RESUMEN
Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).
Asunto(s)
Actinas/metabolismo , Productos del Gen rev/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Factores de Iniciación de Péptidos/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN , Elementos de Respuesta/genética , Actinas/antagonistas & inhibidores , Transporte Activo de Núcleo Celular , Animales , Proteínas Portadoras/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , VIH-1/genética , Células HeLa , Humanos , Virus del Mono Mason-Pfizer/genética , Espectrometría de Masas , Microinyecciones , Microscopía Inmunoelectrónica , Mutación , Membrana Nuclear/química , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestructura , Matriz Nuclear/química , Matriz Nuclear/metabolismo , Matriz Nuclear/ultraestructura , Oocitos/metabolismo , Factores de Iniciación de Péptidos/genética , Unión Proteica , ARN Viral/química , Proteínas Recombinantes de Fusión/metabolismo , Xenopus laevis , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
An understanding of the functional organization of nucleoli, the sites of ribosome biosynthesis, is limited by the present uncertainty about the topological arrangement of the transcribing rRNA genes. Since studies with "standard" nucleoli from somatic cells produced conflicting results, we have examined the amplified nucleoli of Xenopus oocytes. These nucleoli are unique in that they contain high copy numbers of rRNA genes, are not attached to chromosomes, lack non-ribosomal DNA and can be examined in light microscopic spread preparations of nuclear contents. By immunostaining and confocal microscopy we show that in growing stage IV oocytes the sites of rDNA are surrounded by the dense fibrillar component. The rDNA is actively transcribed as revealed by BrUTP injection into oocytes and localization of components of the nucleolar transcription machinery (RNA polymerase I and the transcription factor UBF). At the ultrastructural level, the rDNA sites correlate with the fibrillar centers of amplified nucleoli fixed in situ. The results provide clear evidence that the transcriptionally active rRNA genes are confined to the fibrillar centers of the oocyte nucleoli and open the possibility to analyze the protein composition of almost native, transcriptionally highly active nucleolar chromatin by immunofluorescence microscopy.
Asunto(s)
Nucléolo Celular/ultraestructura , Oocitos/ultraestructura , Animales , Femenino , Microscopía Confocal , Microscopía Electrónica , ARN Ribosómico/genética , ARN Ribosómico/ultraestructura , Xenopus laevisRESUMEN
Glucocorticoid hormones regulate the transcription of nuclear genes by way of their cognate receptors. In addition, these hormones also modulate mitochondrial gene transcription by mechanisms which are as yet poorly understood. Using immunofluorescence labeling and confocal laser scanning microscopy we show that the glucocorticoid receptor of HeLa and Hep-2 cells is specifically enriched at the sites of the mitochondria which were visualized by labeling with the vital dye CMX and antibodies against cytochrome oxidase subunit I. Immunogold electron microscopy demonstrated that the receptor was located within the inner space of the mitochondria. Immunoblotting experiments also revealed the presence of glucocorticoid receptor in mitochondria isolated from HeLa and Hep-2 cells. Finally, living HeLa cells expressing green fluorescent-glucocorticoid receptor fusion protein revealed a distinct mitochondrial GFP fluorescence. Our results support the concept of a receptor-mediated direct action of steroid hormones on mitochondrial gene transcription.
Asunto(s)
Mitocondrias/química , Receptores de Glucocorticoides/análisis , Animales , Western Blotting , Células COS , Fraccionamiento Celular , Dexametasona/farmacología , Complejo IV de Transporte de Electrones/análisis , Complejo IV de Transporte de Electrones/inmunología , Células HeLa , Histocitoquímica/métodos , Humanos , Microscopía Confocal , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Mitocondrias/genética , Mitocondrias/ultraestructura , Receptores de Glucocorticoides/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
The activity of the ribosomal RNA genes generates a distinct subnuclear structure, the nucleolus, which is the site of ribosome biogenesis. The signals that target proteins and snoRNAs (small nucleolar RNAs) to the nucleolus, the nuclear import of ribosomal proteins, the export of the completed ribosomal subunits and the molecular organization of the nucleolus have been the subject of intense research during the past year. Evidence is accumulating that nucleoli functionally interact with coiled bodies and are also involved in the maturation of non-ribosomal RNA species.
