An ultrastructural investigation on the polyethylene glycol-induced adhesion of tobacco nuclei and uptake of micronuclei by soybean protoplasts

Nuclei isolated from tobacco protoplasts were induced to be taken up by soybean protoplasts using a protocol involving polyethylene glycol (PEG), osmotic shock and pH shift. Transmission electron microscopy revealed that PEG treatment condensed the chromatin of the isolated nuclei. Close adhesion of isolated nuclei to the plasma membrane of protoplasts following PEG treatment, was observed by both scanning and transmission electron microscopic methods. Ultrastructural observations were also made on the formation of micronuclei in tobacco cells following the treatment with amiprophosmethyl (APM). Nuclei and micronuclei isolated from APM-treated cells were induced to be taken up by soybean protoplasts. A single case of uptake of an isolated micronucleus was observed by transmission electron microscopy. The observations on the effects of PEG on the isolated nuclei, micronuclei and protoplasts are discussed in relation to the possible mechanism of uptake of nuclei by protoplasts using PEG.     

Structural properties of barley nucleosomes

The structural properties of barley oligonucleosomes are investigated and compared to those of rat liver oligomers. Extraction of barley chromatin was performed using mild nuclease digestion of isolated nuclei leading to a low ionic strength soluble fraction. Oligonucleosomes were fractionated on sucrose gradients and characterized for DNA and histone content. Physico-chemical studies (sedimentation, circular dichroism and electric birefringence) showed that barley oligonucleosomes exhibit properties very close to those of the H1-depleted rat liver counterparts. Moreover, in situ, barley linker DNA was more sensitive to micrococcal nuclease digestion than that of rat liver. These results suggest that barley oligonucleosomes show a less compact structure than their rat liver counterparts and appear to be in contradiction with the very condensed organization of barley chromatin previously suggested.     

Fusion between interphase and mitotic plant protoplasts : Induction of premature chromosome condensation

Morphological changes in interphase nuclei were cytologically studied in heterophasic dinucleate cells formed by the fusion of mitotic and interphase plant protoplasts. Mitotic protoplasts were isolated from a partially synchronized suspension culture of wheat (Triticum monococcum). The mitotic cells were accumulated by colchicine after release of hydroxyurea block. Treatment of protoplast populations with polyethylene glycol-dimethyl sulphoxide solution resulted in metaphase-interphase fusion. Three hours after fusion, the appearance of chromosomes with single chromatid as well as of fragmented, pulverized chromatin in heterophasic cells indicated the induction of premature chromosome condensation (PCC) in somatic wheat cells. Condensation in interphase nuclei of mitotically inactive rice protoplasts was also detected after fusion with mitotic wheat protoplasts.     

Ultrastructure of Amoebophrya sp. and its changes during the course of infection

Amoebophrya is a syndinian parasite that kills harmful bloom forming algae. Previously uncharacterized ultrastructural aspects of infection and development were elucidated. The biflagellate dinospore has two mitochondria, electron-dense bodies, striated strips, trichocysts, and a nucleus with peripherally condensed chromatin. After finding an Akashiwo sanguinea host and adhering to its surface, the parasite penetrates the host surface, apparently using a microfilament based motility and electron-dense bodies within a microtubular basket in the process of parasitophorous vacuole membrane formation. After entering the host nucleus, possibly by a similar mechanism used to enter the host cell, the parasite cytosol expanded substantially prior to mitosis. From 12-36 hours mitochondria were inconspicuous but present. Chromatin condensation was variable. By 36 hours post-infection, parasites had multiple nuclei, a microtubule-supported cytopharynx, and were beginning to form a fully internal mastigocoel. By 48 hours, the characteristic "beehive" appearance was apparent with flagella projecting into a fully developed mastigocoel. The cytoplasm contained trichocysts, elongated mitochondria, and nuclei with peripherally condensed chromatin. Although Amoebophrya lacks an apical complex, its electron-dense bodies show functional similarities to apicomplexan rhoptries. Its lack of permanently condensed chromosomes, but compact dinospore chromatin, supports the idea that dinoflagellate permanently condensed chromosomes may be a remnant of a parasitic ancestor with a compact dispersal stage.     

