Development of Nuclear Vacuoles in Sugar Beet Male Meiocytes

The formation, development and disappearance of nuclear vacuolesin three genetically different lines of Beta vulgaris L. arepresented. Nuclear vacuoles may be generated in zygotene bythe nuclear envelope, providing the membrane necessary for theformation of vacuoles within the nucleus. During subsequentstages of meiotic prophase nuclear vacuole volume increasesrapidly, peaking in pachytene. In diplotene/diakinesis totalnuclear vacuole volume falls gradually, and vacuoles eventuallydisappear in metaphase I. Maximum nuclear vacuole size in pachytenecoincides with a notable increase in nuclear surface. We thereforesuggest that nuclear vacuoles do not perform a mechanical functionin the contraction of the karyoplasm, but instead act as compartmentsin which products of nuclear metabolism may be stored, and subsequentlyparticipate in the elimination of nuclear products to the cytoplasm.These processes may be related to changes occurring preparatoryto the transition from sporophyte to gametophyte generation. Beta vulgaris L, sugar beet, meiocytes, nuclear vacuoles, ultrastructure     

Patterns of Nuclear and Nucleolar Growth in Synchronously Dividing Explants from Tubers of Helianthus tuberosus L.

The nucleus and nucleolus have been examined by phase contrastmicroscopy of isolated fixed nuclei from synchronously dividingcells of Helianthus tuberosus L. tuber explants grown in nutrientmedium on filter paper. The volumes of nuclei and nucleoli werecomputed from their areas and perimeters obtained by digitizingimages projected from film. The nuclei did not show a pattern of growth related to the Sphase but enlarged at times of both de-differentiation and differentiation.There was also rapid post-mitotic nuclear enlargement. The sizeattained by nuclei in the three successive divisions followingcell activation decreased progressively, but started to riseagain at the time of cell differentiation. The changes are discussed in relation to nuclear size regulation,the nuclear matrix and hypotheses relating nuclear growth toDNA, protein and water in the processes of de-differentiation,mitosis and differentiation. Nucleoli showed a clear fusion and growth cycle. The patternof fusion can be used to identify the position of a sample ofcells, though not any particular cell, within the cycle. Nucleolargrowth was different in the succeeding cell cycles that wereinduced in the de-differentiating tissue. Nucleolar enlargementwas slow in the first cycle and continued until mitosis. Therewas rapid nucleolar growth in the second cycle and none in latercycles until the time of cell differentiation. Nucleolar changes are discussed in relation to rRNA gene dosage,replication and polymerase availability. Helianthus tuberosus L. Jerusalem artichoke, isolated-nuclei, tissue culture, cell cycle, nucleolar cycle     

Nuclear architecture of human pachytene spermatocytes: quantitative analysis of associations between nucleolar and XY bivalents

Summary Nucleolar association and heterochromatin coalescence have both been invoked as mechanisms involved in the origin of chromosomal associations between nucleolar bivalents themselves, as well as between these bivalents and the XY pair, during meiotic prophase in human spermatocytes. However, these mechanisms do not satisfactorily explain how associating bivalents meet each other within the nuclear space. To elucidate this problem, we have characterized different types of nucleolar-nucleolar and nucleolar-XY bivalent associations, and their frequencies, in light and electron microscope serial sections of spermatocyte nuclei. In the pachytene nucleus, nucleolar bivalent associations were found to involve only one nucleolar sphere of RNP granules connected through a fibrillar center to a chromatin mass composed of two, or more, nucleolar-bivalent short arms. Structural relationships between these elements were examined using 3D computer models of various nucleolar associations. XY and nucleolar bivalents were usually located towards the nuclear periphery associated with the inner face of the nuclear envelope. Some nucleolar bivalents, whether single or associated appeared beside or over XY chromatin. When nucleolar-bivalent short arms (BK) were found over nucleolar or over XY chromatin, their telomeres were unattached to the nuclear envelope and the corresponding synaptonemal complexes were not observed. Ninety nucleoli were found in sixty pachytene nuclei. Thirty six percent of these nucleoli were bound to associated BKs and the remaining 64% to single BKs. Over 40% of individual spermatocytes showed at least one cluster of associated BKs and about 20% presented single or multiple BKs associated with the XY pair. The frequencies of random BK associations, over the total or restricted areas of the nuclear envelope, were calculated according to a probabilistic nuclear model. A correspondence was found in comparing the observed frequencies of associated BKs with those calculated on the basis of bouquet formation. Such an analysis strongly suggests that the occurrence of associations between nucleolar bivalents may arise at random within the bouquet. Thus, the architecture of the meiocyte nucleus, particularly the organization of the bouquet, may be the primary mechanism by which nucleolar bivalents meet each other and, consequently, become associated either through common nucleolus formation or by heterochromatin coalescence.     

