA, a marker for the apoptotic process, have been reported in retinas from patients with advanced stage RP. In the present study we examine TRPM-2/clusterin expression in two genetically distinct mouse models of RP, the rd (retinal degeneration) and rds (retinal degeneration slow) mice. We establish that in advanced postretinal degenerative stages of the rd mutant the retinal TRPM-2/clusterin mRNA levels are highly elevated, as is seen in the case of human RP. Examination of TRPM-2/clusterin mRNA levels in retina and whole eyes from the rd mouse and morphologically normal controls during the period of retinal degeneration (postnatal days 8-21) in the rd phenotype shows that TRPM-2/clusterin mRNA levels are elevated in the rd animal, and this increase begins just after postnatal day 10 and remains high for the remainder of the time course examined. Northern analysis of rds retina and whole eyes shows a delayed increase in TRPM-2/clusterin mRNA levels relative to the rd profile, coinciding with the known period of rds retinal degeneration (postnatal day 14 to 1 year). In each case, the onset of increased TRPM-2/clusterin mRNA levels coincides with the time of photoreceptor cell death.
Assuntos
Olho/metabolismo , Regulação da Expressão Gênica , Glicoproteínas/genética , Chaperonas Moleculares , Retina/metabolismo , Retinose Pigmentar/metabolismo , Animais , Northern Blotting , Morte Celular , Clusterina , Glicoproteínas/biossíntese , Camundongos , Camundongos Mutantes , RNA Mensageiro/análise , Células Fotorreceptoras Retinianas Bastonetes/citologia , Fatores de TempoRESUMO
In closing, molecular biology has helped us in the study of uveitis, in defining the parts of the IRBP molecule that cause EAU/EAP. Simple northern and Southern blotting experiments can answer significant questions in the study of gene structure and expression and in relating these to genetic eye diseases. Molecular biology has provided answers to several long-standing cell biology question such as: where is IRBP synthesized, where is the gene expressed, and how is the IRBP polypeptide processed? In considering how mother nature invented the IRBP gene, the gene structure suggests some interesting alternative models, and causes us to speculate about how large proteins may have evolved. Finally, the determination of the protein sequence helps to put us in the position to ask and answer questions about the function of IRBP. It also allows us to begin to determine the consequences of a nonfunctional IRBP. We don't know the answers to all these questions yet, but the structural analyses and the isolation of these genes and cDNAs, presented here, united with other powerful biological techniques should provide the answers.
Assuntos
Evolução Biológica , Proteínas do Olho , Proteínas de Ligação ao Retinol/genética , Uveíte/etiologia , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Glândula Pineal/metabolismo , RNA Mensageiro/genética , Retina/metabolismo , Proteínas de Ligação ao Retinol/química , Proteínas de Ligação ao Retinol/metabolismoRESUMO
Interphotoreceptor retinoid-binding protein (IRBP), a glycoprotein which localizes in the retina and pineal gland, induces inflammatory changes in these organs (EAU and EAP, respectively) when injected into various mammals. We have previously identified a determinant (residues 1169-1191) in bovine IRBP which is immunodominant and highly immunogenic and immunopathogenic in Lewis rats. IRBP exhibits a fourfold repeat structure and we report here on the comparison between the active sequence 1179-1191 and its three repeat peptides. Only one of the repeats, 271-283, cross-reacted with 1179-1191 and exhibited immunodominance, albeit of a low level. Peptide 271-283 was also immunogenic and immunopathogenic in Lewis rats, but with a minimal dose approximately 100 times higher than that of 1179-1191. Peptide 880-892, a nondominant determinant, resembled 271-283 in its immunogenicity, but was markedly less immunopathogenic. No immunological activity was detected in the fourth repeat peptide, 579-591. Peptide 1179-1191 was superior to the other repeats also in its antigenicity, i.e., the capacity to stimulate presensitized lymphocytes in culture: the minimal stimulatory concentrations of 1179-1191 was greater than 1000 times lower than those of 271-283 or 880-892. Furthermore, 1179-1191 was stimulatory at concentrations lower than those of 271-283 even when tested with lymphocytes sensitized against 271-283. A correlation was also found between the immunological activities of the repeat peptides and their amphipathicity. This study thus identifies two new immunopathogenic determinants of IRBP and provides additional data to show the association between immunodominance of peptides and their various immunological activities.