Structural features of isolated generative cells and their protoplasts from pollen of some liliaceous plants

Large quantities of intact generative cells and their protoplasts were isolated from pollen protoplasts of four liliaceous plants, and their structural features were investigated. The generative cells, liberated from the vegetative cell cytoplasm of the pollen protoplasts, were initially spindle-shaped with two long, oppositely oriented extensions, and were surrounded by two cell membranes, one on each side of a wall of uniform thickness. The generative nuclei, stained with 4′,6-diamidino-2-phenylindole (DAPI), showed ellipsoidal and highly condensed chromatin, whereas the generative cell cytoplasm, whose quantity was widely different from species to species, showed no fluorescence, suggesting the absence of plastid and mitochondria! DNA, although many mitochondria were present. The isolated generative cells, which were spindle-shaped at first, became spherical in shape in vitro. Immunocytochemistry and transmission electron microscopy revealed that this change was associated with the depolymerization of an axial array of microtubules present in generative cells in situ. These results are discussed in relation to the function of the generative cell within the bicellular pollen of angiosperms.     

Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate

Crude extracts of Xenopus eggs are capable of nuclear assembly around chromatin templates or even around protein-free, naked DNA templates. Here the requirements for nuclear assembly around a naked DNA template were investigated. Extracts were separated by ultracentrifugation into cytosol, membrane, and gelatinous pellet fractions. It was found that, in addition to the cytosolic and membrane fractions, a component of the gelatinous pellet fraction was required for the assembly of functional nuclei around a naked DNA template. In the absence of this component, membrane-bound but functionally inert spheres of lambda DNA were formed. Purification of the active pellet factor unexpectedly demonstrated the component to be glycogen. The assembly of functionally active nuclei, as assayed by DNA replication and nuclear transport, required that glycogen be pre-incubated with the lambda DNA and cytosol during the period of chromatin and higher order intermediate formation, before the addition of membranes. Hydrolysis of glycogen with alpha- amylase in the extract blocked nuclear formation. Upon analysis, chromatin formed in the presence of cytosol and glycogen alone appeared highly condensed, reminiscent of the nuclear assembly intermediate described by Newport in crude extracts (Newport, J. 1987. Cell. 48:205- 217). In contrast, chromatin formed from phage lambda DNA in cytosol lacking glycogen formed "fluffy chromatin-like" structures. Using sucrose gradient centrifugation, the highly condensed intermediates formed in the presence of glycogen could be isolated and were now able to serve as nuclear assembly templates in extracts lacking glycogen, arguing that the requirement for glycogen is temporally restricted to the time of intermediate formation and function. Glycogen does not act simply by inducing condensation of the chromatin, since similarly isolated mitotically condensed chromatin intermediates do not form functional nuclei. However, both mitotic and fluffy interphase chromatin intermediates formed in the absence of glycogen can be rescued to form functional nuclei when added to a second extract which contains glycogen. This study presents a novel role for a carbohydrate in nuclear assembly, a role which involves the formation of a particular chromatin intermediate. Potential models for the role of glycogen are discussed.     

Electron microscope study of chromatin in nuclei of rat hepatocytes during age-related changes in the genome

On ultrathin liver sections, condensed chromatin of rat hepatocyte nuclei was studied. The animals were 2 days, 6 and 28 months old. It was established that neither maturation nor senescence were accompanied by the change of the relative square of total condensed chromatin. Relative square of perimembrane, nucleoplasmic and perinucleolar condensed chromatin were non changed either. Intensively proliferating hepatocytes of nascent animals were characteristic of maximal values of the following parameters (i) the relative length of the perimembrane condensed chromatin boundary with nucleoplasma. (ii) amount of chromatin clumps, (iii) the relative length of the nuclear membrane without condensed chromatin. For mature animals all these parameters are significantly decreased. For old rats as compared with mature ones the following parameters are significantly diminished: (i) the relative length of the perimembrane chromatin boundary with nucleoplasma, (ii) the relative length of the nuclear membrane without condensed chromatin, (iii) the mean square of the nucleolus. So, the known diminishing of the RNA synthesis at senescence is expressed morphologically in margination of condensed chromatin, in smoothing of the condensed chromatin surface responsible for the hnRNA synthesis and also in diminishing of the nucleolus responsible for the rRNA synthesis.     