The Induced Resistance Response of Carrot Root Slices to Heat-killed Conidia and Cell-free Germination Fluid of Botrytis cinerea Pers. ex Pers.: 2. Nuclear Migration, Nucleolar Volume and Uptake of Tritiated Uracil

Sixty-eight per cent of nuclei in the cells of the upper fourlayers of carrot slices treated with heat-killed conidia ofBotrytis cinerea for 6 h followed by inoculation with live sporesfor 18 h, migrated to the cell face nearest to the treated surface,compared with 46 per cent in cells of control slices showinga wound-healing response only. Nucleolar volumes in the surfacecell layers of control slices increased from a mean of 1.0 µm³to 3.8 µm³ over 24 h, and in ‘induced’ slicesto 7.28 µm³. Using a 40 min pulse of [5–³H]uracil,there was an increase within 15 h of slicing in the number oflabelled nuclei in cells from control slices undergoing healing.Within 8 h after treatment of slice surfaces with heat-killedconidia, there was an accelerated incorporation of label into‘nuclear’ RNA. Slices from roots cold-stored for12 months failed to show an induction response and nucleolarvolumes did not increase more than in control slices. Theseresults are discussed in relation to active defence mechanismsin plant tissue. Botrytis cinerea, carrot, induced resistance, nuclear migration, nucleolar volume, RNA incorporation     

Nuclear Changes in the Cotyledons of Pisum arvense L. during Germination

In the cells of the cotyledons of Pisum arvense L. there isa close correlation between cell volume, nuclear volume, andnuclear DNA level. The cells of the epidermis are at the 2Clevel of DNA, those of the hypodermus vary from 2C to 4C, whilethose of the storage tissues range from 4C to 16C. During germinationthe nucler increase in size In the storage tissues there isa three to fourfold increase, which is accompanied by an increasein lobing of the nucler Simultaneously the DNA level of thenucler decreases by about 50 per cent. There are parallel changesin nuclear histone levels. Initially nuclear RNA level is lowbut in any given cell it increases to a maximum at the timethe storage reserves of the cell begin to disappear, after whichit declines. The level of cytoplasmic RNA is high initiallyin the tissues at the abaxial side of the cotyledon. Duringthe first few days of germination it declines until the over-alllevel is uniformly low, after which there is a further smalldecline.     

Number and Nuclear Localisation of Nucleoli in Mammalian Spermatocytes

In seven mammalian species, including man, the position and number of nucleoli in pachytene spermatocyte nuclei were studied from electron microscope (EM) nuclear sections or bivalent microspreads. The number and position of the nucleolar organiser regions (NORs) in mitotic and meiotic chromosomes were also analysed, using silver staining techniques and in situ hybridisation protocols. The general organisation of pachytene spermatocyte nucleoli was almost the same, with only minor morphological differences between species. The terminal NORs of Thylamys elegans (Didelphoidea, Marsupialia), Dromiciops gliroides (Microbiotheridae, Marsupialia), Phyllotys osgoodi (Rodentia, Muridae) and man, always gave rise to peripheral nucleoli in the spermatocyte nucleus. In turn, the intercalated NORs from Octodon degus, Ctenomys opimus (Rodentia, Octodontidae) and Chinchilla lanigera (Rodentia, Cavidae), gave rise to central nucleoli. In species with a single nucleolar bivalent, just one nucleolus is formed, while in those with multiple nucleolar bivalents a variable number of nucleoli are formed by association of different nucleolar bivalents or NORs that occupy the same nuclear peripheral space (Phyllotis and man). It can be concluded that the position of each nucleolus within the spermatocyte nucleus is mainly dependent upon: (1) the position of the NOR in the nucleolar bivalent synaptonemal complex (SC), (2) the nuclear pathway of the nucleolar bivalent SC, being both telomeric ends attached to the nuclear envelope, and (3) the association between nucleolar bivalents by means of their NOR-nucleolar domains that occupy the same nuclear space. Thus, the distribution of nucleoli within the nuclear space of spermatocytes is non-random and it is consistent with the existence of a species-specific meiotic nuclear architecture.     

