RESUMEN
Differentiation of skeletal muscle is affected in myotonic dystrophy (DM) patients. Analysis of cultured myoblasts from DM patients shows that DM myoblasts lose the capability to withdraw from the cell cycle during differentiation. Our data demonstrate that the expression and activity of the proteins responsible for cell cycle withdrawal are altered in DM muscle cells. Skeletal muscle cells from DM patients fail to induce cytoplasmic levels of a CUG RNA binding protein, CUGBP1, while normal differentiated cells accumulate CUGBP1 in the cytoplasm. In cells from normal patients, CUGBP1 up-regulates p21 protein during differentiation. Several lines of evidence show that CUGBP1 induces the translation of p21 via binding to a GC-rich sequence located within the 5' region of p21 mRNA. Failure of DM cells to accumulate CUGBP1 in the cytoplasm leads to a significant reduction of p21 and to alterations of other proteins responsible for the cell cycle withdrawal. The activity of cdk4 declines during differentiation of cells from control patients, while in DM cells cdk4 is highly active during all stages of differentiation. In addition, DM cells do not form Rb/E2F repressor complexes that are abundant in differentiated cells from normal patients. Our data provide evidence for an impaired cell cycle withdrawal in DM muscle cells and suggest that alterations in the activity of CUGBP1 causes disruption of p21-dependent control of cell cycle arrest.
Asunto(s)
Proteínas de Ciclo Celular , Diferenciación Celular , Proteínas de Unión al ADN , Músculo Esquelético/citología , Distrofia Miotónica/patología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/fisiología , Secuencia de Bases , Western Blotting , Proteínas CELF1 , Ciclo Celular , División Celular , Núcleo Celular/metabolismo , Sistema Libre de Células , Células Cultivadas , Clonación Molecular , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Citoplasma/metabolismo , Factores de Transcripción E2F , Eliminación de Gen , Humanos , Microscopía Fluorescente , Modelos Genéticos , Datos de Secuencia Molecular , Distrofia Miotónica/metabolismo , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Ribonucleasas/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo , Rayos Ultravioleta , Regulación hacia ArribaRESUMEN
An RNA CUG triplet repeat binding protein, CUGBP1, regulates splicing and translation of various RNAs. Expansion of RNA CUG repeats in the 3'-untranslated repeat of the mutant myotonin protein kinase (DMPK) mRNA in myotonic dystrophy (DM) is associated with alterations in binding activity of CUGBP1. To investigate whether CUGBP1 is directly affected by expansion of CUG repeats in DM tissues, we examined the intracellular status of CUGBP1 in DM patients as well as in cultured cells over expressing RNA CUG repeats. The analysis of RNA-protein complexes showed that, in control tissues, the majority of CUGBP1 is free of RNA, whereas in DM patients the majority of CUGBP1 is associated with RNA containing CUG repeats. Similarly to DM patients, overexpression of RNA CUG repeats in cultured cells results in the re-allocation of CUGBP1 from a free state to the RNA.protein complexes containing CUG repeats. CUG repeat-dependent translocation of CUGBP1 into RNA-protein complexes is associated with increased levels of CUGBP1 protein and its binding activity. Experiments with cyclohexamide-dependent block of protein synthesis showed that the half-life of CUGBP1 is increased in cells expressing CUG repeats. Alteration of CUGBP1 in DM is accompanied by alteration in translation of a transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta), which has been previously described to be a target of CUGBP1. Analysis of C/EBPbeta isoforms in DM patients with altered levels of CUGBP1 showed that translation of a dominant negative isoform, LIP, is induced by CUGBP1. Results of this paper demonstrate that the expansion of CUG repeats in DM affects RNA-binding proteins and leads to alteration in RNA processing.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , ARN/química , Ribonucleoproteínas/metabolismo , Repeticiones de Trinucleótidos , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteínas CELF1 , Células COS , Distrofia Miotónica/genética , ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/análisis , Ribonucleoproteínas/análisisRESUMEN