Organization of higher-level chromatin structures (chromomere,chromonema and chromatin block) examined using visible light-induced chromatin photo-stabilization

The method of chromatin photo-stabilization by the action of visible light in the presence of ethidium bromide was used for investigation of higher-level chromatin structures in isolated nuclei. As a model we used rat hepatocyte nuclei isolated in buffers which stabilized or destabilized nuclear matrix. Several higher-level chromatin structures were visualized: 100nm globules-chromomeres, chains of chromomeres-chromonemata, aggregates of chromomeres-blocks of condensed chromatin. All these structures were completely destroyed by 2M NaCl extraction independent of the matrix state, and DNA was extruded from the residual nuclei (nuclear matrices) into a halo. These results show that nuclear matrix proteins do not play the main role in the maintenance of higher-level chromatin structures. Preliminary irradiation led to the reduction of the halo width in the dose-dependent manner. In regions of condensed chromatin of irradiated nucleoids there were discrete complexes consisting of DNA fibers radiating from an electron-dense core and resembling the decondensed chromomeres or the rosette-like structures. As shown by the analysis of proteins bound to irradiated nuclei upon high-salt extraction, irradiation presumably stabilized the non-histone proteins. These results suggest that in interphase nuclei loop domains are folded into discrete higher-level chromatin complexes (chromomeres). These complexes are possibly maintained by putative non-histone proteins, which are extracted with high-salt buffers from non-irradiated nuclei.     

Morphology ofEntomophthora muscae protoplasts grownin vitro

Summary Entomophthora muscae (C.) Fres. can be grownin vitro as protoplasts. Light and electron microscopical studies of thein vitro developed protoplasts have demonstrated the absence of an organized wall over the protoplasmic Con A-positive membrane at all stages of growth. The cytological organization is typical of the Entomophthorales with condensed chromatin in the interphase nuclei and small eccentric metaphase spindles. Long strands of endoplasmic reticulum, microubules and vesicles surrounding the plasmalemma may be involved in maintaining the precise shape ofE. muscae protoplast. Starvation of the fungus induces the formation of hyphal bodies after deposition of Con A- and WGA-positive wall material at the plasmalemma surface.Abbreviations Con A concanavalin A - DH Drosophila cell culture medium - FITC fluorescein isothiocyanate - GLEN glucose-lactal-bumin-yeast extract-NaCl culture medium for protoplasts - HBL hyphal body-like protoplasts - MM Mitsuhashi and Maramorosch' insect cell culture medium - PATAg periodic acid-thiocarbohydrazide-silver proteinate technique - PBN phosphate buffer with NaCl - S spherical protoplasts - WGA wheat germ agglutinin     

A cytochemical study of the nonhistone protein content of condensed and extended chromatin

Cytochemical techniques have been used to study the distribution of nonhistone proteins in sections of interphase nuclei and mitotic chromosomes. Condensed chromatin, including the heterochromatin of interphase nuclei from frog liver, and mitotic metaphase and anaphase chromosomes from bovine kidney, show little or no staining for nonhistone protein. Regions of frog liver nuclei which contain extended chromatin (euchromatin) stain intensely for nonhistone protein. These differences in nonhistone staining of condensed and extended chromatin support the suggestion that regions of condensed chromatin contain considerably less nonhistone protein than regions of extended chromatin. The results suggest further that there may be considerably less nonhistone protein associated with chromosomes and interphase heterochromatin than has been reported in most previous analyses of isolated chromatin and chromosome preparations.     

Dynamics of Nuclear DNA Quantities during Zygote Development in Barley

Quantities of DNA were estimated in the nuclei of mechanically isolated egg and zygote protoplasts in two cultivars of barley using 4[prime],6-diamidino-2-phenylindole staining and microfluorometry. Unlike many previous studies on DNA amounts within the sex cells of flowering plants, we obtained consistent and unambiguous results indicating that the egg and sperm nuclei are at the 1C DNA level (basic haploid amount) at the time of karyogamy. Karyogamy was initiated within 60 min postpollination, and the male chromatin became completely integrated into the egg nucleus within 6 to 7 hr postpollination (hpp). Zygotic nuclear DNA levels began to increase at ~9 to 12 hpp in cultivar Alexis and at 12 to 15 hpp in cultivar Igri. The 4C DNA complement was reached in most zygotes by 22 to 26 hpp in cultivar Alexis and by 23 to 29 hpp in cultivar Igri. These data are fundamental to a better understanding of fertilization and zygote maturation in flowering plants. They are also relevant to studies in which the timing of zygotic DNA replication is of interest, such as ongoing investigations on genetic transformations in barley using the microinjection technique.     