Asunto(s)
Nucléolo Celular/fisiología , Nucléolo Celular/ultraestructura , Animales , Humanos , Proteínas Nucleares/fisiología , ARN Nuclear Pequeño/metabolismoRESUMEN
Xenopus early embryos are transcriptionally quiescent until the midblastula transition (MBT). We have examined the question of whether the absence of rRNA synthesis is related to a deficiency in the RNA polymerase I (pol I) transcription machinery. Previously we have demonstrated that the maternally provided pol I transcription factor UBF already binds to the inactive rRNA genes of pre-MBT embryos (P. Bell et al., 1997, J. Cell Sci. 110, 2053-2063). Here we have analyzed the fate of pol I and the TATA box-binding protein (TBP) through immunofluorescence and immunoblotting experiments. Pol I stockpiled in the egg is taken up by in vitro assembled pronuclei and concentrated into numerous distinct nuclear domains. Comparable storage sites of template-free pol I are also seen in nuclei of blastula to neurula stage embryos. In contrast, the amount of TBP is relatively low in oocytes and eggs but increases dramatically during the cleavage stages. Most of the newly synthesized TBP colocalizes with the stored form of pol I in the extranucleolar domains of blastula/gastrula embryos. The amount of TBP per embryo reaches peak values at the blastula/gastrula stage and then rapidly declines to normal somatic levels. The positive correlation of maximal TBP levels with the timing of the MBT suggests that overproduction of TBP is required for the formation of productive transcription complexes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1 , ARN Polimerasa I/metabolismo , Factores de Transcripción/metabolismo , Xenopus laevis/embriología , Animales , Blastocisto , Núcleo Celular/metabolismo , Humanos , Conejos , Proteína de Unión a TATA-Box , Transcripción GenéticaRESUMEN
Using a reconstituted complex of profilin and skeletal muscle actin as an antigen, we generated a monoclonal mouse antibody against actin, termed 2G2. As revealed by immunoblots of proteolytic actin fragments and by pepscan analysis, the antibody recognises a nonsequential epitope on actin which is located within three different regions of the sequence, consisting of aa131-139, aa155-169, and aa176-187. In the actin model derived from X-ray diffraction, these sequences lie spatially close together in the region of the nucleotide-binding cleft, but do not form a coherent patch. In immunoblots, 2G2 reacts with all SDS-denatured actin isoforms and with actins of many vertebrates. In contrast, its immunofluorescence reactivity is highly selective and fixation-dependent. In fibroblasts and myogenic cells, fixed and extracted by formaldehyde/detergent, stress fibres or myofibrils, respectively, remained unstained. Likewise, after microinjection into living cells, 2G2 did not bind to such microfilament bundles. Extraction of myosin and tropomyosin did not alter this pattern indicating that the lack in reactivity is probably not due to epitope-masking by actin-binding proteins. More likely, the reason for the lack of reactivity with filamentous actin is that its epitope is not accessible in F-actin. However, the antibody revealed a distinct pattern of nuclear dots in differentiated myogenic cells but not in myoblasts, and of fibrillar structures in nuclei of Xenopus oocytes. In contrast, after methanol treatment, a 2G2-specific staining of stress fibres and myofibrils was observed, but no nuclear dot staining. We conclude that 2G2, in addition to binding to SDS- and methanol-denatured actin, recognises a specific conformation of native actin which is present in the nucleus and specified by compaction of the antibody-reactive region into a coherent patch. This conformation is apparently present in differentiated myogenic cells and oocytes, but not in cytoplasmic actin filament bundles.
