RESUMO
We have investigated the replication capacity of intact nuclei from quiescent cells using Xenopus egg extract. Nuclei, with intact nuclear membranes, were isolated from both exponentially growing and contact-inhibited BALB/c 3T3 fibroblasts by treatment of the cells with streptolysin-O. Flow cytometry showed that > 90% of all contact-inhibited cells and approximately 50% of the exponential cells were in G0/G1-phase at the time of nuclear isolation. Intact nuclei were assayed for replication in the extract by incorporation of [alpha-32P]dATP or biotin-dUTP into nascent DNA. Most nuclei from exponential cells replicated in the egg extract, consistent with previous results showing that intact G1 nuclei from HeLa cells replicate in this system. In contrast, few nuclei from quiescent cells replicated in parallel incubations. However, when the nuclear membranes of these intact quiescent nuclei were permeabilized with lysophosphatidylcholine prior to addition to the extract, nearly all the nuclei replicated under complete cell cycle control in a subsequent incubation. The ability of LPC-treated quiescent nuclei to undergo DNA replication was reversed by resealing permeable nuclear membranes with Xenopus egg membranes prior to extract incubation demonstrating that the effect of LPC treatment is at the level of the nuclear membrane. These results indicate that nuclei from G1-phase cells lose their capacity to initiate DNA replication following density-dependent growth arrest and suggest that changes in nuclear membrane permeability may be required for the initiation of replication upon re-entry of the quiescent cell into the cell cycle.
Assuntos
Núcleo Celular/metabolismo , Replicação do DNA/fisiologia , Fase G1 , Membrana Nuclear/fisiologia , Células 3T3 , Animais , Proteínas de Bactérias , Fracionamento Celular , Células HeLa , Humanos , Lisofosfatidilcolinas/farmacologia , Camundongos , Membrana Nuclear/efeitos dos fármacos , Óvulo , Permeabilidade , Estreptolisinas/farmacologia , XenopusRESUMO
We have exploited a property of chicken erythrocyte nuclei to analyze the regulation of DNA replication in a cell-free system from Xenopus eggs. Many individual demembranated nuclei added to the extract often became enclosed within a common nuclear membrane. Nuclei within such a "multinuclear aggregate" lacked individual membranes but shared the perimeter membrane of the aggregate. Individual nuclei that were excluded from the aggregates initiated DNA synthesis at different times over a 10-12-h period, as judged by incorporation of biotinylated dUTP into discrete replication foci at early times, followed by uniformly intense incorporation at later times. Replication forks were clustered in spots, rings, and horseshoe-shaped structures similar to those described in cultured cells. In contrast to the asynchronous replication seen between individual nuclei, replication within multinuclear aggregates was synchronous. There was a uniform distribution and similar fluorescent intensity of the replication foci throughout all the nuclei enclosed within the same membrane. However, different multinuclear aggregates replicated out of synchrony with each other indicating that each membrane-bound aggregate acts as an individual unit of replication. These data indicate that the nuclear membrane defines the unit of DNA replication and determines the timing of DNA synthesis in egg extract resulting in highly coordinated triggering of DNA replication on the DNA it encloses.
Assuntos
Replicação do DNA/fisiologia , Membrana Nuclear/fisiologia , Animais , Núcleo Celular/metabolismo , Sistema Livre de Células , Galinhas , Eritrócitos/metabolismo , Oócitos/metabolismo , Fatores de Tempo , Xenopus laevisRESUMO
In several mammalian species, the configuration of germinal vesicle (GV) chromatin correlates with the developmental competence of oocytes. Yet, no study has been published on the configuration of GV chromatin in ferret, nor is it known whether a specific configuration predicts meiotic competence in this species, in spite of the potential importance of ferret cloning to the study of human disease and to species conservation efforts. Here, we report on an analysis of the chromatin configuration in ferret GV oocytes and on how they correlate with meiotic development. Three distinct configurations were identified based on the degree of chromatin condensation: (1) fibrillar chromatin (FC), featuring strands of intertwined chromatin occupying most of the visible GV region; (2) intermediate condensed chromatin (ICC), characterized by dense, irregular chromatin masses throughout the GV; and (3) condensed chromatin (CC), which is highly compact and centered around the nucleolus. We also found that chromatin configuration was related to the extent of association with cumulus cells in cumulus-oocyte complexes; CC-configured oocytes were most often surrounded by a compact cumulus layer and also a compact corona but FC-configured oocytes were associated with neither. In addition, increasing chromatin condensation corresponded to an increase in oocyte diameter. Finally, following in vitro culture, significantly more CC-configured oocytes underwent maturation to meiotic metaphase II than did FC- or ICC-configured oocytes. We conclude that, in ferret, chromatin condensation is related to the sequential achievement of meiotic competencies during oocyte growth and differentiation, and thus can be used as a predictor of competence.