Isolation of rosette-like structures from partially deproteinized chromatin in rat hepatocytes

The structure of partial deproteinized rat hepatocyte chromatin has been studied. Depending on the magnesium concentration the chromatin of isolated nuclei is present in the two conditions: diffuse (at 0-1.5 mM MgCl2) and condensed (at 2-5 mM MgCl2). The main components of nuclei with condensed chromatin are chromomers--globular structures about 100 nm in diameter. By treating such nuclei with heparin and dextransulfate one can observe a rosette-like structure with lateral loops having the following parameters: the length of the loops, 15-20 micron; the number of loops, 15-30. The rosette-like structures are sensitive to endogenous nuclease and DNase 1, but not to RNase. Pronase or higher concentration of polyanions give rise to unfolding of the rosette-like structures. The rosette structures cannot be isolated from the nuclei with diffuse chromatin. On the basis of these observations a hypothesis of chromatin structural organization in the interphase nucleus is proposed, and the connection of the rosette-like structures with some structural levels of chromatin organization is discussed.     

In vitro interaction of 63-nickel(II) with chromatin and DNA from rat kidney and liver nuclei

The interaction of nickel(II) with chromatin was studied in vitro and in isolated nuclei from rat liver and kidney. Nickel(II) bound to chromatin, polynucleosomes (DNA + histone octamer protein complex), and to deproteinized DNA both in intact nuclei and in vitro. The amount of nickel(II) bound depended on the concentration of nickel(II), the presence of chromosomal proteins and the binding sites on DNA which provide a stable coordination environment for nickel(II). The binding of nickel(II) to chromatin and to DNA in whole nuclei was much slower than in vitro indicating that assessibility of the DNA binding sites was influenced by the presence of the nuclear membrane, nuclear matrix and nuclear proteins and/or by the condensed nuclear structure of chromatin. Since DNA containing bound nickel(II) was isolated from chromatin, nickel(II) directly interacted with stable binding sites on the DNA molecule in chromatin. Nickel(II) was associated with the histone and non-histone nuclear proteins as well as the DNA in rat liver and kidney chromatin. Nickel(II) was found to bind to calf thymus histones in vitro. Nickel(II)-nuclear protein and -DNA interactions were investigated by gel electrophoretic analysis of in vitro incubation products. Although nickel-histone and nickel-non-histone protein interactions were completely disrupted by the electrophoretic conditions, fluorography revealed the presence of inert nickel(II)-DNA and/or nickel(II)-DNA-protein complexes.     

Behavior of pericentromere heterochromatin regions under different conditions of chromosome decondensation in isolated nuclei]

In isolated mouse nuclei the chromocenters were shown to be the pericentromeric heterochromatin regions (PCHR). After the decreasing of bivalent ion concentration (0.1 mM Ca2+, 2 mM Mg2+) the main and peripheral parts of the chromatin remained on the contrary as the compact chromatin bodies. The additional ultrasound treatment of isolated nuclei in the presence of 0.1 mM Ca2+ with DNAase II and triton X-100 resulted in the species enriched by the condensed PCHR.     

PHYSICAL STUDIES OF ISOLATED EUCARYOTIC NUCLEI

The degree of chromatin condensation in isolated rat liver nuclei and chicken erythrocyte nuclei was studied by phase-contrast microscopy as a function of solvent pH, K⁺ and Mg⁺⁺ concentrations Data were represented as "phase" maps, and standard solvent conditions selected that reproducibly yield granular, slightly granular, and homogeneous nuclei Nuclei in these various states were examined by ultraviolet absorption and circular dichroism (CD) spectroscopy, low-angle X-ray diffraction, electron microscopy, and binding capacity for ethidium bromide Homogeneous nuclei exhibited absorption and CD spectra resembling those of isolated nucleohistone. Suspensions of granular nuclei showed marked turbidity and absorption flattening, and a characteristic blue-shift of a crossover wavelength in the CD spectra. In all solvent conditions studied, except pH < 2 3, low-angle X-ray reflections characteristic of the native, presumably superhelical, nucleohistone were observed from pellets of intact nuclei. Threads (100–200 A diameter) were present in the condensed and dispersed phases of nuclei fixed under the standard solvent conditions, and examined in the electron microscope after thin sectioning and staining Nuclei at neutral pH, with different degrees of chromatin condensation, exhibited similar binding capacities for ethidium bromide. These data suggest a model that views chromatin condensation as a close packing of superhelical nucleohistone threads but still permits condensed chromatin to respond rapidly to alterations in solvent environment.     

