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At least 19 recordsLinked to original sources

PubMed1995

Nucleoplasmin-mediated decondensation of Mytilus sperm chromatin. Identification and partial characterization of a nucleoplasmin-like protein with sperm-nuclei decondensing activity in Mytilus californianus.

We have been able to induce sperm nuclear decondensation in the mussel Mytilus californianus (mollusc) using either egg extracts or pure nucleoplasmin from Xenopus (amphibian). The nuclear decondensation involves removal of the sperm nuclear basic proteins (SNBPs) which bind to nucleoplasmin. An attempt has been made to isolate an ooplasmic factor from Mytilus with a similar sperm-chromatin decondensing activity. An acidic, thermostable protein with a molecular mass of 58,000 has been purified and partially characterized.

Amino Acids↗

PubMed1986

Xenopus nucleoplasmin: egg vs. oocyte.

Nucleoplasmin has been purified from either oocytes or unfertilized eggs of the frog, Xenopus laevis. We find that the pentameric form of egg nucleoplasmin exhibits an apparent molecular mass approximately 15 000 daltons larger than its oocyte counterpart upon sodium dodecyl sulfate (SDS)-acrylamide gel electrophoresis. Egg nucleoplasmin monomers are more heterogeneous, substantially more acidic, and overall larger in apparent molecular weight than oocyte nucleoplasmin monomers when analyzed by isoelectric focusing or SDS gel electrophoresis. Protease digestions indicate that the structural differences between egg and oocyte nucleoplasmin are primarily confined to the N-terminal halves of the proteins. The structural diversity observed is accompanied by a difference in the ability of nucleoplasmin from the two sources to act as a nucleosome assembly agent in vitro. Egg nucleoplasmin efficiently promotes the formation of nucleosomes onto circular pBR322 DNA in vitro at physiological ionic strength and at physiological histone:DNA ratios, while oocyte nucleoplasmin is markedly deficient in serving as an in vitro chromatin assembly agent under all conditions which we have tested. Treatment of egg nucleoplasmin in vitro with alkaline phosphatase demonstrates that the structural diversity between egg and oocyte nucleoplasmin results primarily from extensive additional phosphorylation of the egg protein. The relevance of nucleoplasmin phosphorylation in leading to differences in the chromatin assembly activity of this protein both in vitro and in vivo is considered.

Animals↗

PubMed1994

Rapid isolation of nuclear transport-competent Xenopus nucleoplasmin produced in Escherichia coli strain BL21(DE3).

Nucleoplasmin is a thermostable karyophilic protein widely used in nuclear transport studies. An expression vector was constructed that contains a string of 10 histidine residues ligated, in frame, to the amino terminal end of the Xenopus nucleoplasmin gene. The vector was then transformed into Escherichia coli strain BL21(DE3). This strain possesses the gene for T7 RNA polymerase under control of the lacUV5 promoter. The induction of the RNA polymerase and subsequent production of nucleoplasmin occurs after exposure to isopropyl-beta-D-thiogalactopyranoside. The nucleoplasmin, produced in milligram quantities per liter of culture, is then isolated by a rapid purification method that includes metal chelation chromatography to purify the oligohistidine-linked nucleoplasmin. Nuclear transport studies indicate that fluorescently labeled nucleoplasmin is translocated to the nuclear interior of permeabilized V79A03 cells, while nucleoplasmin that lacks a nuclear localization signal (core nucleoplasmin) is not imported. The use of this method to produce nuclear transport-competent nucleoplasmin avoids the lengthy purification procedure used to isolate nucleoplasmin from Xenopus laevis oocytes as well as the cost of purchasing and maintaining a toad colony.

Amino Acid Sequence↗

PubMed1995

Nucleoplasmin associates with and is phosphorylated by casein kinase II.