Assuntos
Cromatina/ultraestrutura , Furões , Meiose/fisiologia , Oócitos/crescimento & desenvolvimento , Oócitos/ultraestrutura , Animais , Tamanho Celular , Células Cultivadas , Feminino , Microscopia de Fluorescência , Folículo Ovariano/citologiaRESUMO
Purified DNA can be assembled into structures that closely resemble cell nuclei. The cell-free systems that allow this can be exploited to study assembly pathways for several components of the nucleus. They also offer great opportunities for the experimental analysis of nuclear function.
Assuntos
Núcleo Celular/fisiologia , Animais , Núcleo Celular/ultraestrutura , Sistema Livre de Células , Cromatina/fisiologia , DNA/metabolismoRESUMO
Quiescent nuclei from differentiated somatic cells can reacquire pluripotence, the capacity to replicate, and reinitiate a program of differentiation after transplantation into amphibian eggs. The replication of quiescent nuclei is recapitulated in extracts derived from activated Xenopus eggs; therefore, we have exploited this cell-free system to explore the mechanisms that regulate initiation of replication in nuclei from terminally differentiated Xenopus erythrocytes. We find that these nuclei lack many, if not all, pre-replication complex (pre-RC) proteins. Pre-RC proteins from the extract form a stable association with the chromatin of permeable nuclei, which replicate in this system, but not with the chromatin of intact nuclei, which do not replicate, even though these proteins cross an intact nuclear envelope. During extract incubation, the linker histones H1 and H1(0) are removed from erythrocyte chromatin by nucleoplasmin. We show that H1 removal facilitates the replication of permeable nuclei by increasing the frequency of initiation most likely by promoting the assembly of pre-RCs on chromatin. These data indicate that initiation in erythrocyte nuclei requires the acquisition of pre-RC proteins from egg extract and that pre-RC assembly requires the loss of nuclear envelope integrity and is facilitated by the removal of linker histone H1 from chromatin.
Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Replicação do DNA/fisiologia , Histonas/metabolismo , Proteínas de Xenopus , Células 3T3 , Animais , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Extratos Celulares , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eritrócitos/metabolismo , Eritrócitos/ultraestrutura , Imunofluorescência , Camundongos , Camundongos Endogâmicos BALB C , Componente 7 do Complexo de Manutenção de Minicromossomo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Reconhecimento de Origem , Óvulo , XenopusRESUMO
Somatic histone H1 reduces both the rate and extent of DNA replication in Xenopus egg extract. We show here that H1 inhibits replication directly by reducing the number of replication forks, but not the rate of fork progression, in Xenopus sperm nuclei. Density substitution experiments demonstrate that those forks that are active in H1 nuclei elongate to form large tracts of fully replicated DNA, indicating that inhibition is due to a reduction in the frequency of initiation and not the rate or extent of elongation. The observation that H1 dramatically reduces the number of replication foci in sperm nuclei supports this view. The establishment of replication competent DNA in egg extract requires the assembly of prereplication complexes (pre-RCs) on sperm chromatin. H1 reduces binding of the pre-RC proteins, XOrc2, XCdc6, and XMcm3, to chromatin. Replication competence can be restored in these nuclei, however, only under conditions that promote the loss of H1 from chromatin and licensing of the DNA. Thus, H1 inhibits replication in egg extract by preventing the assembly of pre-RCs on sperm chromatin, thereby reducing the frequency of initiation. These data raise the interesting possibility that H1 plays a role in regulating replication origin use during Xenopus development.
