RESUMEN

The nucleolus is a ubiquitous, mostly spheroidal nuclear structure of all protein-synthesizing cells, with a well-defined functional compartmentalization. Although a number of nonribosomal proteins involved in ribosome formation have been identified, the elements responsible for the shape and internal architecture of nucleoli are still largely unknown. Here, we report the molecular characterization of a novel protein, NO145, which is a major and specific component of a nucleolar cortical skeleton resistant to high salt buffers. The amino acid sequence of this polypeptide with a SDS-PAGE mobility corresponding to M(r) 145,000 has been deduced from a cDNA clone isolated from a Xenopus laevis ovary expression library and defines a polypeptide of 977 amino acids with a calculated mass of 111 kDa, with partial sequence homology to a synaptonemal complex protein, SCP2. Antibodies specific for this protein have allowed its recognition in immunoblots of karyoskeleton-containing fractions of oocytes from different Xenopus species and have revealed its presence in all stages of oogenesis, followed by a specific and rapid degradation during egg formation. Immunolocalization studies at the light and electron microscopic level have shown that protein NO145 is exclusively located in a cage-like cortical structure around the entire nucleolus, consisting of a meshwork of patches and filaments that dissociates upon reduction of divalent cations. We propose that protein NO145 contributes to the assembly of a karyoskeletal structure specific for the nucleolar cortex of the extrachromosomal nucleoli of Xenopus oocytes, and we discuss the possibility that a similar structure is present in other cells and species.

Asunto(s)

Nucléolo Celular/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Oocitos/citología , Oocitos/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis , Secuencia de Aminoácidos , Animales , Nucléolo Celular/química , Nucléolo Celular/ultraestructura , Clonación Molecular , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/ultraestructura , Espectrometría de Masas , Microscopía Confocal , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Oocitos/ultraestructura , Oogénesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Homología de Secuencia de Aminoácido , Proteínas de Xenopus/genética , Proteínas de Xenopus/ultraestructura , Xenopus laevis/genética

RESUMEN

SF3b(155) is an essential spliceosomal protein, highly conserved during evolution. It has been identified as a subunit of splicing factor SF3b, which, together with a second multimeric complex termed SF3a, interacts specifically with the 12S U2 snRNP and converts it into the active 17S form. The protein displays a characteristic intranuclear localization. It is diffusely distributed in the nucleoplasm but highly concentrated in defined intranuclear structures termed "speckles," a subnuclear compartment enriched in small ribonucleoprotein particles and various splicing factors. The primary sequence of SF3b(155) suggests a multidomain structure, different from those of other nuclear speckles components. To identify which part of SF3b(155) determines its specific intranuclear localization, we have constructed expression vectors encoding a series of epitope-tagged SF3b(155) deletion mutants as well as chimeric combinations of SF3b(155) sequences with the soluble cytoplasmic protein pyruvate kinase. Following transfection of cultured mammalian cells, we have identified (i) a functional nuclear localization signal of the monopartite type (KRKRR, amino acids 196--200) and (ii) a molecular segment with multiple threonine-proline repeats (amino acids 208--513), which is essential and sufficient to confer a specific accumulation in nuclear speckles. This latter sequence element, in particular amino acids 208--440, is required for correct subcellular localization of SF3b(155) and is also sufficient to target a reporter protein to nuclear speckles. Moreover, this "speckle-targeting sequence" transfers the capacity for interaction with other U2 snRNP components.

Asunto(s)

Señales de Localización Nuclear/aislamiento & purificación , Fosfoproteínas/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Análisis Mutacional de ADN , Humanos , Fosfoproteínas/química , Fosfoproteínas/genética , Prolina/metabolismo , Estructura Terciaria de Proteína , Factores de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U2/química , Ribonucleoproteína Nuclear Pequeña U2/genética , Ribonucleoproteína Nuclear Pequeña U2/fisiología , Treonina/metabolismo , Células Tumorales Cultivadas