Asunto(s)
Actinas/química , Núcleo Celular/fisiología , Músculo Esquelético/fisiología , Conformación Proteica , Actinas/inmunología , Animales , Anticuerpos Monoclonales , Núcleo Celular/ultraestructura , Células Cultivadas , Pollos , Citoplasma/fisiología , Citoplasma/ultraestructura , Fibroblastos/fisiología , Fibroblastos/ultraestructura , Corazón/fisiología , Ratones , Músculo Esquelético/ultraestructura , Miocardio/ultraestructura , Oocitos/fisiología , Oocitos/ultraestructura , Fragmentos de Péptidos/inmunología , Isoformas de Proteínas/química , Isoformas de Proteínas/inmunología , Xenopus laevisRESUMEN
Amphibian oocytes accumulate a large pool of mRNA molecules for future embryonic development. Due to their association with specific proteins the stored maternal RNAs are translationally repressed. The identification of these RNA-binding proteins and the characterization of their functional domains may contribute to the understanding of the translational repression mechanisms and the subsequent activation processes during early embryogenesis. Here we present the complete Pleurodeles cDNA sequence of a cytoplasmic protein which is present in oocytes, eggs, and very early cleavage stage embryos but undetectable in postcleavage embryo and adult tissues. The predicted molecular mass of the protein is 55 kDa and the apparent molecular mass as determined by SDS-PAGE, 68 kDa. The deduced amino acid sequence reveals proline- and serine-rich domains in the aminoterminal part as well as two RGG boxes which represent characteristic motifs of several RNA-binding proteins. No distinct homologies to the consensus RNA recognition motif were found. The 55-kDa protein was recovered in cytoplasmic ribonucleoprotein (RNP) particles containing poly(A)+ RNA. It was therefore termed RAP55 for mRNA-associated protein of 55 kDa. However, a direct interaction of RAP55 with mRNA could not be demonstrated by UV-crosslinking experiments, indicating that it is bound to mRNP complexes via protein-protein interactions. RAP55 is evolutionarily conserved since antibodies raised against a recombinant Pleurodeles RAP55 fragment recognize the protein from Pleurodeles and Xenopus. The expression pattern and intracellular distribution of RAP55 suggest that it is part of those mRNP particles which are translationally repressed during oogenesis and become activated upon progesterone-induced oocyte maturation.
Asunto(s)
Proteínas del Huevo , Pleurodeles/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Xenopus laevis/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Cromatografía de Afinidad , Clonación Molecular , Citoplasma/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia Molecular , Peso Molecular , Oocitos/metabolismo , Especificidad de Órganos , Pleurodeles/genética , Biosíntesis de Proteínas , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleasas/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Xenopus laevis/embriologíaRESUMEN
The high mobility group 14/17 (HMG-14/-17) proteins form specific complexes with nucleosome core particles and produce distinct footprints on nucleosomal DNA. Therefore, they could be an integral part of the chromatin fiber. Here we show that during the cell cycle these proteins are transiently dissociated from chromatin. They colocalize with the nuclear DNA in interphase and prophase but not in metaphase and anaphase. They relocate into the nucleus and colocalize again with the DNA in late telophase, concomitantly with the appearance of the nuclear envelope. Thus, these nucleosomal binding proteins are not always associated with chromatin. Using reconstituted nuclei and permeabilized cells, we demonstrate that these two small proteins, with a molecular mass <10 kD, are actively imported into the nucleus. We identify the major elements involved in the nuclear import of these chromosomal proteins: HMG-14/-17 proteins contain an intrinsic bipartite nuclear localization signal, and their entry into the nucleus through nuclear pores requires energy and the participation of importin alpha. These findings suggest that the cell cycle-related association of HMG-14/-17 with chromatin is dependent on, and perhaps regulated by, nuclear import processes.