Lipopolysacharide (LPS) induced acute phase response (APR) in mouse liver leads to elevation of the low molecular weight CCAAT/Enhancer binding protein (C/EBP) beta isoform, liver-enriched transcriptional inhibitory protein (LIP). In this paper, we investigate the pathway for LIP induction during APR and the role of LIP in regulation of the C/EBPalpha promoter. The 5' region of C/EBPbeta mRNA has been shown to be involved in the regulation of LIP translation. Our data demonstrate that binding of cytoplasmic proteins to the 5' region of C/EBPbeta mRNA is altered in response to LPS administration. One of the major changes is induced binding of a cytoplasmic protein that is immunologically identical to the previously characterized RNA-binding protein CUGBP1. Induction of CUGBP1 binding activity in liver cytoplasm during APR is accompanied by the elevation of CUGBP1 binding activity on polysomes. CUGBP1 immunoprecipitated from livers of LPS-treated mice, but not from normal animals, is capable of inducing LIP translation in a cell-free translation system. The ability of CUGBP1 to induce LIP translation during APR depends on phosphorylation of CUGBP1. We show that elevation of LIP during APR and after partial hepatectomy leads to increased binding of LIP to the C/EBP consensus site found within the mouse C/EBPalpha promoter. This binding correlates with reduction of C/EBPalpha mRNA levels in both biological situations. Co-transfection experiments showed that full-length C/EBPbeta activates the C/EBPalpha promoter, while LIP blocks this activation. Our data suggest that the dominant negative isoform of C/EBPbeta, LIP, down-regulates the C/EBPalpha promoter in liver and in cultured hepatocytes. Because full-length C/EBPalpha and C/EBPbeta proteins regulate liver proliferation, this function of LIP may be important in liver growth and differentiation.
Asunto(s)
Reacción de Fase Aguda/genética , Arginasa/genética , Proteínas de Unión al ADN/genética , Proteínas Nucleares/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Animales , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Células Cultivadas , Cartilla de ADN , Lipopolisacáridos/administración & dosificación , Hígado/citología , Hígado/metabolismo , Regeneración Hepática , Ratones , Sistemas de Lectura Abierta , Pruebas de Precipitina , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
Comparison of the growing number of disorders known to be associated with triplet repeat expansions reveals both common features and a diversity of molecular pathways. Despite significant progress towards the characterization of proteins coded by the mutant genes, the complex nature of these disorders requires identification of all molecular components of the triplet repeat pathways. In this brief review we will discuss recent progress in determining the molecular mechanisms of disorders with unstable trinucleotide mutations.
Asunto(s)
Repeticiones de Minisatélite , Proteínas de Unión al ARN , Repeticiones de Trinucleótidos , Animales , Apoptosis , Canales de Calcio/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Proteínas de Homeodominio/genética , Humanos , Hierro/metabolismo , Ratones , Modelos Genéticos , Distrofia Miotónica/genética , Distrofia Miotónica/metabolismo , Proteína Quinasa de Distrofia Miotónica , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Péptidos/genética , Péptidos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN/genética , ARN/metabolismoRESUMEN
The transcription factor CCAAT/enhancer binding protein beta, C/EBPbeta, plays a significant role in the regulation of hepatocyte growth and differentiation. A single mRNA coding for C/EBPbeta produces several protein isoforms. Two pathways for generation of low molecular weight C/EBPbeta isoforms have been described: specific proteolytic cleavage and initiation of translation from different AUG codons of C/EBPbeta mRNA. A truncated C/EBPbeta isoform, LIP, is induced in rat livers in response to partial hepatectomy (PH) via the alternative translation mechanism. Here we present evidence that CUG repeat binding protein, CUGBP1, interacts with the 5' region of C/EBPbeta mRNA and regulates translation of C/EBPbeta isoforms. Two binding sites for CUGBP1 are located side by side between the first and second AUG codons of C/EBPbeta mRNA. One binding site is observed in an out of frame short open reading frame (sORF) that has been previously shown to regulate initiation of translation from different AUG codons of C/EBPbeta mRNA. Analysis of cytoplasmic and polysomal proteins from rat liver after PH showed that CUGBP1 is associated with polysomes that translate low molecular weight isoforms of C/EBPbeta. The binding activity of CUGBP1 to the 5' region of C/EBPbeta mRNA shows increased association with these polysomal fractions after PH. Addition of CUGBP1 into a cell-free translation system leads to increased translation of low molecular weight isoforms of C/EBPbeta. Our data demonstrate that CUGBP1 protein is an important component for the regulation of initiation from different AUG codons of C/EBPbeta mRNA.
