RESUMEN
S1 proteins A-D are liberated from thoroughly washed nuclei by mild digestion with DNase I or RNase A, and extracted selectively at pH 4.9 from the reaction supernatants. Here, we characterized the S1 proteins, focusing on protein D2, the most abundant S1 protein in the rat liver, and on protein C2 as well. Using a specific antibody, McAb 351, they were shown to occur in the extranucleolar nucleoplasm, and to be extracted partly in the nuclear soluble fraction. We demonstrate that the S1 proteins in this fraction exist constituting heterogeneous nuclear ribonucleoproteins (hnRNPs), through direct binding to hnRNAs, as revealed by centrifugation on density gradients, immunoprecipitation, and UV cross-linking. In hnRNPs, protein D2 occurred at nuclease-hypersensitive sites and C2 in the structures that gave rise to 40 S RNP particles. By microsequencing, protein D2 was identified with a known protein, CArG box motif-binding factor A (CBF-A), which has been characterized as a transcriptional repressor, and C2 as its isoform protein. In fact, CBF-A expressed from its cDNA was indistinguishable from protein D2 in molecular size and immunoreactivity to McAb 351. Thus, the present results demonstrate that S1 proteins C2 and D2 are novel hnRNP proteins, and suggest that the proteins C2 and D2 act in both transcriptional and post-transcriptional processes in gene expression.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/química , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Ribonucleoproteína Heterogénea-Nuclear Grupo C , Hígado/metabolismo , Proteínas Represoras/química , Ribonucleoproteínas/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular , Línea Celular , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Epitelio/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Datos de Secuencia Molecular , ARN Nuclear Heterogéneo/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Represoras/aislamiento & purificación , Proteínas Represoras/metabolismo , Ribonucleoproteínas/aislamiento & purificación , Ribonucleoproteínas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
Xenopus embryos were exposed to 200 ppb 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 5 days from the 2- to 8-cell stage of cleavage to the early larval stage. Larvae that developed generalized edema were collected at 7 days after the end of TCDD exposure for light and electron microscopic studies. Erythrocytes in the peripheral blood of the edematous larvae were examined. Between 0.3 and 33.9% of identifiable erythrocytes of exposed larvae had dilated perinuclear cisternae. Furthermore, some had extremely condensed nuclear chromatin usually coalesced against 1 pole of the nuclear membrane and overall compacted cytoplasm. The erythrocytes showing nuclear condensation were phagocytosed by macrophages. These features are typical of cells undergoing apoptosis. Anemia is 1 symptom of TCDD toxicity in various animal species, including mammals. In this study, we demonstrate that TCDD induces apoptotic cell death in circulating erythrocytes of Xenopus larvae, which may be 1 cause of anemia in this species.
Asunto(s)
Apoptosis/efectos de los fármacos , Envejecimiento Eritrocítico/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Dibenzodioxinas Policloradas/toxicidad , Animales , Eritrocitos/patología , Eritrocitos/ultraestructura , Larva/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Mitocondrias/ultraestructura , Vacuolas/efectos de los fármacos , Vacuolas/patología , Vacuolas/ultraestructura , XenopusRESUMEN
S1 proteins A-D are hnRNP proteins which were originally isolated from nuclei of various tissues, by selective extraction of pH 4.9 from the supernatants of nuclei mildly treated with DNase I or RNase A. In the present study, a hybridoma was isolated which produced a monoclonal antibody that reacted specifically with S1 proteins C2 and D2. When the antibody was used in indirect immunofluorescence staining of cultured cells, it stained, in addition to the nuclei, the cytoskeleton-like fibrous structures in the cytoplasm. We demonstrate that the cytoskeletal filaments are vimentin intermediate filaments. This is the first report on the hnRNP protein-association with cytoskeleton, and will help to clarify cytoplasmic mRNA localization as well as cytoplasmic distribution of hnRNP proteins.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo C , Filamentos Intermedios/metabolismo , Ribonucleoproteínas/metabolismo , Vimentina/metabolismo , Animales , Anticuerpos Monoclonales , Línea Celular , Técnica del Anticuerpo Fluorescente Indirecta , Ribonucleoproteínas Nucleares Heterogéneas , Hibridomas , Ratas , Coloración y EtiquetadoRESUMEN