Assuntos
Cromatina/fisiologia , Replicação do DNA , Histonas/fisiologia , Espermatozoides/fisiologia , Animais , Extratos Celulares , Linhagem Celular , Núcleo Celular , Masculino , Camundongos , Óvulo , Fase S , Xenopus laevisRESUMO
Starvation of Mouse hepatoma cells for essential amino acids or glucose results in the mono-ADP-ribosylation of the 78 kDa glucose-regulated protein, GRP78. Here we show that the ADP-ribosylated and non-ADP-ribosylated forms of GRP78 are interconvertible during tryptophan starvation and refeeding. In addition, the ADP-ribosylation of GRP78 was shown to be reversible even during nutritional stress. The overexpressed pool of non-ADP-ribosylated GRP78 synthesized during tunicamycin treatment was available for ADP-ribosylation during subsequent amino acid starvation, especially in the absence of tunicamycin. The reversible ADP-ribosylation of GRP78 could be part of a metabolic control mechanism in operation during nutritional stress.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Proteínas de Choque Térmico HSP70 , Proteínas de Membrana/metabolismo , Aminoácidos/metabolismo , Animais , Linhagem Celular , Eletroforese em Gel Bidimensional , Chaperona BiP do Retículo Endoplasmático , Neoplasias Hepáticas Experimentais , Proteínas de Membrana/isolamento & purificação , Camundongos , Peso Molecular , Proteínas de Neoplasias/metabolismo , Tunicamicina/farmacologiaRESUMO
Metacercariae (2,828) obtained from the lens of naturally infected Aplodinotus grunniens were transplanted onto chorioallantoic membranes (CAM's) of 94 eggs (means 30/egg) 5 to 12 days old. Membranes were examined 2 to 8 days later and 381 flukes were recovered. Two hundred fifty-nine chorioallantoic-grown worms were transferred to 28 additional embryos 3 to 8 days after the initial inoculation. These eggs were examined 3 to 11 days later. Fifty-three serially transferred worms were recovered after 7 to 14 days on CAM's. Six stages of development were recognized: stage 1, immature; stage 2, genital rudiment; stage 3, testes; stage 4, follicular ovary; stage 5, vitellaria; stage 6, ovigerous. The intestinal ceca of many worms (except stage 1) contained ingested blood. Trematode eggs oviposited on CAM's were embryonated in tap water, and viable miracidia were observed in 15. No previous study has achieved gonadal development in Diplostomum spathaceum on the CAM. Furthermore, inasmuch as metacercaria matured and produced fertile eggs, this form is shown to be a useful model for the study of trematode differentiation. The development of metacercariae to ovigerous adults capable of producing viable miracidia suggests that the CAM shares characteristics with the intestine of the definitive host, a piscivorous bird.
Assuntos
Alantoide/parasitologia , Córion/parasitologia , Membranas Extraembrionárias/parasitologia , Trematódeos/crescimento & desenvolvimento , Animais , Embrião de Galinha , Parasitologia/métodosAssuntos
Núcleo Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , DNA/metabolismo , Animais , Fracionamento Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Daunorrubicina/metabolismo , Daunorrubicina/farmacologia , Eletroforese em Gel de Poliacrilamida , Feminino , Técnicas In Vitro , Masculino , Nuclease do Micrococo , Óvulo/efeitos dos fármacos , Óvulo/metabolismo , Preparações Farmacêuticas/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Xenopus laevisRESUMO
Recent evidence suggests that the nuclear envelope is directly involved in regulating DNA replication. It does this in at least three ways. First, replication is dependent on assembly of an intact nuclear envelope capable of nuclear transport. Second, the nuclear membrane defines the nucleus as the fundamental unit of replication and determines the timing of initiation. Third, the nuclear membrane is essential for coupling DNA replication to the cell cycle. Thus, regulated DNA replication in eukaryotic cells depends on a structurally intact and functional nuclear envelope.