Asunto(s)
Regiones no Traducidas 5'/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Nucleares/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Sitios de Unión , Proteína beta Potenciadora de Unión a CCAAT , Proteínas Potenciadoras de Unión a CCAAT , Proteínas CELF1 , Sistema Libre de Células , Regulación de la Expresión Génica , Células HeLa , Humanos , Regeneración Hepática , Sistemas de Lectura Abierta , Polirribosomas/metabolismo , Biosíntesis de Proteínas , Isoformas de Proteínas/genética , Conejos , Ratas , Proteínas Represoras/genética , Repeticiones de Trinucleótidos/fisiologíaRESUMEN
Myotonic dystrophy (DM) is a neuromuscular disorder associated with CTG triplet repeat expansion in the myotonin protein kinase gene ( DMPK ). We previously proposed a hypothesis suggesting that the expanded CUG repeats sequester specific RNA-binding proteins and that such a sequestration results in abnormal RNA processing of several RNAs containing CUG repeats in multiple tissues affected in patients with DM. One of the members of the CUG-binding proteins, CUG-BP, has been identified previously. Here we describe the second member of this family, elav -type ribonucleoprotein (ETR-3), which is highly expressed in heart and is able to interact with CUG repeats. Screening of a mouse liver cDNA library with a CUG-BP probe identified two mETR-3 cDNAs. Two additional cDNAs from mouse heart were amplified by RT-PCR. These cDNAs differ by several insertions/deletions and might be generated via alternative splicing. Mouse ETR-3 has a mol. wt of 50 kDa and displays a high level of homology to CUG-BP protein. The organization of the RNA-binding domains (RBDs) within the ETR-3 molecule is similar to one within CUG-BP. A study of mETR-3 RNA-binding activity showed that the mETR-3 binds to (CUG)8repeats. Sequence analysis of mETR-3 indicates the presence of several CUG repeats within the mETR-3 mRNA. Both CUG-BP and mETR-3 bind to mETR-3 mRNA via CUG repeats, suggesting the possible involvement of CUG-BP-like proteins in the regulation of mETR-3 processing. Analysis of the tissue distribution of ETR-3 showed that in human cells, ETR-3 mRNA is highly expressed in heart, but is undetectable in other tissues examined. Our results suggest the existence of a family of proteins that bind to CUG repeats and might be affected in DM by expansion of CUG repeats.
Asunto(s)
Distrofia Miotónica/genética , Distrofia Miotónica/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/genética , ARN/metabolismo , Expansión de Repetición de Trinucleótido , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Proteínas CELF , Cartilla de ADN/genética , ADN Complementario/genética , Células HeLa , Humanos , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Miocardio/metabolismo , Proteínas del Tejido Nervioso , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Myotonic dystrophy (DM) is caused by a CTG expansion in the 3' untranslated region of the DM gene. One model of DM pathogenesis suggests that RNAs from the expanded allele create a gain-of-function mutation by the inappropriate binding of proteins to the CUG repeats. Data presented here indicate that the conserved heterogeneous nuclear ribonucleoprotein, CUG-binding protein (CUG-BP), may mediate the trans-dominant effect of the RNA. CUG-BP was found to bind to the human cardiac troponin T (cTNT) pre-messenger RNA and regulate its alternative splicing. Splicing of cTNT was disrupted in DM striated muscle and in normal cells expressing transcripts that contain CUG repeats. Altered expression of genes regulated posttranscriptionally by CUG-BP therefore may contribute to DM pathogenesis.