S1 proteins A-D constitute a nuclear protein family that are liberated rapidly in a set from chromatin by mild digestion with a DNA or RNA hydrolyzing enzyme. With an anti-S1-protein B antiserum that reacted with B2, C1 and D1, a cDNA clone, pS1-1, was obtained, which encoded a protein of 852 amino acids. The S1-1 protein, encoded within the cells by a mRNA of 3480 nt, was a novel protein and could be distinguished from the S1 proteins B, C and D by their amino acid sequences. The S-1-1 protein synthesized by in vitro translation bound to RNA homopolymers, with a preference for G and U polyribonucleotides and little for poly(A). The protein contained two tandem RNP motifs and several intriguing sequences, such as a novel repeat of five octamers with a consensus sequence DP-S(Q/G)YYY and a potentially perfect amphipathic alpha-helix of five turns with basic and acidic amino acids positioned in an ordered way. The two RNP motif sequences were similar, although homologies were low, to the RNP motif sequences of yeast NSR1 protein, animal nucleolins, Drosophila hnRNP Al and tobacco chloroplast RNP precursor protein, suggesting a functional uniqueness of the S1-1 protein in RNA metabolism and also the evolution of its RNP motif structure before plants and animals diverged. These results indicate that the S1-1 protein encoded by the cDNA is a new class of RNA binding protein.
Asunto(s)
Proteínas Nucleares/genética , Proteínas de Unión al ARN/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Secuencia de Consenso , ADN Complementario/genética , Hígado/química , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , ARN Mensajero/análisis , Proteínas de Unión al ARN/metabolismo , Ratas , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Nuclear S1 proteins are a group of proteins apparently ubiquitous in vertebrate cell nuclei. They were originally isolated at pH 4.9 from the supernatant of rat liver nuclei mildly digested with DNase I. In the present study, under the conditions identical to those employed for vertebrate cells, we identified two S1 proteins in the starfish Asterina Pectinifera. Their molecular weights are 47,200 and 39,000. This finding suggests widespread occurrence of S1 proteins in eukaryotes and their basic function in the cell nucleus.
Asunto(s)
Proteínas Nucleares/aislamiento & purificación , Animales , Electroforesis en Gel de Poliacrilamida , Estrellas de MarRESUMEN
In primary cultures of rat hepatocytes, specific thyroid-hormone-binding activity diminished with time and was hardly detectable at 24 h. In accordance with the loss of 3,5,3'-tri-iodothyronine (T3) binding, responses to the hormone disappeared, as indicated by low induction of the thyroid-hormone-responsive gene S14. In contrast, thyroid hormone receptor proteins were present, as determined by immunostaining with a specific antibody against the receptor. Thus the loss of T3 binding was due to receptor inactivation. After various attempts to restore the T3-binding activity, we found that 2-mercaptoethanol, a reducing agent, when added to the culture medium restored the hormone binding activity in a dose- and time-dependent manner. The observed kinetics and experiments using cycloheximide suggested that mercaptoethanol prevented inactivation of the newly synthesized receptors. Oxidoreductive conditions within cells may have a role in determining the level of activity of thyroid hormone receptors.
Asunto(s)
Hígado/metabolismo , Mercaptoetanol/farmacología , Receptores de Hormona Tiroidea/metabolismo , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Cicloheximida/farmacología , Ditiotreitol/farmacología , Expresión Génica/efectos de los fármacos , Hígado/efectos de los fármacos , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas , Receptores de Hormona Tiroidea/efectos de los fármacos , Triyodotironina/metabolismo , Triyodotironina/farmacologíaRESUMEN
S1 proteins (A, B, C and D) are a group of nuclear proteins, isolated by lowering pH to 4.9 of the reaction supernatant of hepatocyte nuclei that had been mildly digested with DNase I. Protein B, apparently ubiquitous in vertebrate cells, was prepared from rat liver and used to immunize a rabbit. The raised antiserum specifically reacted with S1 proteins; it reacted not only with protein B, but also with C and D. Immunoblotting demonstrated that these proteins occurred exclusively in the nucleus, being absent in the cytosol, microsome and mitochondrial fractions. Indirect immunofluorescence of liver tissue sections confirmed their nuclear localization, and further showed that the antibody selectively stained extranucleolar regions of the cell nucleus. These findings suggest that the anti-S1 antibody is specific to S1 proteins and may be useful for their structural and functional studies.