Nucleoplasmin is a phosphorylated nuclear-accumulating protein. We report herein that the kinetics of its cytoplasm-->nucleus transport are affected by its degree of phosphorylation. Therefore, we sought to identify any protein kinase which specifically associates with nucleoplasmin. We discovered that nucleoplasmin co-isolates by two independent methods (immunoabsorption and chromatography) in a complex including a kinase which phosphorylates nucleoplasmin. The co-purifying kinase is casein kinase II-like because: (i) it phosphorylates casein; (ii) its phospho-transferase activity can be competed out by GTP; (iii) it is stimulated by polylysine; and (iv) it is inhibited by heparin. Moreover, a polyclonal antibody to the alpha (38 kDa) and alpha' (36 kDa) catalytic subunits of casein kinase II specifically recognizes 38 and 36 kDa polypeptides in the nucleoplasmin-complex, and a specific inhibitor of casein kinase II inhibits nucleoplasmin's nuclear transport. Additionally, we found that phosphorylation of nucleoplasmin by its associated casein kinase II is strongly inhibited by histones and that, in addition to nucleoplasmin, another protein (p100) in the nucleoplasmin-complex is phosphorylated by casein kinase II.

Animals↗

PubMed1999

Mechanism of salmon sperm decondensation by nucleoplasmin.

Removal of protamine from DNA-protamine (salmine, protamine from salmon sperm) complexes by nucleoplasmin was examined and compared with that of poly-L-glutamic acid (PLGA) using turbidity and ethidium bromide (EB) treatment methods. When nucleoplasmin or PLGA was added to a DNA-protamine complex solution, turbidity was decreased and the amount of EB intercalated into DNA was increased. These results suggest that nucleoplasmin and PLGA can remove protamine from DNA-protamine complexes. The effect of nucleoplasmin was more potent than that of PLGA. Direct interaction of nucleoplasmin with protamine was confirmed by mixing experiments using circular dichroism (CD) and fluorescence spectroscopies. Results suggest that nucleoplasmin is bound to protamine in a 1:1 ratio and that Trp126 is located near a hydrophilic region containing a polyglutamic acid tract of nucleoplasmin which was obviously influenced by its binding with protamine. It would appear that the polyglutamic acid tract in nucleoplasmin plays a critical role for binding with protamine.

Animals↗

PubMed1988

Expression and segregation of nucleoplasmin during development in Xenopus.

The spatial segregation of informational molecules in the unfertilized egg and embryo has been hypothesized to be a necessary phenomenon for the normal progression of development leading to the determination of cellular phenotypes. This study describes the selection of a monoclonal antibody (Mab: 2G6) that identifies an antigen (Ag: 2G6) which is localized in the germinal vesicle of oocytes and has a discrete pattern of inheritance during embryogenesis. The antigen displayed biochemical and physical characteristics very similar to nucleoplasmin, which is the histone-binding and nucleosome-assembly protein previously described. Immunoblot analysis with purified oocyte nucleoplasmin confirmed this relationship. Indirect immunofluorescence was used to study the temporal expression and spatial distribution of nucleoplasmin. From early cleavage stages through gastrulation, it is preferentially localized in nuclei of blastomeres at the animal pole. By tadpole stages, it was detected only in nuclei of postmitotic cells of the central nervous system and in nuclei of striated muscle. It was not detected in adult tissues. Western blot analysis during embryogenesis revealed at least five immunologically related polypeptides that displayed distinct patterns of expression during development. The different species observed most likely represent different levels of phosphorylation of nucleoplasmin. The more acidic forms, known to be more active in nucleosome assembly, were present during cleavage stages. Analysis of labelled oocyte proteins by two-dimensional immunoblots and autoradiography revealed that synthesis of nucleoplasmin was first detected in stage-2 oocytes, reached 60% maximum levels at stage 3, peaked at stage 4 and was undetectable in stage-6 oocytes. The amount of nucleoplasmin message present does not follow a similar pattern during oogenesis. These results suggest that the message undergoes pronounced changes in translational efficiency during oogenesis. A comparative immunoblot analysis using proteins from a variety of adult tissues revealed that, whereas the polyclonal antisera against amphibian vitellogenic oocyte nucleoplasmin recognized several different, tissue-specific polypeptides, two different monoclonal antibodies (Mab: b7-1D1, Mab: 2G6) failed to recognize any of the adult tissues tested. We conclude that nucleoplasmin is a family of closely related proteins with distinct embryonic and adult members.

Animals↗

PubMed2001

Nucleoplasmin binds to nuclear pore filaments and accumulates in specific regions of the nucleolar cortex.