Asunto(s)
Empalme Alternativo , Distrofia Miotónica/genética , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Repeticiones de Trinucleótidos , Proteínas CELF1 , Línea Celular , Núcleo Celular/metabolismo , Exones , Humanos , Intrones , Músculo Esquelético/citología , Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , Mutación , Distrofia Miotónica/metabolismo , Proteína Quinasa de Distrofia Miotónica , Fosforilación , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/genética , Transcripción Genética , Transfección , Troponina/genética , Troponina TRESUMEN
Myotonic dystrophy (DM) is associated with expansion of CTG repeats in the 3'-untranslated region of the myotonin protein kinase (DMPK) gene. The molecular mechanism whereby expansion of the (CUG)n repeats in the 3'-untranslated region of DMPK gene induces DM is unknown. We previously isolated a protein with specific binding to CUG repeat sequences (CUG-BP/hNab50) that possibly plays a role in mRNA processing and/or transport. Here we present evidence that the phosphorylation status and intracellular distribution of the RNA CUG-binding protein, identical to hNab50 protein (CUG-BP/hNab50), are altered in homozygous DM patient and that CUG-BP/hNab50 is a substrate for DMPK both in vivo and in vitro. Data from two biological systems with reduced levels of DMPK, homozygous DM patient and DMPK knockout mice, show that DMPK regulates both phosphorylation and intracellular localization of the CUG-BP/hNab50 protein. Decreased levels of DMPK observed in DM patients and DMPK knockout mice led to the elevation of the hypophosphorylated form of CUG-BP/hNab50. Nuclear concentration of the hypophosphorylated CUG-BP/hNab50 isoform is increased in DMPK knockout mice and in homozygous DM patient. DMPK also interacts with and phosphorylates CUG-BP/hNab50 protein in vitro. DMPK-mediated phosphorylation of CUG-BP/hNab50 results in dramatic reduction of the CUG-BP2, hypophosphorylated isoform, accumulation of which was observed in the nuclei of DMPK knockout mice. These data suggest a feedback mechanism whereby decreased levels of DMPK could alter phosphorylation status of CUG-BP/hNab50, thus facilitating nuclear localization of CUG-BP/hNab50. Our results suggest that DM pathophysiology could be, in part, a result of sequestration of CUG-BP/hNab50 and, in part, of lowered DMPK levels, which, in turn, affect processing and transport of specific subclass of mRNAs.
Asunto(s)
Distrofia Miotónica/genética , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Repeticiones de Trinucleótidos , Animales , Proteínas CELF1 , Homocigoto , Humanos , Ratones , Ratones Noqueados , Proteína Quinasa de Distrofia Miotónica , Fosforilación , Proteínas de Unión al ARN/genéticaRESUMEN
Several human disorders are now known to be caused by expansion of unstable trinucleotide repeat sequences, including fragile X syndrome (FRAX), myotonic dystrophy (DM), spinal and bulbar muscular atrophy (SBMA, also known as Kennedy disease), Huntington disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), spinocerebellar ataxia type 1 (SCA1), Machado-Joseph disease (MJD), and Friedreich ataxia. As these diseases are studied in more detail, important differences have emerged in the nature of the unstable repeats and the mechanism by which the repeat expansions cause disease symptoms. There are already animal models of some of these disorders, and these are important resources for studying pathology and therapeutic strategies. Diagnostic procedures for these disorders are only beginning to be standardized, and effective therapy will have to wait for further information on disease mechanisms. Much has been learned since discovery of the fragile X syndrome gene in 1991, but much remains to be done.