The Nuclear Endoreduplication Cycle in Metaxylem Cells of Primary Roots of Zea mays L.

The nuclear DNA content of metaxylem cells in roots of Zea mayscv. Golden Bantam reaches 16C or 32C by successive rounds ofDNA endoreduplication. Each phase of endoreduplication (endo-S)is separated by a non-DNA synthetic phase (endo-G). These phasesseem to occur in zones at fixed distances from the root tip.The duration of the phases in two of the endoreduplication cycles(4C–8C, 8C–16C) has been estimated in two ways.The first makes use of the rate of movement of cells throughthe positions along the root where the different phases of thecycle are occurring, the second uses labelling with methyl-[³H]thymidineand autoradiography. Both methods indicate that the endo-S phaseswhich cause the nuclear DNA content to rise from 4C to 8C andfrom 8C to 16C last 8–10 h, and that the intervening endo-Gphase lasts 8–12 h. DNA endoreduplication keeps pace withthe increase of nuclear volume; cell volume increases at a morerapid rate, however. Comparison of the endoreduplication cyclein the metaxylem with the mitotic cycle in the adjoining filesof parenchyma cells shows that the mitotic cells complete theircycle more slowly. DNA synthesis, endoreduplication cycle, mitotic cycle, root apex, Zea mays     

Cell and Nuclear Size in Vicia faba Roots: changes during Germination and in Response to Levels of Ambient Water

Changes in cell and nuclear size were studied in roots of germinatingseeds of Vicia faba in order to answer the following questions:(1) When is growth of cells and nuclei initiated? (2) Is theratio, nucleus: cell size, constant over the first two to threecell cycles, i.e. are nuclear and cell growth closely coordinatedor can they vary independently of one another? Answers to thesequestions should contribute to our knowledge of the relationbetween cell and nuclear growth and the systems that regulategrowth of actively proliferating cells. The results show thatmean cell area increased very little during the first cell cycleand then decreased over the subsequent two to three cell cycles,i.e. until roots reached a steady state condition. Nuclei, onthe other hand, increased in size from 30 to 50 h after seedswere sown, i.e. when most nuclei were in S or G₂ The mean andthe range in nuclear volumes reached in the first two mitoticcycles during germination are maintained when root meristemsachieve a steady state condition. Thus nuclei maintain a constantmean size over the first few cycles, while cells decrease insize. As a, result of this difference, the ratio, nucleus: cellarea, increases over the few first cell cycles from 0·157to 0·222. The ratio increases in both interphase andprophase cells. Increases in size of cells and nuclei are notabsolutely coordinated; this suggests that nuclear and cellgrowth are, to some extent, regulated independently of eachother. Vicia faba L, broad bean, nuclear volume, cell area, root meristem, germination, water level     

Nuclear Dry Mass and Area in Epidermal Cells of Senescing Soybean Cotyledons

The nuclear dry mass and area of epidermal cells of senescingsoybean cotyledons were measured with an interference microscopeand found to decrease until cotyledon abscission. Decapitationafter germination prevented yellowing and abscission of cotyledonsand the decrease of both nuclear dry mass and area. The meansof nuclear dry mass and area for normal and decapitated cotyledonswere highly correlated in two experiments, r=0.0.76 and 0.90respectively.     

Nuclear transporters in a multinucleated organism: functional and localization analyses in Aspergillus nidulans

Nuclear transporters mediate bidirectional macromolecule traffic through the nuclear pore complex (NPC), thus participating in vital processes of eukaryotic cells. A systematic functional analysis in Aspergillus nidulans permitted the identification of 4 essential nuclear transport pathways of a hypothetical number of 14. The absence of phenotypes for most deletants indicates redundant roles for these nuclear receptors. Subcellular distribution studies of these carriers show three main distributions: nuclear, nucleocytoplasmic, and in association with the nuclear envelope. These locations are not specific to predicted roles as exportins or importins but indicate that bidirectional transport may occur coordinately in all nuclei of a syncytium. Coinciding with mitotic NPC rearrangements, transporters dynamically modified their localizations, suggesting supplementary roles to nucleocytoplasmic transport specifically during mitosis. Loss of transportin-SR and Mex/TAP from the nuclear envelope indicates absence of RNA transport during the partially open mitosis of Aspergillus, whereas nucleolar accumulation of Kap121 and Kap123 homologues suggests a role in nucleolar disassembly. This work provides new insight into the roles of nuclear transporters and opens an avenue for future studies of the molecular mechanisms of transport among nuclei within a common cytoplasm, using A. nidulans as a model organism.     

