Nuclear reassembly in vitro is independent of nucleosome/chromatin assembly

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Nuclear reassemblyin vitro is independent of nucleosome/chromatin assembly

It was show11 that nuclear reassembly was induced by small pieces of DNA fragments in cell-free extracts ofXenopus. In an attempt to learn the relationship between the nuclear reassembly and nucleosome/chromatin assembly, limited amounts of CM-Cellulose are used to eliminate the capacity of the egg extract S-150 to assemble chromatin. while the forming of nucleosomes is checked with DNA supercoiling by plasmid DNA pBR322 incubated in the extract, and further analysed by micrococcal nuclease digestion. This depleted extract is then used to induce nuclear reassembly around demembraned sperms with membrane vesicles. It is found that CM-Cellulose depletes histones H2A and H2B efficiently and blocks the assembly of nucleosomes, the demembraned sperms are yet reconstituted into nuclei in the treated S-150, although the chromatin in reassembled nuclei does not produce protected DNA fragments when digested with micrococcal nuclease. It suggests that in the cell-free system ofXenopus, DNA can be formed into nuclei without assembly of nucleosomes or chromatin.     

Nuclear reconstitution of demembranatedOrychophragmus violaceus sperm inXenopus laevis egg extracts

The cell-free extracts from animalXenopus laevis egg could induce chromatin decondensation and pronuclear formation from demembranated plant (Orychophragmus violaceu) sperm. The demembranatedOrychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.     

Assembly of correct kinetochore architecture in Xenopus egg extract requires transition of sperm DNA through interphase

Xenopus egg extracts provide a powerful tool for studying the formation and function of chromosomes. Two alternative protocols are generally used to obtain mitotic chromosomes. The first one uses a direct chromatin assembly from sperm nuclei in cytostatic factor (CSF)-arrested meiotic extracts, while the second is based on transition of sperm DNA through a replication step with subsequent reestablishment of CSF arrest. In this study we show that general kinetochore structure is disrupted in chromosomes assembled directly in CSF egg extracts: The amounts of outer kinetochore proteins such as Bub1, BubR1, and Dynactin subunit p150^glued are reduced and the components of the inner centromeric region (Aurora B kinase and Survivin) show compromised recruitment to centromeres. On the contrary, kinetochores on chromosomes assembled according to the second protocol closely resemble those in somatic cells. Our results indicate that the transition of sperm nuclei through interphase is an essential step for proper kinetochore assembly.     

Attachment of DNA Loops to an Artificial Matrix Does Not Affect the Replication Origin Specificity in Early Development of Xenopus laevis

Replication initiation proceeds in a random fashion in early development of Xenopus laevis. The replication origins become fixed only at later stages of development after the mid-blastula transition. Specification of replication origins occurs at the same time with the specification of the DNA attachment to the nuclear matrix. Replication origins of many species coincide or are located in the vicinity of sites of DNA attachment to the nuclear matrix. The present work was dedicated to development of an experimental system where DNA loops were specifically attached to an artificial matrix and a study of an effect of this attachment on specificity of DNA replication initiation in extracts of Xenopus laevis oocytes. We have found that DNA attachment to the artificial matrix increases the efficacy of DNA replication as compared to the control, but does not affect the replication specificity. It is likely that the transition from non-specific to specific replication is determined by a combination of several factors, and specificity of DNA attachment to a matrix alone is not sufficient for specification of a replication origin.     

Daunomycin disrupts nuclear assembly and the coordinate initiation of DNA replication in Xenopus egg extracts

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 μM. However, a high concentration of daunomycin 150 μM) 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 μM. 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 that 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. J. Cell. Biochem. 64:476–491. © 1997 Wiley-Liss, Inc.     

Distribution of Lectin Binding Sites in Xenopus laevis Egg Jelly

Eggs from the anuran Xenopus laevis are surrounded by a thick jelly coat that is required during fertilization. The jelly coat contains three morphologically distinct layers, designated J1, J2, and J3. We examined the lectin binding properties of the individual jelly coat layers as a step in identifying jelly glycoproteins that may be essential in fertilization. The reactivity of 31 lectins with isolated jelly coat layers was examined with enzyme-linked lectin-assays (ELLAs). Using ELLA we found that most of the lectins tested showed some reactivity to all three jelly layers; however, two lectins showed jelly layer selectivity. The lectin Maackia amurensis (MAA) reacted only with J1 and J2, while the lectin Trichosanthes kirilowii (TKA) reacted only with J2 and J3. Some lectins were localized in the jelly coat using confocal microscopy, which revealed substantial heterogeneity in lectin binding site distribution among and within jelly coat layers. Wheat germ agglutinin (WGA) bound only to the outermost region of J3 and produced a thin, but very intense, band of fluorescence at the J1/J2 interface while the remainder of J2 stained lightly. The lectin MAA produced an intense fluorescence-staining pattern only at the J1/J2 interface. Several lectins were also tested for the ability to inhibit fertilization. WGA, MAA, and concanavalin A significantly inhibited fertilization and WGA was found to block fertilization by preventing sperm from penetrating the jelly. Using Western blotting, we identified high-molecular-weight components in J1 and J2 that may be important in fertilization.     