Assuntos
Replicação do DNA , Membrana Nuclear/fisiologia , Animais , Transporte Biológico , Fase S , Xenopus/embriologia , Zigoto/metabolismoRESUMO
Starvation of a mouse hepatoma cell line, Hepa, for any essential amino acid results in the mono-ADP-ribosylation of an 80-kDa protein, P80. The ADP-ribose acceptor and its putative precursor were identified in two-dimensional gel patterns and isolated by electroelution. Amino-terminal sequence analysis showed they were the 78-kDa glucose-regulated protein, GRP78. Starvation of Hepa cells for tryptophan or glucose stimulated the relative rate of synthesis, and the ADP-ribosylation of GRP78. Inhibition of N-linked glycosylation by treatment with tunicamycin, 2-deoxy-D-glucose or glucosamine stimulated the synthesis of non-ADP-ribosylated GRP78 up to sixfold with relatively little effect on its ADP-ribosylation. Both forms were identified in mouse liver, lung, heart, kidney, spleen and brain.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico , Neoplasias Hepáticas Experimentais/metabolismo , Chaperonas Moleculares , Distúrbios Nutricionais/metabolismo , ADP Ribose Transferases , Sequência de Aminoácidos , Animais , Proteínas de Transporte/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Chaperona BiP do Retículo Endoplasmático , Camundongos , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
We describe a cell-free extract derived from the oocytes of Xenopus laevis. The oocyte extract is capable of decondensing sperm chromatin and of replicating single-stranded DNA in a semiconservative, aphidicolin-sensitive manner. In addition, oocyte extract supports the elongation phase of DNA synthesis in nuclei that have been preinitiated for replication. All of these properties are shared by previously described egg extracts. However, oocyte extracts differ from egg extracts in two important ways. First, they cannot support nuclear assembly, as visualised by phase-contrast, fluorescence and electron microscopy. Second, they do not initiate replication on chromatin or nuclei de novo. Crude low-speed supernatants can be partially fractionated into soluble and vesicular components by high-speed centrifugation. Such fractions from eggs can be functionally reconstituted, but the oocyte soluble fraction does not acquire the ability to assemble nuclei, or replicate them, even when supplemented with the egg vesicular fraction. Similarly, oocyte vesicles cannot substitute for egg vesicles on reconstitution with the egg soluble fraction. When the requirement for nuclear assembly is bypassed by using preformed, quiescent nuclei, replication is observed in egg but not oocyte extracts. However, the oocyte extract is not inhibitory for initiation of replication, as it does not prevent replication of sperm nuclei when mixed with egg extract. We suggest that the different capabilities of egg and oocyte extracts could provide the basis of an assay system for identifying factors involved in the initiation of DNA replication.
Assuntos
Extratos Celulares/farmacologia , Núcleo Celular/fisiologia , Replicação do DNA/fisiologia , Oócitos/fisiologia , Óvulo/fisiologia , Animais , Núcleo Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Fase G1/fisiologia , Técnicas In Vitro , Masculino , Espermatozoides/fisiologia , Moldes Genéticos , Xenopus laevisRESUMO
Starvation of mouse hepatoma cells for essential amino acids or glucose results in the ADP-ribosylation of the molecular chaperone BiP/GRP78. Addition of the missing nutrient to the medium reverses the reaction. The signal mediating the response to environmental nutrients involves the translational efficiency. An inhibitor of proteins synthesis, cycloheximide, or reduced temperature, both of which reduce translational efficiency, stimulate the ADP-ribosylation of BiP/GRP78. Inhibition of N-linked glycosylation of proteins results in the overproduction of BiP/GRP78. The over produced protein is not ADP-ribosylated suggesting that this is the functional form of BiP/GRP78. The over produced BiP/GRP78 can, however, be ADP-ribosylated if the cells are starved for an essential amino acid. BiP/GRP78 resides in the lumen of the endoplasmic reticulum where it participates in the assembly of secretory and integral membrane proteins. ADP-ribosylation of BiP/GRP78 during starvation is probably part of a nutritional stress response which conserves limited nutrients by slowing flow through the secretory pathway.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico , Cadeias Pesadas de Imunoglobulinas/metabolismo , Chaperonas Moleculares/metabolismo , Fenômenos Fisiológicos da Nutrição Animal , Animais , Chaperona BiP do Retículo Endoplasmático , Células Tumorais CultivadasRESUMO
Nucleoplasmin is necessary and sufficient for the initial stage of Xenopus sperm decondensation in egg extracts. In this article we show that sperm decondensation is accompanied by loss of two sperm-specific basic proteins (X and Y) and gain of histones H2A and H2B, resulting in nucleosome formation. Purified nucleoplasmin alone removes X and Y and assembles purified H2A and H2B on decondensing sperm chromatin, forming nucleosome cores. Immunodepletion of nucleoplasmin from extract prevents removal of X and Y and addition of H2A and H2B, while adding back nucleoplasmin restores decondensation and X and Y removal. Thus, nucleoplasmin acts as both an assembly and a disassembly factor for remodeling sperm chromatin at fertilization.