Asunto(s)
Ciclo Celular , Núcleo Celular/metabolismo , Cromosomas/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Células 3T3 , Animales , Transporte Biológico Activo , Cromatina/metabolismo , Femenino , Interfase , Masculino , Metafase , Ratones , Mitosis , Membrana Nuclear/fisiología , Nucleosomas/metabolismo , Oocitos/fisiología , Espermatozoides/fisiología , Extractos de Tejidos , XenopusRESUMEN
Chromosomal proteins HMG-14/-17 are nucleosomal binding proteins, which alter the structure of the chromatin fiber and enhance transcription, but only from chromatin templates. Here we show that in tissue culture cells, HMG-17 protein colocalizes with sites of active transcription. Incubation of permeabilized cells with a peptide corresponding to the nucleosomal binding domains of HMG-14/-17 specifically arrested polymerase II-dependent transcription. In these cells the peptide displaces HMG-17 from chromatin and reduces the cellular content of the protein. These results suggest that the presence of HMG-14/-17 in chromatin is required for efficient polymerase II transcription. In non-permeabilized, actively transcribing cells, the protein is dispersed in a punctate pattern, throughout the nucleus. Upon transcriptional inhibition by alpha-amanitin or actinomycin D, the protein gradually redistributes until it localizes fully to interchromatin granule clusters, together with the splicing factor SC35. The results suggest that the association of HMG-17 with chromatin is dynamic rather than static, and that in the absence of transcription, HMG-17 is released from chromatin and accumulates in interchromatin granule clusters. Thus, the intranuclear distribution of chromosomal proteins which act as architectural elements of chromatin structure may be dynamic and functionally related to the transcriptional activity of the cell.
Asunto(s)
Proteínas del Grupo de Alta Movilidad/metabolismo , Transcripción Genética , Sitios de Unión , Transporte Biológico , Núcleo Celular/metabolismo , Humanos , Nucleosomas , Péptidos , ARN Polimerasa II , Células Tumorales CultivadasRESUMEN
To explore the potential of recombinant vectors based on recombinant adeno-associated virus (rAAV) for cancer vaccination, we investigated the transduction efficiency of rAAV into cancer cells ex vivo. Infection of human epithelial cancer cell lines with rAAV carrying reporter genes encoding beta-galactosidase (rAAV/LacZ) or luciferase (rAAV/Luc) resulted in high levels of reporter gene expression (>90% positive cells). In marked contrast, rAAV poorly transduced all murine tumor cell lines, as well as human hematopoietic cell lines. Either irradiation or adenovirus infection of tumor cells prior to rAAV infection induced a 10- to 100-fold increase of reporter gene expression. To determine the transduction efficiency of rAAV into primary cancer cells, freshly isolated, irradiated tumor cells from malignant melanoma and ovarian carcinoma patients were infected with rAAV/Luc, resulting in up to 6.9-fold higher levels of gene expression than in a HeLa tumor cell line. Time course experiments with freshly isolated tumor cells infected with rAAV/Luc showed maximal levels of luciferase expression between days 3 and 9 posttransduction. Simultaneous infection of primary tumor cells with up to three rAAV vectors containing genes encoding the immunostimulatory proteins B7-2 (CD86), p35 subunit of IL-12, and p40 subunit of IL-12 resulted in high expression of B7-2 in more than 90% of the tumor cells and in the secretion of high levels of IL-12. Taken together, our results demonstrate that rAAV efficiently transduces freshly isolated human, epithelial tumor cells and might therefore be a potent tool to produce improved, gene-modified cancer vaccines.