In vitro reassembly of nuclear envelopes and organelles in Xenopus egg extracts

We reconstituted bilayer nuclear membranes, multilayer membranes, and organelles from mixtures ofXenopus laevis egg extracts and demembranatedXenopus sperm nuclei. Varying proportions of the cytosolic and vesicular fractions from the eggs were used in the reconstitution mixtures. A cytosol：vesicle ratio of 10：1 promoted reassembly of the normal bilayer nuclear membrane with inserted nuclear pore complexes around the decondensed Xenopus sperm chromatin. A cytosol： vesicle ratio of 5：1 caused decondensed and dispersed sperm chromatin to be either surrounded by or divided by unusual multilayer membrane structures with inlaid pore complexes. A cytosol：vesicle ratio of 2.5：1 promoted reconstitution of mitochondria, endoplasmic reticulum networks, and Golgi apparatus. During reassembly of the endoplasmic reticulum and Golgi apparatus, vesicular fragments of the corresponding organelles fused together and changed their shape to form flattened cistemae, which were then stacked one on top of another.     

The binding of ORC2 to chromatin from terminally differentiated cells

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.     

Nuclear reconstitution of demembranatedOrychophragmus violaceus sperm inXenopus laevis egg extracts

The cell-free extracts from animal Xenopus laevis egg could induce chromatin decon- densation and pronuclear formation from demembranated plant (Orychophragmus violaceus)sperm. The demembranated Orychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.     

Analysis of nucleosome arrangement on satellite DNA of rat liver chromatin

The arrangement of nucleosomes on the nucleotide sequence of satellite DNA of Oceanian rat (Rattus rattus) has been studied. Nucleosome cores were prepared from rat liver nuclei with micrococcal nuclease, exonucleaseIII and nuclease Sl. From the total population of core DNA fragments, the satellite-containing fragments were selected by molecular cloning and the complete nucleotide sequence of these clones was determined. The data show that nucleosomes occupy a number of preferred positions on satellite DNA. These positions are strictly defined. Thus location of nucleosomes along the satellite sequence is non-random. Such finding may have important biological significance.     

Role of DnaA protein during replication of plasmid pBR322 in Escherichia coli

Summary The in vivo role of the Escherichia coli protein DnaA in the replication of plasmid pBR322 was investigated, using a plasmid derivative carrying an inducible dnaA ⁺ gene. In LB medium without inducer, the replication of this plasmid, like that of pBR322, was inhibited by heat inactivation of chromosomal DnaA46 protein so that plasmid accumulation ceased 1 to 2 h after the temperature shift. This inhibition did not occur when the plasmid dnaA ⁺ gene was expressed in the presence of the inducer isopropyl-1-thin--d-galactopyranoside (IPTG). Inhibition was also not observed in glycerol minimal medium or in the presence of low concentrations of rifampicin or chloramphenicol. Deletion of the DnaA binding site and the primosome assembly sites (pas, rri) downstream of the replication origin did not affect the plasmid copy number during exponential growth at 30° C, or after inactivation of DnaA by a shift to 42° C in a dnaA46 host, or after oversupply of DnaA, indicating that these sites are not involved in a rate-limiting step for replication in vivo. The accumulation of the replication inhibitor, RNAI, was independent of DnaA activity, ruling out the possibility that DnaA acts as a repressor of RNAI synthesis, as has been suggested in the literature. Changes in the rate of plasmid replication in response to changes in DnaA activity (in LB medium) could be resolved into an early, rom-dependent, and a late, rom-independent component. Rom ^– plasmids show only the late effect. After heat inactivation of DnaC, plasmid replication ceased immediately. These results, together with previously published reports, suggest that DnaA plays no specific role during in vivo replication of ColE1 plasmids and that the gradual cessation of plasmid replication after heat inactivation of DnaA in LB medium results from indirect effects of the inhibition of chromosome replication and the ensuing saturation of promoters with RNA polymerase under nonpermissive growth conditions.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

Heart formative factor(s) is localized in the anterior endoderm of early Xenopus neurula

To elucidate the mechanisms of early heart morphogenesis in Xenopus laevis, we examined the effect of endoderm on heart morphogenesis in the early Xenopus neurula. Explants of anterior ventral (presumptive heart) mesoderm from early neurula were cultured alone or in combination with endoderm dissected from various regions. Heart formation was scored by an original heart index based on morphology. These explant studies revealed that anterior ventral endoderm plays a critical role in heart morphogenesis. Furthermore, we found that it was possible to confer this heart-forming ability on posterior ventral endoderm by the injection of poly(A)⁺ RNA from stage 13 anterior endoderm. These results imply that the heart formative factor(s) is localized in the anterior endoderm of the early neurula and that at least part of this activity is encoded by mRNA(s).     