Asunto(s)
Síndrome del Cromosoma X Frágil/genética , Enfermedad de Huntington/genética , Atrofia Muscular Espinal/genética , Distrofia Miotónica/genética , Degeneraciones Espinocerebelosas/genética , Repeticiones de Trinucleótidos , Animales , Modelos Animales de Enfermedad , Síndrome del Cromosoma X Frágil/terapia , Humanos , Enfermedad de Huntington/terapia , Atrofia Muscular Espinal/terapia , Mutación , Distrofia Miotónica/terapia , Degeneraciones Espinocerebelosas/terapiaRESUMEN
Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease that is associated with a (CTG)n repeat expansion in the 3'-untranslated region of the myotonin protein kinase (Mt-PK) gene. This study reports the isolation and characterization of a (CUG)n triplet repeat pre-mRNA/mRNA binding protein that may play an important role in DM pathogenesis. Two HeLa cell proteins, CUG-BP1 and CUG-BP2, have been purified based upon their ability to bind specifically to (CUG)8 oligonucleotides in vitro. While CUG-BP1 is the major (CUG)8-binding activity in normal cells, nuclear CUG-BP2 binding activity increases in DM cells. Both CUG-BP1 and CUG-BP2 have been identified as isoforms of a novel heterogeneous nuclear ribonucleoprotein (hnRNP), hNab50. The CUG-BP/hNab50 protein is localized predominantly in the nucleus and is associated with polyadenylated RNAs in vivo. In vitro RNA-binding/photocrosslinking studies demonstrate that CUG-BP/hNab50 binds to RNAs containing the Mt-PK 3'-UTR. We propose that the (CUG)n repeat region in Mt-PK mRNA is a binding site for CUG-BP/hNab50 in vivo, and triplet repeat expansion leads to sequestration of this hnRNP on mutant Mt-PK transcripts.
Asunto(s)
Distrofia Miotónica/genética , Proteínas de Unión al ARN/química , Ribonucleoproteínas/química , Repeticiones de Trinucleótidos , Secuencia de Aminoácidos , Proteínas CELF1 , Células HeLa , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Precursores del ARN/química , Precursores del ARN/aislamiento & purificación , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoAsunto(s)
Distrofia Miotónica/metabolismo , Proteínas Serina-Treonina Quinasas , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Repeticiones de TrinucleótidosRESUMEN
While an unstable CTG triplet repeat expansion is responsible for myotonic dystrophy, the mechanism whereby this genetic defect induces the disease remains unknown. To detect proteins binding to CTG triplet repeats, we performed bandshift analysis using as probes double-stranded DNA fragments having CTG repeats [ds(CTG)6-10] and single-stranded oligonucleotides having CTG repeats ss(CTG)8 or RNA CUG triplet repeats (CUG)8. The source of protein was nuclear and cytoplasmic extracts of HeLa cells, fibroblasts and myotubes. Proteins binding to the double-stranded DNA repeat [ds(CTG)6-10], were inhibited by nonlabeled ds(CTG)6-10, but not by a non-specific DNA fragment (USF/AD-ML). Another protein binding to ssCTG probe and RNA CUG probe was inhibited by nonlabeled (CTG)8 and (CUG)8. Nonlabeled oligos with different triplet repeat sequences, ss(CAG)8 or ss(CGG)8, did not inhibit binding to the ss(CTG)8 probe. However, when labeled as probes, the (CAG)8 and (CGG)8 bound to proteins distinct from the CTG proteins and binding was inhibited by nonlabeled (CAG)8 or (CGG)8 respectively. The protein binding only to the RNA repeat (CUG)8 was inhibited by nonlabeled (CUG)8 but not by nonlabeled single- or double-stranded CTG repeats. Furthermore, the CUG-BP exhibited no binding to an RNA oligonucleotide of triplet repeats of the same length but having a different sequence, CGG. The CUG binding protein was localized to the cytoplasm, whereas dsDNA binding proteins were localized to the nuclear extract. Thus, several trinucleotide binding proteins exist and their specificity is determined by the triplet sequence. The novel protein, CUG-BP, is particularly interesting since it binds to triplet repeats known to be present in myotonin protein kinase mRNA which is responsible for myotonic dystrophy.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Distrofia Miotónica/genética , Proteínas de Unión al ARN/metabolismo , Repeticiones de Trinucleótidos , Secuencia de Bases , Unión Competitiva , Células Cultivadas , ADN/genética , ADN/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/química , Fibroblastos , Células HeLa , Humanos , Datos de Secuencia Molecular , Peso Molecular , Fibras Musculares Esqueléticas , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/químicaRESUMEN