Nucleoplasmin is a karyophilic protein that is involved in nucleosome formation and decondensation of chromatin, although other precise functions and modes of action of this molecule are still poorly understood. In the present paper we describe a novel nucleocytoplasmic transport assay that has enabled us to study the nuclear distribution of nucleoplasmin following its transport into the nucleus. Single Xenopus laevis oocyte nuclei were isolated and incubated with Xenopus egg extract containing colloidal gold-conjugated nucleoplasmin. After a period of incubation, each individual nucleus was processed for electron microscopy. The nuclear accumulation of nucleoplasmin was dependent upon the karyophilic properties of the protein, since BSA-conjugated gold particles did not enter the nuclear interior under the same experimental conditions. Once inside the nucleus, nucleoplasmin was detected in tracks emanating from the nuclear pores and reaching the nucleolus. Additionally, we found a striking accumulation of nucleoplasmin in specific areas of the nucleolar cortex. These perinucleolar regions were surrounded by areas of electron density similar to that of the fibrillar centers. Our results indicate that nucleoplasmin may play an important role in the transcription of ribosomal precursors. Moreover, this nucleocytoplasmic transport assay will enable the determination of the precise intranuclear localization of other karyophilic proteins.

Animals↗

PubMed1990

Association of gold-labelled nucleoplasmin with the centres of ring components of Xenopus oocyte nuclear pore complexes.

We have used heavy-metal shadowing to study the interaction of morphological components of Xenopus oocyte nuclear pore complexes with nucleoplasmin conjugated to colloidal gold. When microinjected into Xenopus oocytes, gold-labelled nucleoplasmin accumulated on the axis of the pores. Envelopes partially disrupted by treatment with low ionic strength buffer produced isolated islands of pores together with substantial quantities of rings deriving from the cytoplasmic and nucleoplasmic faces of the pores. In preparations from oocytes in which nucleoplasmin-gold had been microinjected, most (238/288) of the rings examined had also been labelled and, in the majority of these (60%), the label was located centrally within isolated rings. The central positioning of the nucleoplasmin-gold in isolated rings indicated that these morphological components of the pores were probably involved in the transport of nucleoplasmin into the nucleus, either by way of the initial binding of the molecule or by way of its translocation across the nuclear envelope. Although more work is required to resolve the precise stage at which the rings are involved, a number of lines of evidence suggested that they were more likely to be involved in the translocation step rather than in initial binding of nucleoplasmin.

Animals↗

PubMed1997

Conformation of nucleoplasmin and its interaction with DNA-protamine complex as a simple model of fish sperm nuclei.

Nucleoplasmin was isolated from Xenopus laevis eggs and purified by an improved method using an open column. Its conformation was investigated spectrophotometrically by UV, CD and fluorescence. It was shown that alpha-helix content of nucleoplasmin was 30-40%, and one of the two tryptophan residues in nucleoplasmin located in the hydrophobic surroundings and the other in the relatively hydrophilic surroundings. The isolated nucleoplasmin was found to decondense sperm nuclei of salmon also, suggesting a possibility of the existence of nucleoplasmin-like protein in fish as well. Collapse of the protamine (salmine)-DNA complex as a simple model for fish sperm nuclei by nucleoplasmin was directly observed by measuring OD320 of aqueous protamine-DNA mixtures. This is a molecular level observation for the removal of protamine from DNA-protamine complex.

Amino Acid Sequence↗

PubMed1996

Hyperphosphorylation of nucleoplasmin facilitates Xenopus sperm decondensation at fertilization.

Previous studies showed that the nuclear phosphoprotein nucleoplasmin performs the first stage of chromatin decondensation of Xenopus sperm at fertilization. It binds and removes sperm basic proteins replacing them with histones. We now show that this activity depends upon the massive hyperphosphorylation of nucleoplasmin that occurs when oocytes mature into eggs. Egg extracts or purified hyperphosphorylated egg nucleoplasmin decondense sperm chromatin and remove sperm basic proteins much faster than oocyte extracts or hypophosphorylated oocyte nucleoplasmin. Furthermore, dephosphorylation of egg nucleoplasmin slows sperm decondensation and prevents basic protein removal from sperm chromatin. We conclude that hyperphosphorylation of nucleoplasmin is used to modulate the rapid changes in chromatin structure that accompany early development in Xenopus.