Assuntos
Epitopos/análise , Proteínas do Olho/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas de Ligação ao Retinol/imunologia , Sequência de Aminoácidos , Animais , Doenças Autoimunes/etiologia , Bovinos , Reações Cruzadas , Relação Dose-Resposta Imunológica , Masculino , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos Lew , Retinite/etiologia , Uveíte/etiologiaRESUMO
We have determined the sequence of the human interphotoreceptor retinoid-binding protein mRNA from three separately isolated cDNAs. The sequence is 4.28 kb long and encodes a protein of 1247 amino acids (aa) including a putative signal peptide and propeptide. The sequence is shorter (by about 1.67 kb) than the bovine mRNA with the major difference in the lengths located in the 3'-untranslated region. We suggest that this resulted from an insertion in the bovine gene or a large deletion from the human gene. The insertion/deletion is flanked on either side by sequences that are similar in the bovine and human sequences. Like the bovine polypeptide, the deduced protein sequence from the human cDNA contains a fourfold repeat, with each repeat containing about 300 aa. Among the four repeats, the identity is about 30-40%. The identity between the complete bovine and human polypeptide sequences is 84%. The identity between the nucleotide sequences is 83% (excluding the major insertion/deletion). Comparison with the bovine gene indicates that the human sequence may lack about 5-10 bp at the 5' end of the cDNA; it, however, includes a poly(A) tail at the 3' end. Thus, the human sequence is virtually full length, is similar to the bovine sequence, and contains a striking fourfold repeat.
Assuntos
Clonagem Molecular , Proteínas do Olho/genética , Proteínas de Ligação ao Retinol/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Códon , DNA/genética , Proteínas do Olho/metabolismo , Glicosilação , Humanos , Íntrons , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas/genética , RNA Mensageiro/genética , Sequências Repetitivas de Ácido Nucleico , Proteínas de Ligação ao Retinol/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
We have extended the cDNA sequence of bovine interphotoreceptor retinoid-binding protein (IRBP) and subcloned one of the sequenced cDNA fragments into an expression vector. The nucleotide (nt) sequences of four bovine IRBP cDNA clones have been determined. These sequences when assembled cover the 3' proximal 3629 nt of the IRBP mRNA and encode the C-terminal 551 amino acids (aa) of IRBP. This cDNA sequence validates the intron: exon boundaries predicted from the gene. A 2-kb EcoRI insert from lambda IRBP2, one of the clones sequenced, encoding the C-terminal 136 aa of IRBP was subcloned into the expression vector pWR590-1. Escherichia coli carrying this plasmid construction, pXS590-IRBP, produced a fusion protein containing 583 N-terminal aa of beta-galactosidase, three linker aa residues, 136 C-terminal aa of IRBP and possibly a number of additional C-terminal residues due to suppressed termination. This 86-kDa fusion protein, purified by detergent/chaotrope extraction followed by reverse-phase high-performance liquid chromatography, cross-reacted with anti-bovine IRBP on Western blots. This protein induced an experimental autoimmune uveo-retinitis and experimental autoimmune pinealitis in Lewis rats indistinguishable from that induced by authentic bovine IRBP. Thus, it is evident that biological activity of this region of IRBP, as manifested by immuno-pathogenicity, is retained by the fusion protein.
Assuntos
Proteínas do Olho/genética , Proteínas Recombinantes de Fusão/genética , Proteínas de Ligação ao Retinol/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA/genética , Escherichia coli/genética , Olho/imunologia , Olho/patologia , Oftalmopatias/etiologia , Oftalmopatias/imunologia , Oftalmopatias/patologia , Proteínas do Olho/imunologia , Masculino , Dados de Sequência Molecular , Plasmídeos , Ratos , Proteínas Recombinantes de Fusão/imunologia , Proteínas de Ligação ao Retinol/imunologiaRESUMO
The gene for bovine interphotoreceptor retinoid-binding protein (IRBP) has been cloned, and its nucleotide sequence has been determined. The IRBP gene is about 11.6 kilobase pairs (kb) and contains four exons and three introns. It transcribed into a large mRNA of approximately 6.4 kb and translated into a large protein of 145,000 daltons. To prove the identity of the genomic clone, we determined the protein sequence of several tryptic and cyanogen bromide fragments of purified bovine IRBP protein and localized them in the protein predicted from its nucleotide sequence. There is a 4-fold repeat structure in the protein sequence with 30-40% sequence identity and many conservative substitutions between any two of the four protein repeats. The third and fourth repeats are the most similar pair. All three of the introns in the IRBP gene fall in the fourth protein repeat. Two of the exons, the first and the fourth, are large, 3173 and 2447 bases, respectively. The introns are each about 1.5-2.2 kb long. The human IRBP gene has a sequence that is similar to one of the introns from the bovine gene. The unexpected gene structure and protein repeat structure in the bovine gene lead us to propose a model for the evolution of the IRBP gene.