Characterization of Proteins of Nuclear Envelopes Prepared from Mung Bean Hypocotyls

The polypeptide composition of nuclear envelopes prepared fromhypocotyls of mung bean (Vigna radiata) was investigated. Thetissue was homogenized in the presence of Triton X-100 and nucleiwere isolated by differential and discontinuous Percoll gradientcentrifugation. The nuclei were subjected to sonication in 2M KC1 or 50 mM lithium diiodosalicylate and then the nuclearenvelopes were collected by centrifugation. Proteins in theenvelope fraction were analyzed by sodium dodecylsulfate-polyacrylamidegel electrophoresis and blotting techniques. When the envelopefraction was incubated with [-³²P]ATP, 10 to 15 polypeptideswere labeled and the intensity of labeling of some of thesepolypeptides was enhanced by the addition of calcium ions. Theresults suggest the presence of a protein-phosphorylation systemin nuclear envelopes. Three polypeptides of 100, 42, and 40kDa stained blue with the cationic carbocyanine dye "Stains-all",and they were labeled with ⁴⁵Ca²⁺ on a transfer membrane. Thelectin concanavalin A recognized glycoproteins that migratedas polypeptides of 50, 49, 47, 43, 35 and 32 kDa, respectively.Of these polypeptides the two larger ones were prominent andwere solubilized by treatment of the envelope fraction withKCl at 2 M but not at less than 100 mM. These results suggestthat the mung bean nuclear envelope contains some calcium-bindingproteins and glycoproteins. These newly identified proteinsmay become useful as characteristic markers of the nuclear envelope. (Received July 16, 1993; Accepted December 15, 1993)     

Nuclear Chromatin Structure in Relation to Cell Differentiation and Cell Activation in the Cap and Quiescent Centre of Zea mays L.

Barlow, P. W. 1985. Nuclear chromatin structure in relationto cell differentiation and cell activation in the cap and quiescentcentre of Zea mays L —J. exp. Bot. 36: 1492–1503.Nuclear chromatin structure has been analysed by electron microscopyof thin sections of cells in four zones of the root cap—meristem,central, slime-secreting and outermost cells—and alsoin the quiescent centre of the root before and after decapping.The chromatin pattern has been related to the DNA and RNA syntheticactivity of the nuclei. During cap cell maturation there wasa progressive condensation of the chromatin and this was accompaniedby some reduction of RNA synthesis. The degree of condensationwas estimated from the area and number of pieces of electrondense chromatin which increased and decreased, respectively,during cap maturation. The volume fraction of condensed chromatinwas also estimated but, in the cap, was not found to be a goodindicator of nuclear activity. The outermost cells of the capshowed the greatest degree of chromatin condensation but werestill active in RNA synthesis. Microdensitometry of their nuclearDNA contents gave an indication of loss of DNA in some of thenuclei. Decapping activated DNA and RNA synthesis in the quiescentcentre and also stimulated a decondensation of chromatin: thenumber of condensed pieces of chromatin increased, and theirsize and volume fraction both decreased 4 h after decapping.The number of pores per unit length of nuclear envelope profilewas also estimated. In the cap this number increased duringcap maturation; in the activated quiescent centre the numberremained constant except for a small rise 4 h after decapping Key words: Zea mays, chromatin, root cap, quiescent centre     

Nuclear transplantation in bovine embryo: fine structural and autoradiographic studies

Nucleolar fine structure, "blebbing" activity of nuclear envelope, and activation of heterogeneous nuclear RNA (hnRNA) synthesis were studied in bovine reconstructed embryos obtained by electrofusion of a single eight-cell blastomere with an enucleated oocyte. Developmental progress of nucleolar fine structure and hnRNA synthesis are arrested during three cell cycles following fusion. The activation of both appears during the eight-cell stage of the reconstructed embryo, after the same number of cell cycles after fusion as in nonmanipulated bovine embryos after fertilization. "Blebbing" activity of nuclear envelope, which is already absent in original blastomeres, reappears after fusion and continues for the next two cell cycles. From the present results, it can be concluded that the donor nuclei are arrested after fusion in morphology and function. Their reactivation corresponds to the developmental pattern typical for normal bovine embryos.     