Animals↗

PubMed1986

Association of nucleoplasmin with transcription products as revealed by immunolocalization in the amphibian oocyte.

The oocyte nucleus of Pleurodeles waltlii contains a major 32,000-mol-wt acidic protein which is called nucleoplasmin. Rabbit antibodies were raised against total nuclear proteins from Pleurodeles oocytes. Affinity-purified antibodies directed against nucleoplasmin were prepared using antigens bound to nitrocellulose paper. The specificity of the antibody was controlled on two-dimensional electrophoretic gels of nuclear proteins. The intranuclear distribution of nucleoplasmin was analyzed by indirect immunofluorescence and the immunogold technique in light and electron microscopy. The antibody was tested on a spread of the nuclear content prepared in the presence of calcium, on the nuclear content spread in the presence of phalloidin so that an actin network appeared, and on a spread of nuclei from oocytes previously treated by actinomycin D. In all cases, nucleoplasmin appeared to be localized on the lampbrush loops, i.e., on the sites of transcription and, more specifically, on the ribonucleoprotein (RNP) particles; this protein was also associated with the RNP particles of the nuclear sap (free or inserted in the actin network). Nucleoplasmin was localized on large RNP particles that appeared when transcription was blocked. We never found this protein on the chromosome axis. These results suggest that nucleoplasmin may play a role in transcriptional activity.

Animals↗

PubMed1987

Cloning of nucleoplasmin from Xenopus laevis oocytes and analysis of its developmental expression.

Nucleoplasmin is the most abundant protein in the nucleus of Xenopus laevis oocytes. We cloned a cDNA coding for nucleoplasmin from an expression library of immature Xenopus laevis oocytes. The deduced amino acid sequence shows that the carboxyl terminus is very hydrophilic and contains an unusual stretch of 12 glutamic acid residues, which is consistent with one of the proposed functions of nucleoplasmin--that of promoting chromatin assembly. The last 50 amino acids are lysine- and alanine-rich and contain short stretches of homology to histone H1. These regions could be involved in interactions with nucleosomes. The levels of nucleoplasmin mRNA and protein during oogenesis and embryogenesis were investigated using Northern blots, Western blots, and in situ hybridization to oocyte sections. The mRNA is detected during oogenesis but not during embryogenesis, suggesting that nucleoplasmin may be an exclusively maternally expressed gene. However, the protein is present throughout embryogenesis and undergoes pronounced changes in its level of phosphorylation during maturation of the oocyte and just after midblastula transition. These results support the notion that nucleoplasmin is not only important in oocytes but also plays a major role during the rapid cleavages of early embryogenesis.

Amino Acid Sequence↗

PubMed1995

Stimulation of transcription factor binding and histone displacement by nucleosome assembly protein 1 and nucleoplasmin requires disruption of the histone octamer.

To investigate the mechanisms by which transcription factors invade nucleosomal DNA and replace histones at control elements, we have examined the response of the histone octamer to transcription factor binding in the presence of histone-binding proteins (i.e., nucleosome assembly factors). We found that yeast nucleosome assembly protein 1 (NAP-1) stimulated transcription factor binding and nucleosome displacement in a manner similar to that of nucleoplasmin. In addition, disruption of the histone octamer was required both for the stimulation of transcription factor binding to nucleosomal DNA and for transcription factor-induced nucleosome displacement mediated by nucleoplasmin or NAP-1. While NAP-1 and nucleoplasmin stimulated the binding of a fusion protein (GAL4-AH) to control nucleosome cores, this stimulation was lost upon covalent histone-histone cross-linking within the histone octamers. In addition, both NAP-1 and nucleoplasmin were able to mediate histone displacement upon the binding of five GAL4-AH dimers to control nucleosome cores; however, this activity was also forfeited when the histone octamers were cross-linked. These data indicate that octamer disruption is required for both stimulation of factor binding and factor-dependent histone displacement by nucleoplasmin and NAP-1. By contrast, transcription factor-induced histone transfer onto nonspecific competitor DNA did not require disruption of the histone octamer. Thus, histone displacement in this instance occurred by transfer of complete histone octamers, a mechanism distinct from that mediated by the histone-binding proteins nucleoplasmin and NAP-1.