There are some genes whose expression increases in non-proliferating hepatocytes after whole-body X-irradiation. We suppose that some of them participate in cell division regulation. To verify our postulate we have studied expression of autonomously replicating sequence pDARC1 in normal, irradiated and dividing rat hepatocytes. We have succeeded earlier in cloning the autonomously replicating sequence pDARC1 and have shown that it could be transcribed in X-irradiated hepatocytes. Here we report on the results of analysis of this sequence expression in dividing mammalian cells. Hybridization with the pDARC1 sequence reveals 4.0 kb mRNA in non-proliferating rat hepatocytes after X-irradiation: this mRNA is absent in normal hepatocytes. Induction of 4.0 kb mRNA is a function of radiation dose; 4.0 kb mRNA content is maximum 6-24 h following whole-body 6 Gy X-irradiation. The 4.0 kb mRNA is also found in dividing rat hepatocytes during the pre-replicative period.
Asunto(s)
Expresión Génica , Hígado/efectos de la radiación , ARN Mensajero/genética , Animales , Northern Blotting , Southern Blotting , División Celular , Humanos , Hígado/citología , Dosis de Radiación , Ratas , Transcripción Genética , Irradiación Corporal TotalRESUMEN
A ubiquitous mammalian transcription factor, Oct-1 (also known as OTF-1, NF-A1, OBP100, or NFIII), stimulates the initiation of replication of adenovirus DNA, and may also be involved in the activation of some chromosomal replication origins. If this is true, binding sites for Oct-1 should be present within regions responsible for the initiation of DNA replication. In this study such a binding site has been identified within a 340bp fragment that was originally isolated from a minor fraction of DNA associated with a complexed form of DNA polymerase alpha from nonregenerating rat liver, and which shows autonomous replication sequence activity in a transient transfection assay. Northern blot analysis was used to show that Oct-1 mRNA is induced in regenerating rat liver 6-14 h after hepatectomy.
Asunto(s)
ADN Polimerasa II/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Hígado/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Northern Blotting , Desoxirribonucleasa I/metabolismo , Hepatectomía , Factor C1 de la Célula Huésped , Hígado/enzimología , Regeneración Hepática , Datos de Secuencia Molecular , Factor 1 de Transcripción de Unión a Octámeros , RatasRESUMEN
Individual mRNA coding for proteins of the alpha-polymerase complex were isolated from replicating hepatocytes. cDNAs were synthesized by reverse transcriptase. Three clones were identified by colony hybridization of the S period cDNA library. The sequences of these clones were complementary to the investigated mRNAs. Two clones named pr12 and pr167 revealed increased expressions in the S period. The level of mRNA pr127 does not change during liver regeneration. The amount of pr127 mRNA was about 1% of the total mRNA population. pr12, pr127 and pr167 mRNAs were isolated by the hybrid-selection method and were translated in a cell-free system. The products of translation were analysed by "activity" gel. It was shown that pr167 mRNA coded for protein 140 kDa with DNA polymerase activity. pr12 protein is an unknown component of the alpha-polymerase complex. We suggested that this protein participates in the initiation of DNA replication.