Asunto(s)
Vacunas contra el Cáncer , Dependovirus , Células Epiteliales/metabolismo , Técnicas de Transferencia de Gen , Antígenos CD/genética , Antígeno B7-2 , Femenino , Células HT29 , Células HeLa , Humanos , Melanoma , Glicoproteínas de Membrana/genética , Neoplasias Ováricas , Recombinación Genética , Células Tumorales Cultivadas , Rayos XRESUMEN
When nuclei (pronuclei) were assembled from sperm chromatin in Xenopus egg extract and examined by immunofluorescence microscopy, UBF was concentrated at a single intranuclear dot-like or more extended necklace-like structure. These UBF-foci contained rDNA as demonstrated by in situ hybridization and hence represent the chromosomal nucleolus organizing regions (NORs). Besides UBF, other components of the transcription machinery such as the TATA-box binding protein (TBP) and RNA polymerase I (pol I) as well as several nucleolar proteins could not be detected at the NORs. Immuno-depletion experiments indicated the UBF is maternally provided and taken up by the pronuclei. Essentially the same results were obtained when we examined the NORs of early Xenopus embryos up to the midblastula stage. After this stage, when transcription of the rRNA genes has begun, nucleoli developed and the NORs acquired TBP and pol I. Our results support the hypothesis that UBF is an architectural element which converts the rDNA chromatin into a transcriptionally competent form.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Región Organizadora del Nucléolo/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Operón de ARNr/fisiología , Animales , Especificidad de Anticuerpos , Blastocisto/química , Núcleo Celular/química , Núcleo Celular/genética , Cromatina/química , ADN Ribosómico/análisis , ADN Ribosómico/metabolismo , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/inmunología , Femenino , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Región Organizadora del Nucléolo/metabolismo , ARN Polimerasa I/análisis , ARN Polimerasa I/inmunología , Espermatozoides/química , Proteína de Unión a TATA-Box , Factores de Transcripción/análisis , Factores de Transcripción/inmunología , Transcripción Genética/fisiología , Xenopus laevisRESUMEN
We describe novel nucleolar structures, observed by thin section electron microscopy in oocyte nuclei of the grasshopper Locusta migratoria, which we interpret, based on morphological and compositional criteria, as rDNA transcription units. Morphologically they resemble the condensed and foreshortened "Christmas trees" seen in Miller spreads of nucleolar chromatin prepared from the same biological material. They contain DNA and rRNA as shown by immunocytochemistry and in situ hybridization and are concentrated in several intranucleolar cavities. The presumptive rDNA transcription units extend throughout the interior of these nucleolar pockets or are selectively enriched at their outermost zones in close contact with the surrounding fibrillarin-positive dense component. We suggest that the nucleolar pockets of Locusta oocytes are equivalent to the fibrillar centers of somatic nucleoli and discuss possible implications for the current understanding of the functional organization of nucleoli.
Asunto(s)
Nucléolo Celular/fisiología , Nucléolo Celular/ultraestructura , Oocitos/fisiología , Animales , Proteínas Cromosómicas no Histona/inmunología , Proteínas Cromosómicas no Histona/metabolismo , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Femenino , Saltamontes/fisiología , Oocitos/ultraestructura , ARN Ribosómico/análisis , ARN Ribosómico/metabolismoRESUMEN
Nuclei assembled in Xenopus egg extract contain numerous spherical aggregations or nuclear bodies. Previously we have shown that they closely resemble prenucleolar bodies (PNBs), both at the compositional and ultrastructural level. Subsequently, coilin was also identified and for this reason they were called coiled bodies. Here we present morphological and immunocytochemical evidence that the in vitro nuclear bodies resemble authentic PNBs and are different from coiled bodies. In particular we show that coilin, previously considered as the defining protein constituent of coiled bodies, is also present in PNBs of cultured cells. In contrast, the PNB-associated nucleolar proteins nucleolin and B23/NO38 are not detectable in coiled bodies and may thus serve as suitable markers for PNBs. Our results suggest that PNBs are primary assembly structures which contribute to the formation of both nucleoli and coiled bodies and thus offer an explanation for the frequently observed structural association of coiled bodies with nucleoli. To gain some insight into the assembly process of PNBs in vitro, specific nucleolar proteins were removed from Xenopus egg extract. Quite surprisingly, the immuno-depleted extracts still promoted the assembly of nuclear bodies which lacked either fibrillarin, nucleolin, xNopp180 or B23/NO38. Only after fibrillarin-depletion fewer PNBs were seen as compared to controls. Digestion of the extract with RNase followed by northern blot analysis revealed that U3 small nucleolar RNA is not required for the formation and structural maintenance of PNBs in vitro.