Base Sequence↗

PubMed1990

Nucleoplasmin: the archetypal molecular chaperone.

Nucleoplasmin was the first protein to be described as a molecular chaperone. Studies of nucleoplasmin have resulted in advances in two areas of cell biology. Firstly, the pathway of nucleosome assembly in Xenopus oocytes and eggs has been elucidated and is the only assembly pathway known in detail. Nucleosome assembly represents the major chaperoning function of nucleoplasmin. Secondly, nucleoplasmin has been used to elucidate the transport of proteins into the nucleus, revealing a selective entry mechanism for nuclear proteins, passage through the nuclear pore complex, and a two-step mechanism of transport. The properties and functions of nucleoplasmin are reviewed, together with other proteins which are related either structurally or functionally to nucleoplasmin.

Animals↗

PubMed1987

The phosphorylation of nucleoplasmin by casein kinase-2 is resistant to heparin inhibition.

Highly purified preparations of casein kinase-2 from the nuclei of Xenopus laevis oocytes and from calf thymus can phosphorylate in vitro purified nucleoplasmin from X. laevis oocytes and eggs. The phosphorylation of nucleoplasmin by both kinase preparations is quite insensitive to heparin in contrast with casein phosphorylation which is completely abolished by heparin concentrations above 10 micrograms/ml. However, the phosphorylation of nucleoplasmin and casein are inhibited in a very similar fashion by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a well characterized specific inhibitor of casein kinase-2. Similarly, nucleoplasmin phosphorylation by the oocyte enzyme can be stimulated several-fold by spermine, another characteristic of this enzyme. These findings indicate that the phosphorylation of nucleoplasmin by purified casein kinase-2, while showing typical response to DRB and spermine, exhibits anomalous behavior in its resistance to heparin inhibition. It is possible that the large clusters of acidic amino acids in nucleoplasmin permit this substrate to interact with the enzyme more efficiently than other protein substrates. Heparin is generally considered a potent and specific inhibitor of casein kinase-2. This study, however, questions the validity of utilizing heparin inhibition as a criterion for casein kinase-2 involvement.

Animals↗

PubMed1987

Purification of a novel, nucleoplasmin-like protein from somatic nuclei.

We have purified a nucleoplasmin-like protein from the nuclei of somatic Xenopus laevis cells. This protein possesses a number of the distinctive features of nucleoplasmin isolated from oocytes or unfertilized eggs. The protein is recognized by both monoclonal and polyclonal antisera raised against egg nucleoplasmin. The protein has an oligomeric structure, which must be heated in SDS to completely dissociate, is acidic, phosphorylated and efficiently promotes the in vitro formation of chromatin. We have partially characterized this novel protein and because of its resemblance to nucleoplasmin isolated from oocytes or unfertilized eggs we have named this protein nucleoplasmin S.

Amino Acids↗

PubMed1980

Assembly of nucleosomes: the reaction involving X. laevis nucleoplasmin.

We analyze the nucleosome core assembly reaction which is mediated in vitro by a protein previously purified from Xenopus laevis eggs, now named nucleoplasmin in reference to its occurrence in the soluble phase of the nucleus of a wide range of vertebrate cell types. Nucleoplasmin is present in solution as a pentamer. We use nuclease digestion analysis to show that the protein assembles bona fide nucleosome cores in vitro from purified histones and DNA. Nucleoplasmin itself binds neither to DNA nor to the nucleoprotein particles which it assembles in vitro. However, it interacts with histones in vitro in such a way that histones no longer adhere to negatively charged surfaces. We have found no evidence for sterically specific interactions with particular histones. The initial rate of the nucleosome core assembly reaction mediated by purified nucleoplasmin in vitro is essentially identical with the rate of the nucleosome assembly reaction which occurs in the cell-free extracts of Xenopus eggs from which nucleoplasmin was purified. This rate is sufficient to account for the rate of nucleosome assembly required during the early development of Xenopus embryos.

Amino Acids↗

PubMed1991

Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin.

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.

Animals↗