Asunto(s)
ADN Polimerasa II/genética , Replicación del ADN , Expresión Génica , Hígado/metabolismo , Animales , ADN/genética , ADN Polimerasa II/metabolismo , Immunoblotting , Hígado/citología , Hígado/enzimología , Hibridación de Ácido Nucleico , Plásmidos , Biosíntesis de Proteínas , ARN Mensajero/genética , RatasRESUMEN
A complex from of DNA polymerase alpha was isolated from the nuclear membrane of hepatocytes. DNA fragments were shown to be among components of the complex under study. In this paper we present evidence that DNA from the alpha-polymerase complex from quiescent hepatocytes (DNA-G) differs in its nucleotide composition from its counterpart (DNA-S) isolated from hepatocytes synthesizing DNA. As judged by dot hybridization, DNA-G0 does not contain nucleotide sequences which are complementary to ribosomal or messenger RNA, whereas the abovementioned sequences are present in DNA-S. At the same time DNA-G0 is found to contain sequences which are homologous to both SV40 DNA and yeast TRPI-ARS1 DNA. The difference in nucleotide sequences between DNA-G0 and DNA-S indicates that in the process of replication DNA is being stretched across the multienzyme complex located on the nuclear membrane.
Asunto(s)
ADN Polimerasa II/metabolismo , ADN/aislamiento & purificación , Hígado/enzimología , Animales , ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Membrana Nuclear/enzimología , Hibridación de Ácido Nucleico , RatasRESUMEN
The library of cDNAs synthesized on poly(A)+ mRNA derived from rat liver after total X-irradiation of animals has been obtained. cDNA-clones (p gamma clones) representing sequences inducibly transcribed as a result of radiation treatment were isolated by differential screening. The increased expression of p gamma mRNAs was observed during the period from 6 to 24 h after irradiation by 3-6 Gy dose. It was concluded that p gamma mRNAs do not code for the liver-specified proteins because these mRNAs are also present in the rat fibroblasts. We suggest that p gamma genes are involved in the pathways of DNA-repair system.
Asunto(s)
Clonación Molecular , ADN/genética , Expresión Génica , Hígado/efectos de la radiación , Animales , Células Cultivadas , ADN/análisis , Biblioteca Genómica , Hígado/citología , Regeneración Hepática , Hibridación de Ácido Nucleico , Plásmidos , Ratas , Transcripción GenéticaRESUMEN
The method of hybridization in a dot was used to study the dynamics of expression of pRL genes inducible in dividing cells of the regenerating rat liver and c-myc oncogene after whole-body X-irradiation. As overall incorporation of mRNA into a polyribosomal fraction was inhibited the level of mRNA in the most studied pRL genes and c-myc oncogene within the translated mRNA increased. The maximum induction of pRL gene expression occurred 6-12 h following X-irradiation depending on radiation dose. It is suggested that X-radiation induction of pRL gene expression is related to the increased transcription of the corresponding mRNA since their level does not change in conditions of cycloheximide blockade of the protein synthesis.
Asunto(s)
Regulación de la Expresión Génica/efectos de la radiación , Hígado/efectos de la radiación , Animales , Regeneración Hepática , Oncogenes , Genética de Radiación , Ratas , Irradiación Corporal TotalRESUMEN
A library of double-stranded cDNA was prepared using poly (A) + RNA from regenerating rat liver 20 h after partial hepatectomy. Differential screening of 350 recombinant clones with cDNA-G0 and cDNA-S identified eleven cDNA clones (pRL), the sequences of which were preferentially expressed during the DNA replication period. Levels of mRNAs complementary to these clones were 2--10-fold higher in the S-period, than in G0. Using plasmid cDNAs to different mRNA, pRL we have investigated the changes in the levels of mRNA pRL during liver regeneration. The level of mRNA mRL2 and pRL79 was increased just before DNA replication. mRNA pRL35 accumulates after partial hepatectomy with the maximum at 6 h. The augment of two other mRNA concentrations was expressed to a lesser extent. Northern-blot analysis allowed to determine the individual dual mRNAs corresponding to each of the three clones with their sizes ranging from about 1650 to 3900 bases. Three mRNAs (pRL35, 67 and 79) were shown (by hybrid-selected translation) to code for proteins of about 100, 140 and 120 kDa, respectively.