RESUMEN

We report the identification, cDNA cloning, and molecular characterization of a novel, constitutive nucleolar protein. The cDNA-deduced amino acid sequence of the human protein defines a polypeptide of a calculated mass of 61.5 kDa and an isoelectric point of 9.9. Inspection of the primary sequence disclosed that the protein is a member of the family of "DEAD-box" proteins, representing a subgroup of putative ATP-dependent RNA helicases. ATPase activity of the recombinant protein is evident and stimulated by a variety of polynucleotides tested. Immunolocalization studies revealed that protein NOH61 (nucleolar helicase of 61 kDa) is highly conserved during evolution and shows a strong accumulation in nucleoli. Biochemical experiments have shown that protein NOH61 synthesized in vitro sediments with approximately 11.5 S, i.e., apparently as homo-oligomeric structures. By contrast, sucrose gradient centrifugation analysis of cellular extracts obtained with buffers of elevated ionic strength (600 mM NaCl) revealed that the solubilized native protein sediments with approximately 4 S, suggestive of the monomeric form. Interestingly, protein NOH61 has also been identified as a specific constituent of free nucleoplasmic 65S preribosomal particles but is absent from cytoplasmic ribosomes. Treatment of cultured cells with 1) the transcription inhibitor actinomycin D and 2) RNase A results in a complete dissociation of NOH61 from nucleolar structures. The specific intracellular localization and its striking sequence homology to other known RNA helicases lead to the hypothesis that protein NOH61 might be involved in ribosome synthesis, most likely during the assembly process of the large (60S) ribosomal subunit.

Asunto(s)

Nucléolo Celular/química , ARN Helicasas/química , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Línea Celular , Nucléolo Celular/metabolismo , Centrifugación por Gradiente de Densidad , Cromatografía en Gel , Clonación Molecular , ARN Helicasas DEAD-box , Células HeLa , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Peso Molecular , Oocitos , ARN Helicasas/aislamiento & purificación , Ribosomas/química , Transfección , Xenopus laevis

RESUMEN

We report the identification and molecular characterization of a novel type of constitutive nuclear protein that is present in diverse vertebrate species, from Xenopus laevis to human. The cDNA-deduced amino acid sequence of the Xenopus protein defines a polypeptide of a calculated mass of 146.2 kDa and a isoelectric point of 6.8, with a conspicuous domain enriched in the dipeptide TP (threonine-proline) near its amino terminus. Immunolocalization studies in cultured cells and tissues sections of different origin revealed an exclusive nuclear localization of the protein. The protein is diffusely distributed in the nucleoplasm but concentrated in nuclear speckles, which represent a subnuclear compartment enriched in small nuclear ribonucleoprotein particles and other splicing factors, as confirmed by colocalization with certain splicing factors and Sm proteins. During mitosis, when transcription and splicing are downregulated, the protein is released from the nuclear speckles and transiently dispersed throughout the cytoplasm. Biochemical experiments have shown that the protein is recovered in a approximately 12S complex, and gel filtration studies confirm that the protein is part of a large particle. Immunoprecipitation and Western blot analysis of chromatographic fractions enriched in human U2 small nuclear ribonucleoprotein particles of distinct sizes (12S, 15S, and 17S), reflecting their variable association with splicing factors SF3a and SF3b, strongly suggests that the 146-kDa protein reported here is a constituent of the SF3b complex.

Asunto(s)

Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Empalmosomas/metabolismo , Secuencia de Aminoácidos , Animales , Biomarcadores/análisis , Bovinos , Línea Celular , Centrifugación por Gradiente de Densidad , Embrión de Pollo , Cromatografía en Gel , Clonación Molecular , ADN Complementario/aislamiento & purificación , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Peso Molecular , Proteínas Nucleares/química , Proteínas Nucleares/aislamiento & purificación , Especificidad de Órganos , Proteínas de Unión al ARN/metabolismo , Ratas , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Coloración y Etiquetado , Xenopus laevis

RESUMEN

We report the discovery and molecular characterization of a small and very acidic nucleolar protein of an SDS/PAGE mobility corresponding to Mr 29,000 (NO29). The cDNA-deduced sequence of the Xenopus laevis protein defines a polypeptide of a calculated molecular mass of 20,121 and a pI of 3.75, with an extended acidic region near its C terminus, and is related to the major nucleolar protein, NO38, and the histone-binding protein, nucleoplasmin. This member of the nucleoplasmin family of proteins was immunolocalized to nucleoli in Xenopus oocytes and diverse somatic cells. Protein NO29 is associated with nuclear particles from Xenopus oocytes, partly complexed with protein NO38, and occurs in preribosomes but not in mature ribosomes. The location and the enormously high content of negatively charged amino acids lead to the hypothesis that NO29 might be involved in the nuclear and nucleolar accumulation of ribosomal proteins and the coordinated assembly of pre-ribosomal particles.