Assuntos
Cromatina/metabolismo , Proteínas Nucleares/metabolismo , Óvulo/metabolismo , Fosfoproteínas , Espermatozoides/metabolismo , Animais , Extratos Celulares , Eletroforese em Gel Bidimensional , Feminino , Histonas/metabolismo , Masculino , Nucleoplasminas , Nucleossomos/metabolismo , Testes de Precipitina , Interações Espermatozoide-Óvulo , Xenopus laevisRESUMO
We have used Xenopus egg extracts to investigate the effects of the antitumor drug daunomycin on DNA replication in vitro. Xenopus sperm nuclei replicated nearly synchronously in our egg extracts, thereby allowing us to determine the effects of the drug on both replication initiation and elongation. Titration experiments demonstrated that daunomycin effectively inhibited replication in the extract, with 50% inhibition at a total drug concentration of 2.7 microM. However, a high concentration of daunomycin (50 microM) also inhibited nuclear envelope assembly, a prerequisite for the initiation of replication in this system. Therefore, to bypass the effects of daunomycin on nuclear envelope assembly, sperm nuclei were preassembled in extract prior to drug addition. Initiation of replication in preassembled nuclei was also inhibited by daunomycin, with 50% inhibition at a drug concentration of 3.6 microM. At low drug concentrations, where replication did occur, the synchrony of initiations within individual nuclei was lost. This drug-induced disruption of initiation events may provide important clues regarding the mechanism(s) by which these events are coordinated in eukaryotic cells. Daunomycin also inhibited replication elongation in preassembled, preinitiated nuclei. However, the concentration of drug required for 50% inhibition of elongation was nearly fourfold higher than the required for inhibition of initiation. Taken together, these data demonstrate that Xenopus egg extract can be used to investigate the effects of DNA-binding antitumor drugs on a number of interrelated cellular processes, many of which are less tractable in whole cell systems.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Replicação do DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Daunorrubicina/farmacologia , Animais , DNA/biossíntese , Replicação do DNA/genética , Relação Dose-Resposta a Droga , Óvulo , XenopusRESUMO
We have used Xenopus egg extract to investigate the requirements for reactivation of DNA replication in nuclei isolated from terminally differentiated chicken erythrocytes. Previous work has shown that reactivation of erythrocyte nuclei in egg extract is accompanied by chromatin decondensation, nuclear envelope reformation, and the accumulation of egg lamin, LIII. However, in those studies, erythrocyte nuclei were prepared by methods that were not designed to maintain the selective permeability of the nuclear membrane, and as such, it is not clear if loss of nuclear membrane integrity played a role in the reactivation process. Therefore, the purpose of this study was to determine if changes in nuclear membrane permeability are required for reactivation of erythrocyte nuclei in egg extract. Nuclei with intact nuclear membranes were prepared from erythrocytes with streptolysin O and permeable nuclei by treatment of intact nuclei with the detergent Nonidet-P40. Like permeable nuclei, most intact nuclei decondensed, imported nuclear protein, and accumulated lamin LIII from the extract. However, unlike permeable nuclei, which replicated extensively in the extract, few intact nuclei initiated replication under the same conditions. These data demonstrate that permeabilization of the nuclear membrane is required for reactivation of DNA replication in terminally differentiated erythrocyte nuclei by egg extract and suggest that loss of nuclear membrane integrity may be a general requirement for replication of quiescent cell nuclei by this system.
Assuntos
Diferenciação Celular , Extratos Celulares/farmacologia , Replicação do DNA , Membrana Nuclear/fisiologia , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Galinhas/sangue , Detergentes/farmacologia , Proteínas do Ovo/metabolismo , Eritrócitos/ultraestrutura , Membrana Nuclear/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Octoxinol , Oócitos , Permeabilidade , Polietilenoglicóis/farmacologia , Xenopus laevisRESUMO
Nuclear membrane permeabilization is required for replication of quiescent (G0) cell nuclei in Xenopus egg extract. We now demonstrate that establishment of replication competence in G0 nuclei is dependent upon a positive activity present in the soluble egg extract. Our hypothesis is that G0 nuclei lose the license to replicate following growth arrest and that this positive activity is required for relicensing DNA for replication. To determine if G0 nuclei contain licensed DNA, we used the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), to prepare egg extracts that are devoid of licensing activity. Intact nuclei, isolated from mammalian cells synchronized in G1-phase (licensed), G2-phase (unlicensed), and G0 were permeabilized and assayed for replication in 6-DMAP-treated and untreated extracts supplemented with [alpha-32P]dATP or biotinylated-dUTP. Very little radioactivity was incorporated into nascent DNA in each nuclear population; however, nearly all nuclei in each population incorporated biotin in 6-DMAP extract. The pattern of biotin incorporation within these nuclei was strikingly similar to the punctate pattern observed within nuclei incubated in aphidicolin-treated extract, suggesting that initiation events occur within most replication factories in 6-DMAP extract. However, density substitution and alkaline gel analyses indicate that the incorporated biotin within these nuclei arises from a small number of active origins which escape 6-DMAP inhibition. We conclude that 6-DMAP-treated egg extract cannot differentiate licensed from unlicensed mammalian somatic cell nuclei and, therefore, cannot be used to determine the "licensed state" of G0 nuclei using the assays described here.