Asunto(s)
Nucléolo Celular/fisiología , Núcleo Celular/fisiología , Proteínas Nucleares/metabolismo , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN , Animales , Biomarcadores , Nucléolo Celular/química , Nucléolo Celular/ultraestructura , Núcleo Celular/química , Núcleo Celular/ultraestructura , Sistema Libre de Células , Células Cultivadas , Femenino , Técnica del Anticuerpo Fluorescente , Immunoblotting , Microscopía Electrónica , Nucleofosmina , Óvulo , Fosfoproteínas , Xenopus laevis , NucleolinaRESUMEN
Using hight-titer recombinant adeno-associated viral vectors (rAAV), we have investigated the feasibility of cancer vaccines from tumor explants. In a first set of experiments, rAAV vectors expressing firefly luciferase reporter genes were used to transduce different human tumor cell lines. At day three post transduction, all of the human tumor cell lines tested showed high levels of luciferase expression. To further evaluate rAAV-mediated gene transfer efficiency into primary tumor cells, we transduced freshly isolated tumor cells from malignant melanoma and ovarian carcinoma patients. As a remarkable result, reporter gene expression in primary tumor cells was significantly higher than in the tested established tumor cell lines. These data could also be reproduced with a rAAV/lacZ vector, since the portion of successfully transduced primary tumor was higher than 90%. Taken together, our data demonstrate that rAAV-mediated gene transfer is a very efficient method for the transduction of freshly isolated human tumor cells and may allow the generation of potent autologous cancer vaccines.
Asunto(s)
Vacunas contra el Cáncer/genética , Dependovirus/genética , Vectores Genéticos , Transducción Genética , Adenoviridae/genética , Animales , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/aislamiento & purificación , Escarabajos/enzimología , Escarabajos/genética , Expresión Génica , Técnicas de Transferencia de Gen , Genes Reporteros , Humanos , Operón Lac , Luciferasas/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Sobreinfección , Transducción Genética/efectos de los fármacos , Transducción Genética/efectos de la radiación , Células Tumorales CultivadasRESUMEN
Chromosomal proteins HMG-14 and HMG-17 are nucleosome binding proteins which can function as architectural elements to alter the structure of the chromatin fiber and enhance transcription from chromatin templates. Here we study the spatial organization of these HMG proteins in the nucleus and the distribution of nucleosomes containing HMG-17 in the chromatin fiber. By confocal immunofluorescence microscopy we find that HMG-14/17 proteins are clustered into foci containing either HMG-14 or HMG-17. These results suggest that HMG-14/17 proteins segregate into distinct nuclear domains. Indeed, immunofractionation of defined length oligonucleosomes, with affinity pure antibodies to HMG-17, indicates that oligonucleosomes containing HMG-17 are devoid of HMG-14. Quantitative analysis indicates that in cellular chromatin nucleosomes containing HMG-17 are clustered. The average size of the cluster is six contiguous HMG-17-containing nucleosomes. The nucleosomes in this cluster contain either two or zero molecules of HMG-17 and a complete set of four core histones. We suggest that HMG-14/17 proteins modify the nucleosomal organization of the 30 nm chromatin fiber, to unfold the higher order chromatin structure and facilitate access to the underlying DNA sequence. Clustering of architectural elements, such as HMG proteins and linker histone subtypes into distinct domains, may lead to structural and functional heterogeneity along the chromatin fiber.