Asunto(s)

Nucléolo Celular/química , Núcleo Celular/química , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/química , Oocitos/fisiología , Fosfoproteínas/biosíntesis , Fosfoproteínas/química , Secuencia de Aminoácidos , Animales , Fraccionamiento Celular , Línea Celular , Nucléolo Celular/ultraestructura , Núcleo Celular/ultraestructura , Clonación Molecular , Femenino , Humanos , Riñón/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Proteínas Nucleares/análisis , Nucleoplasminas , Oocitos/química , Oocitos/ultraestructura , Fosfoproteínas/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Ribosomas/química , Ribosomas/ultraestructura , Alineación de Secuencia , Transfección , Xenopus laevis

RESUMEN

Using a specific monoclonal antibody (mAb AND-1/23-5-14) we have identified, cDNA-cloned and characterized a novel DNA-binding protein of the clawed toad, Xenopus laevis, that is accumulated in the nucleoplasm of oocytes and various other cells. This protein comprises 1,127 amino acids, with a total molecular mass of 125 kDa and a pI of 5.27. It is encoded by a mRNA of approximately 4 kb and contains, in addition to clusters of acidic amino acids, two hallmark motifs: the amino-terminal part harbours seven consecutive 'WD-repeats', which are sequence motifs of about 40 amino acids that are characteristic of a large group of regulatory proteins involved in diverse cellular functions, while the carboxy terminal portion possesses a 63-amino-acid-long 'HMG-box', which is typical of a family of DNA-binding proteins involved in regulation of chromatin assembly, transcription and replication. The DNA-binding capability of the protein was demonstrated by DNA affinity chromatography and electrophoretic mobility shift assays using four-way junction DNA. Protein AND-1 (acidic nucleoplasmic DNA-binding protein) appears as an oligomer, probably a homodimer, and has been localized throughout the entire interchromatinic space of the interphase nucleoplasm, whereas during mitosis it is transiently dispersed over the cytoplasm. We also identified a closely related, perhaps orthologous protein in mammals. The unique features of protein AND-1, which is a 'natural chimera' combining properties of the WD-repeat and the HMG-box families of proteins, are discussed in relation to its possible nuclear functions.

Asunto(s)

Proteínas de Unión al ADN/genética , Proteínas del Grupo de Alta Movilidad/genética , Proteínas Recombinantes de Fusión/genética , Proteínas de Xenopus , Animales , Anticuerpos Monoclonales , Núcleo Celular/química , Reacciones Cruzadas , ADN Complementario/análisis , ADN Complementario/aislamiento & purificación , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/inmunología , Femenino , Proteínas del Grupo de Alta Movilidad/química , Proteínas del Grupo de Alta Movilidad/inmunología , Humanos , Ratones , Datos de Secuencia Molecular , Oocitos/química , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Secuencias Repetitivas de Ácidos Nucleicos/genética , Homología de Secuencia de Aminoácido , Solubilidad , Especificidad de la Especie , Xenopus laevis

RESUMEN

To identify the element(s) in nucleolar proteins which determine nucleolus-specific topogenesis, we have used different kinds of cDNA constructs encoding various chimeric combinations of mutants of the constitutive nucleolar protein NO38 (B23): 1) with an amino terminally placed short "myc tag"; 2) with two different carboxyl terminally attached large alpha-helical coiled coil structures, the lamin A rod domain or the rod domain of vimentin; 3) with the sequence-related nucleoplasmic histone-binding protein nucleo-plasmin; and 4) with the soluble cytoplasmic protein pyruvate kinase. To avoid the problem of formation of complexes with endogenous wild-type (wt) molecules and "piggyback" localization, special care was taken to secure that the mutants and chimeras used did not oligomerize as is typical of protein NO38 (B23). Using microinjection and transfection of cultured cells, we found that the segment comprising the amino-terminal 123 amino acids (aa) alone was sufficient to effect nucleolar accumulation of the construct molecules, including the chimeras with the entire rod domains of lamin A and vimentin. However, when the amino-terminal 109 aa were deleted, the molecules still associated with the nucleolus. The results of further deletion experiments and of domain swaps with nucleoplasmin all point to the topogenic importance of two independent molecular regions located at both the amino- and carboxyl-terminal end. Our definition of dominant elements determining the nucleolar localization of protein NO38 (B23) as well as of diverse nonnucleolar proteins will help to identify its local binding partner(s) and functions, the construction of probes examining other proteins or sequence elements within the nucleolar microenvironment, and the generation of cells with an altered nuclear architecture.