THE HISTONES ASSOCIATED WITH CONDENSED AND EXTENDED CHROMATIN OF MOUSE LIVER

Histones were extracted from isolated mouse liver nuclei, and from mouse liver condensed and extended chromatin. Mouse liver histones were found to be very similar to those of calf thymus in their solubility properties, relative electrophoretic mobilities, and molecular weights as determined on SDS-polyacrylamide gels. Quantitative analysis by high-resolution gel electrophoresis demonstrated a remarkable similarity between the histones of condensed chromatin and those of extended chromatin. However, minor differences were found. A unique subspecies was found only in condensed chromatin histone and the relative amounts of fractions F2A1 and F2A2 differed in the two types of chromatin. The ratio of the parental to the acetylated form of F2A1 was identical in the two chromatin samples. Since DNA extracted from the condensed chromatin fraction consisted of approximately 50% satellite DNA, the general similarities between the histones of condensed and extended chromatin make it likely that even this simple, highly repetitive DNA is complexed with a number of histone subfractions.     

Localization of the loosely bound nuclear proteins of rat brain: an immunocytochemical ultrastructural study

Antibodies against the loosely bound subnuclear protein fraction (0.35 M NaCl-extractable subnuclear fraction) of rat brain were raised in rabbits, and the ultrastructural distribution of the antigenic determinants in rat cerebellum was studied using the unlabeled antibody peroxidase-antiperoxidase (PAP) method. A localization restricted to the nucleus was observed. Both neuronal and neuroglial nuclei exhibited antigens, whereas nuclei of pericytes and endothelial cells did not. The immunoreaction product was homogeneously distributed in dispersed chromatin and was absent from condensed chromatin, suggesting that the antigens were confined to the active regions of the genoma. The outer nuclear membrane and the nucleolus appeared to be free of the antigens, while a perinucleolar ring of immunoreaction was detectable. Liver preparations showed a nuclear reaction markedly weaker than the one for brain nuclei. Adsorption of the serum with isolated liver nuclei nullified the reactivity in liver tissue, whereas a sharp reaction was still observed in the cerebellum, indicating the subcellular reaction under examination to contain antigens specifically concentrated in the nervous system or unique to the brain.     

The study of chromatin and chromosome structure on preparations of interphase nucleus derivatives resulting from nuclear wall removal. IV. Structural heterogeneity of stretched chromatin in membrane-free cell nuclei from human peripheral lymphocytes

Densely aggregated chromatin of mature human or animal peripheral lymphocytes is inaccessible for structural investigation on preparations of both intact cell and conventionally spread chromatin. Giemsa- and DAPI-positive "free chromatin" structures, in addition to amembraneous nuclei, were isolated from intact lymphocytes gently treated with Triton-X-100. Surface stretching of both these nuclei and structures, shortly fixed in methanol-glacial acetic acid (3:1), revealed three main types of these "free chromatin" structures: dense chromatin structures (DCS), loose chromatin structures (LCS) and nuclear spreads (NS). The share of each nuclear derivative may be shifted by changing either detergent concentration and(or) the time of incubation in detergent solution. Each DSC consists of condensed "residual" nucleus, similar in from and size with an intact lymphocyte nucleus, and involves 1-15 uni- or olygonemic chromatin sprouts of different length. LSC contain heterogeneously loosened spindle-shape or drop-like nuclei, being several times longer and wider than DCS-nuclei, and 1-3 long uni- or olygonemic chromatin tail-pieces and incidentally observed lateral chromatin sprouts. The majority of LCS contain either a chromocenter of different number of end-to-end associated spindle-shape domains of condensed chromatin. The latter reached 2-5 x 1.5 microns being cross-striated or spiral in structure. NS represent spread chromatin fibrillar structures varying from 150 to 500 microns in length and from 1.5 to almost 50 microns in width. NS consist of 0.3-0.4 micron smooth and 0.4-0.8 micron beaded chromatin fibres. Thin fibres produce web-like domains of NS. and thick fibres form olygonemic bundles or end-to-end association of unit chromatin fibres within NS. Some portion of thick unit fibres of NS gave rise to local splitting into two thin fibres with a similar bead patterns. Thick argyrophilic fibers of the nucleolus also displayed a beaded structure and commonly spread hand-in-hand with the basic chromatin fibre aggregations.