Asunto(s)
Anticuerpos/aislamiento & purificación , Proteínas Nucleares/inmunología , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos , Western Blotting , Núcleo Celular/análisis , Técnica del Anticuerpo Fluorescente , Hígado/análisis , Proteínas Nucleares/aislamiento & purificación , Conejos , Ratas , Ratas EndogámicasRESUMEN
S1 proteins are present in the nuclear structures sensitive to DNases and RNase. To examine localization of these proteins, an antibody was raised in a rabbit. Indirect immunofluorescence staining revealed that S1 proteins located in the extranucleolar nuclear regions of quiescent myocardial and cerebellar cells as well as actively duplicating mouse 3T3 fibroblasts. They located in euchromatin regions of thymus lymphocytes, with a characteristic aster-like immunofluorescence pattern, and on the border of condensed chromatin areas by deposition of immunogold particles in ultrathin sections of thymus. Thus, S1 proteins may be in a nuclear function assigned to the border of heterochromatin areas, and other than synthesis of DNA or of ribosomal RNA. Possible involvement of S1 proteins in the extranucleolar RNA synthesis is discussed.
Asunto(s)
Proteínas Nucleares/inmunología , Animales , Anticuerpos/inmunología , Técnica del Anticuerpo Fluorescente , Immunoblotting , Inmunohistoquímica , Ratones , ConejosRESUMEN
A quick and accurate method for estimating tissue water content by gas chromatography was developed. Age-related changes in tissue water content in the neocortex of rat brain were easily determined by this method.
Asunto(s)
Química Encefálica , Agua/análisis , Factores de Edad , Animales , Cromatografía de Gases , Femenino , RatasRESUMEN
Indirect and peroxidase anti-peroxidase (PAP) immunoenzymatic methods were used to detect terminal deoxynucleotidyl transferase (TdT) in imprints and formalin-fixed paraffin sections of normal rat thymus. TdT is found in the nuclei of small lymphocytes in imprint samples from neonatal and adult rat thymus, showing granular or circular patterns of peroxidase reaction products. Diffuse brown reaction products of peroxidase are located in both the nuclei and cytoplasm of medium and large lymphocytes. Indirect measurements show that, as age progresses, the percentage of peroxidase-positive cells decreases in all types of lymphocytes, from 72.4% on the 11th day to 54.8% in the 5th month, whereas that of negative cells increases from 14.4% to 39.4%. In formalin-fixed paraffin sections, peroxidase-positive lymphocytes are found mainly in the cortex and cortico-medullary boundary, and only rarely in the medulla.
Asunto(s)
Timo/enzimología , Envejecimiento , Animales , ADN Nucleotidiltransferasas/metabolismo , Histocitoquímica , Técnicas para Inmunoenzimas , Ratas , Timo/crecimiento & desarrolloRESUMEN
In the previous report, we had developed a simultaneous competitive enzyme immunoassay for hCG, using sheep red blood cells as the solid phase. The serum sample was not checked because of serum interference. In this report we have developed sequential competitive enzyme immunoassay. The sequential method is preferred because serum interference is removed and silicone rods are used as the solid phase instead of sheep red blood cells, which would be easily washed away in that procedure. The working range of this procedure is from 0.5 mIU/ml to 200 mIU/ml. That value for serum hCG correlates well with that by RIA (n = 0.904) Coefficients of variation are satisfied (14.2%: within assay and 15.2%: between assay).
Asunto(s)
Gonadotropina Coriónica/sangre , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Técnicas para Inmunoenzimas , Lactante , Embarazo , RadioinmunoensayoRESUMEN
A simultaneous competitive enzyme immunoassay (SICEIA) for hCG was developed using beta-D-galactosidase (beta-Gal) as a labelled enzyme and anti-hCG antibody coated sheep red blood cells (SRBC) as a solid phase. In this report, a new coupling agent, MCAE, was used to couple beta-Gal with hCG. The sensitivity was improved to the degree of 2.5 mIU/ml, equal to that of RIA. The present procedure was safer and rapider than RIA. The value of hCG in urine by our procedure had good correlation with that by RIA.