Asunto(s)

Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Animales , Sitios de Unión , Análisis Mutacional de ADN , Epítopos , Humanos , Lamina Tipo A , Laminas , Microinyecciones , Mutación , Proteínas Nucleares/genética , Nucleofosmina , ARN Mensajero , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Xenopus laevis

RESUMEN

Primary transcripts in eukaryotic cells undergo several processing steps within the nucleus, and resulting mature RNA molecules are selectively exported to the cytoplasm. Nucleo-cytoplasmic mRNA transport is an active process that likely involves RNA-protein interactions. To identify specific RNA-binding proteins, we designed a novel approach, which allows the analysis of interactions between mRNAs and proteins along the transport pathway. The method consists of inducing in vivo a covalent binding between nuclear proteins and microinjected mRNAs. Using such a procedure, we were able to detect a direct interaction between nucleoporin p62 with mRNA during export. The formation of the mRNA-p62 complex was inhibited by wheat-germ agglutinin, an inhibitor of mRNA export. Antibodies directed against p62 caused a substantial reduction in the rate of mRNA export from the nucleus.

Asunto(s)

Núcleo Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Oocitos/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Cinética , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/aislamiento & purificación , Microinyecciones , Peso Molecular , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/aislamiento & purificación , Unión Proteica , ARN Mensajero/efectos de los fármacos , ARN Mensajero/aislamiento & purificación , Receptores de Transferrina/biosíntesis , Transcripción Genética , Aglutininas del Germen de Trigo/farmacología , Xenopus laevis

RESUMEN

Proteins that shuttle between nucleus and cytoplasm are implicated in transport and signal transduction processes. Using assays based on interspecies heterokaryons and microinjection of Xenopus oocytes, we examined what structural features determine nuclear export of shuttling proteins. Three classes of proteins were studied: first, wild-type and mutant forms of nucleolin, one of the first shuttling proteins identified; second, artificial nuclear reporter proteins derived from cytoplasmic pyruvate kinase; and third, wild-type and mutant lamins differing in their abilities to be incorporated into the lamina. Our results show that a protein does not require positively acting export signals to be transported from nucleus to cytoplasm; instead, its shuttling ability is limited primarily by intranuclear interactions. We conclude that nucleocytoplasmic shuttling is a general phenomenon not restricted to proteins involved in nucleocytoplasmic transport.

Asunto(s)

Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Oocitos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN , Secuencia de Aminoácidos , Animales , Fusión Celular , Pollos , Citoplasma/metabolismo , Femenino , Humanos , Cinética , Laminas , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Fosfoproteínas/biosíntesis , Fosfoproteínas/genética , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Transducción de Señal , Transfección , Xenopus laevis , Nucleolina

RESUMEN

Nucleolin, a major nucleolar phosphoprotein, is presumed to function in rDNA transcription, rRNA packaging and ribosome assembly. Its primary sequence was highly conserved during evolution and suggests a multi-domain structure. To identify structural elements required for nuclear uptake and nucleolar accumulation of nucleolin, we used site-directed mutagenesis to introduce point- and deletion-mutations into a chicken nucleolin cDNA. Following transient expression in mammalian cells, the intracellular distribution of the corresponding wild-type and mutant proteins was determined by indirect immunofluorescence microscopy. We found that nucleolin contains a functional nuclear localization signal (KRKKEMANKSAPEAKKKK) that conforms exactly to the consensus proposed recently for a bipartite signal (Robbins, J., Dilworth, S.M., Laskey, R.A. and Dingwall, C. (1991) Cell 64, 615-623). Concerning nucleolar localization, we found that the N-terminal 250 amino acids of nucleolin are dispensible, but deletion of either the centrally located RNA-binding motifs (the RNP domain) or the glycine/arginine-rich C terminus (the GR domain) resulted in an exclusively nucleoplasmic distribution. Although both of these latter domains were required for correct subcellular localization of nucleolin, they were not sufficient to target non-nucleolar proteins to the nucleolus. From these results we conclude that nucleolin does not contain a single, linear nucleolar targeting signal. Instead, we propose that the protein uses a bipartite NLS to enter the nucleus and then accumulates within the nucleolus by virtue of binding to other nucleolar components (probably rRNA) via its RNP and GR domains.