Ultrastructural studies of H-1 parvovirus replication : III. Intracellular localization of viral antigens with immunocytochrome c

Immunoelectron microscopy with cytochrome c conjugated anti-H-1 IgG was used to localize antigens of the parvovirus H-1 within synchronized human NB cells. Since glutaraldehyde destroyed H-1 antigenicity, a fixative containing formaldehyde was developed which preserved both cellular ultrastructure and antigenic function. The earliest H-1 specific staining occurred on the heterochromatin bordering the nuclear envelope at 8 h post infection (p.i.). At 10 h p.i., labeling was found on the chromatin associated with the nucleolar surface and tufts of heterochromatin distributed throughout the nucleoplasm. Except for this H-1 labeling, the chromatin appeared indistinguishable from that of uninfected cells. By 12 h p.i., however, coinciding with the abrupt rise in synthesis of H-1 hemagglutinin and infectious virus, H-1 labeled intranuclear chromatin had condensed and migrated toward the nuclear membrane. Also, trabeculae of intranuclear chromatin were tagged with anti-H-1 conjugate as contraction of nucleolar chromatin and disintegration of nucleolar ultrastructure began. Condensation of the nucleolar associated chromatin and nuclear heterochromatin appeared complete by 18–36 h p.i. when thick zones of this H-1 labeled material were observed at the nucleolar and nuclear periphery. Our results indicate that the binding of unassembled H-1 proteins to specific regions of chromatin is associated with their condensation and margination, resulting in early nucleolar destruction and subsequent nuclear damage during H-1 infection.     

Nuclear envelope transport capacity and the cell cycle in yeast (Saccharomyces cerevisiae).

Changes in the nuclear envelope transport capacity, as measured by the number of nuclear pore complexes/unit nuclear volume/cell, were followed during the Saccharomyces cerevisiae cell cycle using data obtained by freeze-fracture electron microscopy. Pore number per unit nuclear volume decreased sharply in early G0, remained steady from mid-GO through S to G2, and showed a further slight decrease at M and G1. These periods of decline apparently resulted from nuclear enlargement without sufficient formation of new nuclear pore complexes to maintain the pore number to nuclear volume ratio. However, marked nuclear pore formation did accompany both increases in nuclear volume. The significance of these changes in relation to other events in the cell cycle is discussed. The validity of using nuclear pore number/unit nuclear volume and other pore number data as indices of nuclear envelope transport capacity and cell activity is critically examined.     

Vesicle trafficking maintains nuclear shape in Saccharomyces cerevisiae during membrane proliferation

The parameters that control nuclear size and shape are poorly understood. In yeast, unregulated membrane proliferation, caused by deletion of the phospholipid biosynthesis inhibitor SPO7, leads to a single nuclear envelope "flare" that protrudes into the cytoplasm. This flare is always associated with the asymmetrically localized nucleolus, which suggests that the site of membrane expansion is spatially confined by an unknown mechanism. Here we show that in spo7Δ cells, mutations in vesicle-trafficking genes lead to multiple flares around the entire nucleus. These mutations also alter the distribution of small nucleolar RNA-associated nucleolar proteins independently of their effect on nuclear shape. Both single- and multi-flared nuclei have increased nuclear envelope surface area, yet they maintain the same nuclear/cell volume ratio as wild-type cells. These data suggest that, upon membrane expansion, the spatial confinement of the single nuclear flare is dependent on vesicle trafficking. Moreover, flares may facilitate maintenance of a constant nuclear/cell volume ratio in the face of altered membrane proliferation.     

Changes in Nuclear DNA Content of Endosperm Cells During Grain Development in Rice (Oryza sativa)

Morphological, cytological and quantitative DNA changes associatedwith endosperm development in rice caryopsis were investigated.Following a brief free-nuclear phase, the endosperm became cellularby the 4th d after anthesis. While the mean length and breadthof grain attained maximum values at about 10, d after anthesis,f. wt of the whole grain, and of the endosperm separately, continuedto increase until about 16 d after anthesis. Cell divisionsin the endosperm continued until 10 d and following stabilizationof the cell number, the nuclei attained irregular shapes. Thesize of the nuclei and nuclei and the amount of nuclear DNAvaried considerably during endosperm development. The endospermnuclei did not retain the expected 3C–6C DNA level afterthe first few rounds of division and nuclei having more than30C DNA were frequent 8 d past anthesis. The highest C valuerecorded was 74C in a 16-d-old endosperm cell. Oryza sativa, rice, caryopsis, endosperm, cell number, nuclear area, nuclear DNA content, endoreduplication