Assuntos
Evolução Biológica , Proteínas do Olho , Genes , Células Fotorreceptoras/metabolismo , Proteínas de Ligação ao Retinol/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , Dados de Sequência Molecular , Conformação Proteica , Sequências Repetitivas de Ácido Nucleico , Mapeamento por RestriçãoRESUMO
The gene locus for bovine IRBP, as well as several kilobases of sequence flanking the gene on either end, has been cloned. Two of the several clones seem to contain full-length copies of the protein encoding portion of the gene. Using these clones and cDNA clones, we have determined that there are one or perhaps two copies of the IRBP gene per haploid genome in several species. The gene is compact considering the large size of the protein (145,000 daltons) and its large mRNA (about 6.5 kb). Surprisingly, the gene is no more than 14 kb, being fully contained on lambda clones of maximum packaging size 20 kb. Small parts of the gene were sequenced for the purpose of proving the identity of the genomic clones. DNA sequencing of one of the IRBP gene clones demonstrates the existence of an intron in the gene. The sequence analysis of another fragment identified the N-terminus which has been sequenced at the protein level. The DNA sequence analysis showed the existence of a putative signal sequence and the potential existence of a short five amino acid sequence between the signal sequence and the authentic N-terminus of the secreted extracellular IRBP. This confirms and validates the finding of the extra five amino acid sequence that is present on 40-50% of the polypeptides in monkey and human IRBP which have been isolated from the subretinal space. The presence of the appropriate gene sequence for the pentapeptide but its absence in bovine IRBP indicates differences in processing among the vertebrate IRBPs.
Assuntos
Proteínas do Olho/genética , Genes , Proteínas de Ligação ao Retinol/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Bovinos , Mapeamento Cromossômico , Clonagem Molecular , DNA/análise , Proteínas do Olho/biossíntese , Íntrons , Dados de Sequência Molecular , Peso Molecular , Sinais Direcionadores de Proteínas , Proteínas de Ligação ao Retinol/biossínteseRESUMO
Interphotoreceptor retinoid-binding protein (IRBP), a retinal specific antigen, induces experimental autoimmune uveoretinitis (EAU) when injected into Lewis rats. Here we report that certain cyanogen bromide fragments of IRBP are capable of inducing EAU. Bovine IRBP, reduced and S-carboxymethylated, was subjected to cyanogen bromide cleavage. This CNBr digest was subjected to reversed-phase high performance liquid chromatography. Three fragments were purified to apparent homogeneity. These three fragments were subjected to gas-phase amino-terminal sequencing analysis. All three yielded single sequences, confirming their purity. On the basis of this amino-terminal sequencing and sequencing of cDNAs encoding bovine IRBP, two of these sequences, named CB-58 and CB-71, were localized to the C-terminal one-third of the IRBP molecule, whereas the third, a subfragment thought to result from cleavage at a tryptophan residue and named CB-47, was localized to the N-terminal one third of the protein. CB-71 and CB-47 shared a strong homology, suggesting a putative internal gene duplication event in the evolution of IRBP. All three of these fragments when injected into Lewis rats caused moderately severe EAU with early onset at relatively low doses. The histopathologic changes induced were indistinguishable from those caused by the intact protein. It would seem, therefore, that bovine IRBP contains multiple uveitogenic sites.
Assuntos
Doenças Autoimunes/imunologia , Brometo de Cianogênio , Fragmentos de Peptídeos/imunologia , Células Fotorreceptoras/metabolismo , Retinite/imunologia , Proteínas de Ligação ao Retinol/imunologia , Uveíte/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Cromatografia , Dados de Sequência Molecular , Peso Molecular , Ratos , Ratos Endogâmicos Lew , Retinite/patologia , Proteínas de Ligação ao Retinol/genética , Proteínas de Ligação ao Retinol/metabolismo , Uveíte/patologiaRESUMO
Upon lens removal, the adult Eastern Spotted newt, Notophthalmus viridescens, has the capacity to regenerate an ocular lens. Crystallins, proteins characteristic of the vertebrate lens, were studied from normal and 3-month regenerated adult newt lenses. When separated by high-performance liquid chromatography (HPLC) or Sephadex G-200SF column chromatography, the crystallins from normal and regenerated lenses were fractionated into what appear to be the classical four groups: alpha, beta High, beta Low, and gamma. Upon further examination by immunoelectrophoresis, the first peak contains both alpha and beta crystallins. This study provides evidence that most of the crystallins from the regenerated lenses share biochemical properties with those of the normal lens crystallins based on their native molecular weight, isoelectric point, and the molecular wt of their constituent polypeptides, indicating that the fidelity of gene expression in reactivated iris tissue is high. Some differences are found between normal and regenerated lens crystallins and are most obvious in the beta-crystallin region: the proportion of beta crystallins is decreased in regenerated lenses when the total proteins are fractionated by column chromatography and some of the beta-crystallin polypeptide chains found in normal lenses are missing from regenerated lenses. Iris epithelial cells are normally withdrawn from the cell cycle and are synthesizing a tissue-specific product, melanin. After lentectomy these cells dedifferentiate, redifferentiate into lens cells, and their progeny then synthesize different tissue-specific proteins, crystallins. Little is known about the specific mechanism(s) for the activation of gene expression in eukaryotes, but the regenerating lens suggests itself as a good model in which to study this biological problem.