Asunto(s)

Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico Activo , Pollos , Secuencia de Consenso , ADN Complementario/genética , Células HeLa , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Fosfoproteínas/genética , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Nucleolina

RESUMEN

Small nucleolus-related bodies which occur in the nucleoplasm of "micronuclei" lacking nucleolar organizers have been studied by immunofluorescence microscopy. These bodies stained specifically with three different antibodies directed against proteins that are normally associated with the dense fibrillar component of functional nucleoli, but not with antibodies specific for certain proteins of the granular component or the fibrillar centers. Our data show that, in the absence of rRNA genes, the various constituent proteins characteristic of the dense fibrillar component spontaneously assemble into spherical entities but that the subsequent fusion of these bodies into larger structures is prevented in these micronuclei. The similarity between these nucleolus-related bodies of micronuclei and the prenucleolar bodies characteristic of early stages of nucleologenesis during mitotic telophase is discussed.

Asunto(s)

Nucléolo Celular/ultraestructura , Animales , Línea Celular , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Microscopía de Contraste de Fase

RESUMEN

Using a cDNA probe encoding the nucleolar protein NO38 of Xenopus laevis, we have isolated clones that code for the corresponding mammalian protein from cDNA libraries of mouse embryonal carcinoma and fetal liver cells. The murine cDNA-derived amino acid sequence defines a polypeptide of 292 amino acids (including the initial methionine) of a total molecular weight of 32,560 and identifies a single approximately 1.5 kb mRNA on Northern blot hybridization. This polypeptide, which is highly homologous to the Xenopus protein NO38, displays an organization in three major domains: (1) an aminoterminal portion of 119 amino acids, which shows a striking homology to nucleoplasmin of Xenopus; (2) a central portion of 68 amino acids that contains two extended acidic domains, a shorter of 13 residues and a longer of 29 residues, separated by an interval enriched in positively charged amino acids; (3) a carboxyterminal portion of 105 amino acids, which is almost identical to the reported partial amino acid sequence of human and rat nucleolar protein termed B23. The sequence comparisons show that the murine protein is the mammalian counterpart to the nucleolar protein NO38 of Xenopus and is compatible with the idea that both proteins NO38 represent the amphibian and murine equivalents to the human and rat nucleolar phosphoprotein B23. Special sequence features and predicted conformations of this protein are discussed in relation to the specific localization and the possible functions of this major nucleolar protein.

Asunto(s)

Antígenos/genética , Nucléolo Celular/metabolismo , Clonación Molecular , Proteínas Nucleares/genética , Péptidos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/genética , ADN/aislamiento & purificación , Feto , Hígado/metabolismo , Ratones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Nucleofosmina , Conformación Proteica , Homología de Secuencia de Ácido Nucleico , Xenopus laevis

RESUMEN

Using monoclonal antibodies we have localized a polypeptide, appearing on gel electrophoresis with a Mr of approximately 38,000 and a pI of approximately 5.6, to the granular component of the nucleoli of Xenopus laevis oocytes and a broad range of cells from various species. The protein (NO38) also occurs in certain distinct nucleoplasmic particles but is not detected in ribosomes and other cytoplasmic components. During mitosis NO38-containing material dissociates from the nucleolar organizer region and distributes over the chromosomal surfaces and the perichromosomal cytoplasm; in telophase it re-populates the forming nucleoli. With these antibodies we have isolated from a X. laevis ovary lambda gt11 expression library a cDNA clone encoding a polypeptide which, on one- and two-dimensional gel electrophoresis, co-migrates with authentic NO38. The amino acid sequence deduced from this clone defines a polypeptide of 299 amino acids of mol. wt 33,531 which is characterized by the presence of two domains exceptionally rich in aspartic and glutamic acid, one of them flanked by two putative karyophilic signal heptapeptides. Comparison with other protein sequences shows that NO38 is closely related to the histone-binding, karyophilic protein nucleoplasmin: the first 124 amino acids have 58 amino acid positions in common. Protein NO38 also shows striking homologies to the phosphopeptide region of rat nucleolar protein B23 and the carboxyterminal region of human B23. We propose that protein NO38, which forms distinct homo-oligomers of approximately 7S and Mr of approximately 230,000, is a member of a family of karyophilic proteins, the 'nucleoplasmin family'. It is characterized by its specific association with the nucleolus and might be involved in nuclear accumulation, nucleolar storage and pre-rRNA assembly of ribosomal proteins in a manner similar to that discussed for the role of nucleoplasmin in histone storage and chromatin assembly.