Asunto(s)
Cromatina/química , Proteínas del Grupo de Alta Movilidad/química , Proteínas del Grupo de Alta Movilidad/metabolismo , Nucleosomas/química , Animales , Núcleo Celular/metabolismo , Dimerización , Histonas/química , Histonas/metabolismo , Humanos , Ratones , Microscopía Fluorescente , Nucleosomas/metabolismo , Pruebas de PrecipitinaRESUMEN
Pore complexes are not confined to the nuclear envelope but can also be found in the cytoplasm of numerous cell types in the form of annulate lamellae (AL). We have induced formation of AL by exposure of rat cells (line RV) to sublethal doses of the antimitotic drug vinblastine sulfate, and compared the distribution of several nuclear pore complex proteins (nucleoporins) in the nuclear envelope and AL by immunocytochemistry, cytochemical lectin binding studies and immunoblot analyses of nuclear and AL-enriched fractions. All the antibodies used yielded punctate nuclear surface staining in immunofluorescence microscopy which is characteristic for nuclear pore complex components. When we applied antibodies against the nucleoporin p62, AL were visualized as numerous cytoplasmic dot-like structures. Immunogold electron microscopy confirmed the correspondence of the cytoplasmic bodies with stacks of AL. Antibodies to constituents of the cytoplasmic (nup180) and nucleoplasmic (nup153) filaments extending from both sides of nuclear pore complexes also stained the AL, indicating that pore complexes are intrinsically asymmetric assemblies independent of their specific intracellular topology. By contrast, AL were negative with five different antibodies against the transmembrane nuclear pore glycoprotein gp210 and the lectin concanavalin A (ConA) known to bind to the oligosaccharide side chains of gp210. Similarly, there was no staining of the AL with antibodies to the other nuclear pore membrane protein so far known in higher eukaryotes, POM121. Immunoblot analyses confirmed the presence of p62, nup180 and nup153 in both the nuclear and AL fractions and the absence of gp210 and POM121 from AL. Our results do not support the generally held view that gp210 and POM121 function in anchoring the pore complex scaffold to the pore membrane. Rather, they point to a role for these proteins in transport processes through the nuclear pore complexes. Since AL are not involved in nucleocytoplasmic transport processes they may lack components of the transport machinery.
Asunto(s)
Núcleo Celular/ultraestructura , Citoplasma/ultraestructura , Músculo Liso Vascular/ultraestructura , Porinas/análisis , Animales , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Inmunohistoquímica , Microscopía Inmunoelectrónica , RatasRESUMEN
Xenopus oocytes express a 165 kDa variant of DNA topoisomerase I (topo I) as opposed to the canonical 110 kDa form of somatic cells (Richard and Bogenhagen, Dev. Biol. 146: 4-11, 1991). By immunofluorescence microscopy using variant-specific antibodies we show that this high molecular weight form is associated with lampbrush chromosome loops and the inner regions of the amplified nucleoli. Inhibition of topo I-activity by either Camptothecin-treatment or microinjection of neutralizing antibodies resulted in loop retraction and the condensation of chromosomes and amplified nucleoli. These data indicate that the oocyte-specific 165 kDa form of topo I is involved in transcriptional processes mediated by RNA polymerase I and II and is therefore functionally equivalent to the somatic cell 110 kDa counterpart.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , Oocitos/enzimología , Xenopus laevis/metabolismo , Animales , Nucléolo Celular/enzimología , Cromosomas/enzimología , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/inmunología , Femenino , Microscopía Fluorescente , Peso MolecularRESUMEN
By immunizing Balb/c mice with oocyte nuclei of Pleurodeles waltl we obtained a monoclonal antibody, mAb 4A6, that labels distinct globular domains of the lampbrush chromosomal axes of Pleurodeles. These domains are found at corresponding sites of homologous chromosomes, often at telomeric and putative centromeric regions, and appear to be devoid of DNA. Because of these characteristic features it is most likely that the mAb 4A6-positive domains correspond to the central part of the "axial granules" of urodelan lampbrush chromosomes. In immunoblotting analyses mAb 4A6 reacts with a nuclear antigen of approximately Mr 180000 and a structurally nonrelated cytoplasmic protein of Mr 98000, which was not characterized any further. Comparative immunofluorescence and immunoblotting studies with mAb 4A6 and an antiserum against DNA topoisomerase II (topo II) as well as immunodepletion experiments demonstrated that the nuclear 4A6 antigen is topo II. Our results indicate that topo II is not a constituent of a continuous, loop-anchoring scaffold in lampbrush chromosomes of Pleurodeles but, rather, is restricted to the axial granules.