Xenopus laevis Egg Jelly Contains Small Proteins That Are Essential to Fertilization

The eggs of Xenopus laevis are surrounded by investment layers of egg jelly that interact with the sperm immediately prior to fertilization. Components of these egg jelly layers are necessary for the fertilization of the egg by incoming sperm. Eggs which are stripped of their jelly layers are refractile to fertilization by sperm, but the addition of solubilized jelly promotes fertilization. We have shown previously that the egg jelly layers are composed of a fibrous network of glycoconjugates which loosely hold smaller diffusible components. Extracts of these diffusible components were prepared by incubation of freshly ovulated eggs in high-salt buffers for 12 h at 4°C. This diffusible component extract, when incubated with sperm, promoted the sperm's ability to fertilize dejellied eggs in a dose-dependent manner. In contrast, the high-molecular-weight “structural” glycoconjugates of jelly that remain after extraction of the diffusible components did not increase fertilization efficiency of dejellied eggs nor did nonspecific proteins, carbohydrate polymers, or organic polymers. The diffusible components, analyzed by SDS–PAGE, consisted of a mixture of proteins from 4 to 180 kDa. The protein responsible for fertilization rescue appeared to be <50 kDa and appeared to self-aggregate or to bind to larger proteins. This protein component was required during sperm binding to the egg, its action required an intact egg vitelline envelope, and its action was independent of large soluble polymers such as Ficoll.     

Contributions to nucleosome dynamics in chromatin from interactive propagation of phosphorylation/acetylation and inducible histone lysine basicities

The effect of phosphorylation on the basicities of amines in histone H3 peptides and their acetylation kinetics is probed with a mild chemical acetylating agent. Phosphorylation of Ser‐10 lowers the rate of chemical acetylation of Lys‐9, Lys‐14, and Lys‐18 by methyl acetyl phosphate in that order consistent with a higher pK_a of these Lys residues induced by phosphorylation; basicities increase up to 3 pK_a units as a function of distance from Ser‐10 phosphate. Enzymic acetylation of Lys residues with high pK_a values in nucleosomes is also expected to be enhanced by phosphorylation, consistent with the known mechanism involving binding of protonated amines to N‐acetyltransferases; fetal hemoglobin has a related linkage of increased basicity at a specific site, its acetylation, and a resulting decrease in subunit interaction strength. In the absence of a phosphate on Ser‐10, the amines of Lys‐9, Lys‐14, and Lys‐18 have lowered pK_a values. Chemical acetylation of glycine and glycinamide have analogous kinetic profiles to the histone peptides but the phosphate inductive effect in histone H3 is more potent since the linkage between phosphorylation and acetylation is propagated with a range extending 9–10 amino acids in either direction from the phosphorylation site enhancing protonation of amino groups. We conclude that lysine amine basicities in histone tails are not static but inducible and variable due to a dynamic and immediate interaction between phosphorylation/acetylation that may contribute to inactive heterochromatin by compaction through such Ser phosphate–Lys amine electrostatic interactions and their relaxation by acetylation in euchromatin.     

Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell—free extracts of Xenopus laevis eggs

Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation,nuclear envelope assembly,and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle.The assembled nuclei,being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii.However,incubation of dinoflagellate Cyrthecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinium cohnii cells does not induce nuclear reconstitution.     

Integration of repeated sequences (pBR322) in the Bacillus subtilis 168 chromosome without affecting the genome structure

The Escherichia coli plasmid pBR322 sequence (4363 bp) was integrated at the met, pro, or leuB locus of the Bacillus subtilis chromosome without duplication of the flanking chromosomal regions. The integrated pBR322 was stably maintained as part of the chromosome regardless of its orientation or location. It was found that a DNA segment as large as 17 kb cloned in pBR322 can be readily transferred to the B. subtilis chromosome by transformation. It was demonstrated that a second pBR322 sequence could be effectively introduced at different regions of the chromosome by sequential transformation using chromosomal DNA isolated from a strain that had already acquired a pBR322 sequence at a different locus. Similarly, a third pBR322 sequence could be introduced. By this method, two or three pBR322 sequences can be incorporated at unlinked loci without affecting the overall structure of the B. subtilis genome.