Asunto(s)

Nucléolo Celular/ultraestructura , Proteínas Nucleares/genética , Fosfoproteínas , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Clonación Molecular , ADN/análisis , Femenino , Técnica del Anticuerpo Fluorescente , Microscopía Electrónica , Datos de Secuencia Molecular , Proteínas Nucleares/análisis , Nucleoplasminas , Oocitos/ultraestructura , Xenopus laevis

RESUMEN

A monoclonal murine antibody (No-114) is described which reacts specifically with a polypeptide of molecular weight (Mr) 180 000 present in low-speed nuclear pellets from oocytes and somatic cells of Xenopus laevis and X. borealis and in isolated amplified nucleoli. Two-dimensional gel electrophoresis has revealed the acidic nature of this polypeptide (isoelectric at pH of ca 4.2 in the presence of 9.5 M urea). A relatively large proportion of the protein is extracted at elevated ionic strength (i.e., at 0.4-0.5 M alkali salt) in a form sedimenting at approx. 7-8S, compatible with a monomeric state. It is also extracted by digestion with RNase but not with DNase. In immunofluorescence microscopy, antibody No-114 stains intensely nucleoli of oocytes and all somatic cells examined, including the residual nucleolar structure of Xenopus erythrocytes which are transcriptionally inactive. During mitosis the antigen does not remain associated with the nucleolar organizer regions (NOR) of chromosomes but is released and dispersed over the cytoplasm until telophase when it re-associates with the reforming interphase nucleoli. At higher resolution the immunofluorescent region is often resolved into a number of distinct subnucleolar components of varied size and shape. Immunoelectron microscopy using colloidal gold-coupled secondary antibodies reveals that the Mr 180 000 protein is confined to the dense fibrillar component of the nucleolus. This conclusion is also supported by its localization in the fibrillar part of segregated nucleoli of cells treated with actinomycin D. We conclude that nucleoli contain a prominent protein of Mr 180 000 which contributes to the general structure of the dense fibrillar component of the interphase nucleolus, independent of its specific transcriptional activity.

Asunto(s)

Nucléolo Celular/análisis , Proteínas/análisis , Animales , Anticuerpos Monoclonales , Reacciones Cruzadas , Electroforesis , Femenino , Punto Isoeléctrico , Microscopía Electrónica , Microscopía Fluorescente , Peso Molecular , Oocitos/análisis , Xenopus , Xenopus laevis

RESUMEN

We have investigated the existence of structural components in the nucleus of the oocyte of Xenopus laevis and other amphibia that are insoluble in non-denaturing detergents and buffers of low and high ionic strength. These cells are particularly suitable for such studies as they have a high frequency of extrachromosomal amplified nucleoli and pore complexes of the nuclear envelope. Using biochemical and immunological techniques, we have shown these structures to contain only two major proteins. These are a polypeptide of Mr 145000, which is located in a meshwork of filaments specific to the nucleolar cortex, and certain nucleoplasmic bodies probably derived therefrom, and a polypeptide of Mr 68000, which is the predominant constituent of the lamina-pore complex structure. We show that the latter protein is related to, but not identical to, lamina proteins ('lamins') of somatic cells, indicating cell type-specificity of the expression of polypeptides of the lamin family. In addition, we describe a protein of Mr 180000, which is the major constituent of the dense fibrillar component of the nucleolus. This can be partially solubilized in buffers of moderately high ionic strength. We interpret proteins of this category as karyoskeletal components involved in the architectural organization of specific functional topology within the nucleus. In contrast to previous reports for other cell types we have found no other prominent high-salt-insoluble structures in the nuclear interior, indicating the absence of an extended internal nuclear matrix in this kind of nucleus.

Asunto(s)

Núcleo Celular/análisis , Nucleoproteínas/análisis , Oocitos/análisis , Animales , Nucléolo Celular/análisis , Electroforesis en Gel de Poliacrilamida , Femenino , Técnicas Inmunológicas , Microscopía Electrónica , Microscopía Fluorescente , Membrana Nuclear/análisis , Oocitos/ultraestructura , Xenopus laevis