Assuntos
Adenina/análogos & derivados , Replicação do DNA/efeitos dos fármacos , Inibidores de Proteínas Quinases , Células 3T3 , Adenina/farmacologia , Animais , Ciclo Celular , Extratos Celulares , Núcleo Celular , Cromatina , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Mamíferos , Camundongos , Camundongos Endogâmicos BALB C , Óvulo , XenopusRESUMO
We have used synchronized HeLa cells to investigate the role of the nuclear membrane in preventing rereplication in a single cell cycle. Nuclei were prepared with intact nuclear membranes using streptolysin-O or digitonin and assayed for replication in Xenopus egg extracts. Intact G1 nuclei replicate semiconservatively, but intact G2 nuclei do not replicate in egg extract. However, permeabilizing the nuclear membranes of G2 nuclei by treatment with NP-40 allows them all to replicate in egg extract under cell cycle control, suggesting that integrity of the nuclear membrane is required to distinguish G2 from G1 human nuclei and to prevent rereplication within a single cell cycle. The results are discussed in terms of the previously proposed licensing factor model.
Assuntos
Núcleo Celular/fisiologia , Fase G1/fisiologia , Fase G2/fisiologia , Membrana Nuclear/fisiologia , Animais , Proteínas de Bactérias , Sistema Livre de Células , Replicação do DNA/fisiologia , Digitonina , Células HeLa , Humanos , Octoxinol , Polietilenoglicóis , Estreptolisinas , XenopusRESUMO
At fertilization, sperm chromatin decondenses in two stages, which can be mimicked in extracts of Xenopus eggs. Rapid, limited decondensation is followed by slower, membrane-dependent decondensation and swelling. Nucleoplasmin, an acidic nuclear protein, occurs at high concentration in Xenopus eggs and has a histone-binding role in nucleosome assembly. Immunodepleting nucleoplasmin from egg extracts inhibits the initial rapid stage of sperm decondensation, and also the decondensation of myeloma nuclei, relative to controls of mock depletion and TFIIIA depletion. Readdition of purified nucleoplasmin recues depleted extracts. A physiological concentration of purified nucleoplasmin alone decondenses both sperm and myeloma nuclei. We conclude that nucleoplasmin is both necessary and sufficient for the first stage of sperm decondensation in Xenopus eggs.
Assuntos
Cromatina/fisiologia , Proteínas Nucleares/fisiologia , Óvulo/fisiologia , Fosfoproteínas , Interações Espermatozoide-Óvulo , Espermatozoides/fisiologia , Animais , Western Blotting , Núcleo Celular/fisiologia , Núcleo Celular/ultraestrutura , Cromatografia de Afinidade , Citoplasma/fisiologia , Feminino , Masculino , Camundongos , Proteínas Nucleares/isolamento & purificação , Nucleoplasminas , XenopusRESUMO
Nuclei from terminally differentiated Xenopus erythrocytes lack essential components of the prereplication complex, including the origin recognition complex (ORC) proteins XORC1 and XORC2. In Xenopus egg extract, these proteins are able to bind erythrocyte chromatin from permeable nuclei, but not from intact nuclei, even though they are able to cross an intact nuclear envelope. In this report we use both permeable and intact erythrocyte nuclei to investigate the role of cyclin-dependent kinase activity in modulating the binding of XORC2 to chromatin. We find that elevating the level of cyclin A-dependent kinase in egg extract prevents the binding of XORC2 to chromatin from permeable nuclei and that kinase inhibition reverses this effect. We also observe a nuclear transport-dependent accumulation of H1 kinase activity within intact nuclei incubated in the extract. However, inhibiting this kinase activity does not facilitate the binding of XORC2 to chromatin, suggesting that other molecules and/or mechanisms exist to prevent association of XORC proteins with replication origins within intact nuclei from terminally differentiated cells.