Asunto(s)
Anticuerpos Monoclonales , Cromosomas/enzimología , ADN-Topoisomerasas de Tipo II/análisis , Animales , Antígenos Nucleares , Cromatina/química , Cromosomas/ultraestructura , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/inmunología , Femenino , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Peso Molecular , Proteínas Nucleares/análisis , Proteínas Nucleares/química , Oocitos , Pleurodeles , Proteínas/análisis , Proteínas/química , Xenopus laevisRESUMEN
A partial cDNA clone coding for the mouse homologue of the human Ran-GTP binding protein, RanBP2, has been isolated by screening of a murine expression library with antibodies to nup180, a previously identified nuclear pore complex protein (nucleoporin). Whether the antibodies cross-reacted with the polypeptide encoded by the cDNA clone or, alternatively, nup180 is proteolytically related to RanBP2, has not been determined. The 3795-bp open reading frame of the cDNA encodes a polypeptide consisting of 1265 amino acids with three Ran-GTP binding domains (RanBD) that are almost identical with published partial amino acid sequences of human RanBP2 as deduced from several partial cDNA clones of other authors. Sequence analysis further revealed that murine RanBP2 contains tandemly repeated zinc fingers of Cys2-Cys2 type and multiple copies of the FXFG nucleoporin "signature" motif clustered in regions preceding the RanBDs. Antibodies raised against a synthetic peptide of the derived amino acid sequence decorated the cytoplasmic rings of nuclear pore complexes (NPCs) as shown by immunogold electron microscopy. We suggest that the cytoplasmically disposed nucleoporin RanBP2 provides docking sites for import substrate-receptor complexes and, further, that the affinity of these sites to the transport substrate is modulated in a Ran-dependent fashion.
Asunto(s)
Citoplasma/química , Proteínas de Unión al ADN/análisis , Membrana Nuclear/química , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Humanos , Ratones , Chaperonas Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ratas , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Dedos de Zinc , Proteína de Unión al GTP ranRESUMEN
When cells enter mitosis, RNA synthesis ceases. Yet the RNA polymerase I (pol I) transcription machinery involved in the production of pre-rRNA remains bound to the nucleolus organizing region (NOR), the chromosome site harboring the tandemly repeated rRNA genes. Here we examine whether rDNA transcription units are transiently blocked or "frozen" during mitosis. By using fluorescent in situ hybridization we were unable to detect nascent pre-rRNA chains on the NORs of mouse 3T3 and rat kangaroo PtK2 cells. Appropriate controls showed that our approach was sensitive enough to visualize, at the light microscopic level, individual transcriptionally active rRNA genes both in situ after experimental unfolding of nucleoli and in chromatin spreads ("Miller spreads"). Analysis of the cell cycle-dependent redistribution of transcript-associated components also revealed that most transcripts are released from the rDNA at mitosis. Upon disintegration of the nucleolus during mitosis, U3 small nucleolar RNA (snoRNA) and the nucleolar proteins fibrillarin and nucleolin became dispersed throughout the cytoplasm and were excluded from the NORs. Together, our data rule out the presence of "frozen Christmas-trees" at the mitotic NORs but are compatible with the view that inactive pol I remains on the rDNA. We propose that expression of the rRNA genes is regulated during mitosis at the level of transcription elongation, similarly to what is known for a number of genes transcribed by pol II. Such a mechanism may explain the decondensed state of the NOR chromatin and the immediate transcriptional reactivation of the rRNA genes following mitosis.