Somatic nuclear division in the sporidia of Ustilago violacea. III. Ultrastructural observations.

The paper provides detailed ultrastructural observations on nuclear division in the smut fungus Ustilago violacea and is based on previous light-microscopic work outlining the division in living and stained cells. The division as in many other Basidiomycetes is not intranuclear, but occurs within a partially disrupted membrane. The division takes place after migration of most of the nucleus into the bud cell, after limited breakdown of the nuclear membrane, and after the formation of a spindle between two spindle-pole bodies (SPB). The remaining part of the nucleus containing the nucleolus is left behind in the parent cell and degenerates there. The SPB, as in other Basidiomycetes, is a dome-shaped relatively structureless body, quite distinct from the flat plaques of many Ascomycetes and the elaborat centrioles of Phycomycetes. The SPB divides shortly before migration into the daughter cell and invariably is located at the apex of the migrating nucleus. Nuclear division is completed when the two masses of chromatin clustered about each of the SPB's are separated as the spindle elongates. One daughter neculeus reforms in the bud and the other is reformed in the mother cell. Cells fixed and stained by conventional light-microscopic methods were examined in the light of the electron-microscopic observations to determine whether these procedures induce artefacts. Aceto-orcein and Giemsa when used cold were found to produce relatively artefact-free preparations. However, previous results in which the cells were warmed gently in these stains are now seen to contain artefacts in the form of contracted chromatinic granules often arranged in chains. These artefacts may provide useful information but clearly they must be interpreted cautiously until the nature of the changes induced by heating are known.     

CYTOLOGY OF HELMINTHOSPORIUM TURCICUM AND ITS ASCIGEROUS STAGE,TRICHOMETASPHAERIA TURCICA

Knox- Davies , P. S., and J. G. Dickson . (U. Wisconsin, Madison.) Cytology of Helmintho sporium turcicum and its ascigerous stage, Trichometasphaeria turcica . Amer. Jour. Bot. 47(5) : 328—339. Illus. 1960.–The cells of the vegetative hyphae were generally multinucleate. Interphase nuclei resembled those of higher organisms, with a matrix of thread-like chromatin material surrounding a spherical nucleolus. “Beaked” nuclei frequently associated with anastomosing hyphae were interpreted as migrating nuclei. Nuclear division in the vegetative hyphae was rapid. Various division stages were distinguished but it was difficult to make accurate chromosome counts. The nucleoli were discarded at prophase or prometaphase and were reorganized in daughter nuclei at telophase. An outstanding feature of nuclear division was that all the nuclei in a cell divided simultaneously. Conidiophores and conidia were occasionally joined by wide cytoplasmic connections. They were multinucleate throughout their development. Mechanisms therefore exist for the perpetuation of heterokaryons through the conidium. Ascus development was studied in a hybrid between a dark and an albino isolate. Crozier formation was typical and nuclear fusion occurred in the young ascus. Four nuclear divisions were completed in the ascus before there was evidence of ascospore delimitation. Further nuclear division took place in the ascospores whose cells were multinucleate. The occurrence of less than 8 ascospores in an ascus appeared to follow degeneration of nuclei rather than the incorporation of a number of division-Ill nuclei in a single ascopore. Chromosome counts and irregularities in the appearance and behavior of nuclei and chromosomes in the asci indicate that aneuploidy occurs in Trichometasphaeria turcica. It is suggested that aneuploidy is a common phenomenon in the conidial stage of the fungus H. turcicum, and possibly also in other imperfect fungi.     

VEGETATIVE NUCLEAR DIVISION IN NEUROSPORA

A re-examination of the mode of vegetative nuclear division in Neurospora crassa was facilitated by the availability of the mutant “clock” which produces definite growth bands. Since the growth rhythm is correlated with nuclear divisions, stained mycelial mats of this mutant prepared at intervals from the beginning of a growth period provided a sequence of stages of division. In a 28-hour period the following broad features of nuclear behavior were observed: In the early part of the period during rapid mycelial growth, dividing elongated nuclei predominated. At the end of the period the mycelium contained mostly rounded resting nuclei. In the middle of a growth period nuclear forms of various degrees of annularity occurred along with elongated and rounded nuclei. Elongated and rounded nuclei completed division cycles without change in form, although the corresponding stages of the two types were similar. Elongated nuclei assumed a spiral form at the beginning of division. As division proceeded, relaxation of the nuclear gyres was accompanied by a visible duplication of the chromatin thread and the appearance of chromomere-like bodies on the daughter threads. One of the chromomere-like bodies became displaced and was interpreted to be a chromosome or a segment of a chromosome that acts as a mitotic center. All the chromosomes were found to be interconnected and to act as a unit throughout the division cycle. Only after the separation of the daughter chromatin threads could seven chromosomes be counted. Electron microscopic studies complemented the observations with the light microscope. On the basis of the evidence it was concluded that the vegetative nuclear division in Neurospora differs from the classical mitotic pattern in the following respects: (1) absence of visible centrioles, (2) the presence of interconnected chromosomes, (3) the comparatively late appearance of countable chromosomes, and (4) the frequent presence of interzonal connections between separating chromatin threads.     

A NOVEL PATTERN OF APICAL CELL POLYPLOIDY,SEQUENTIAL POLYPLOIDY REDUCTION AND INTERCELLULAR NUCLEAR TRANSFER IN THE RED ALGA POLYSIPHONIA

Nearly a century ago, Rosenvinge published a now-classic paper reporting nuclear transfer between cells of Polysiphonia during secondary pit connection (SPC) formation. While reinvestigating this phenomenon, we discovered that the uninucleate apical cell, which is the progenitor of all cells in the plant, has many times (ca. 64–128 ×) the level of nuclear DNA characteristic of nuclei of gametes or mature pericentral cells. Via a regular sequence of cell divisions, the polyploid apical cell gives rise to tiers of cells, each composed of a number of pericentral cells which surround a single central cell. A large proportion of the nuclear divisions are not accompanied by DNA replication. Thus, as the number of nuclei within elongating pericentral cells increases, the DNA level of nuclei in these cells “cascades” down to the DNA level expected for the particular life history generation (i.e., gametophyte or tetrasporophyte). In mature pericentral cells, the number of nuclei is proportional to the volume of the cell. The pattern of nuclear division, reduction in ploidy level and the timing of intercellular nuclear transfer via SPC formation is regular and characteristic of a species. Nuclei transferred from one cell to an adjacent cell participate in the further nuclear divisions of the recipient cell. The degree of polyploidy in apical cells may determine the number of cells in a “determinant” branch or even the number of cells in “indeterminant” axes. In addition, the highly polyploid state of the germinating spore and its pattern of development may provide for the rapid initial growth so characteristic of this taxon.     

Morphological apoptotic characteristics of the post-meiotic micronuclei in Paramecium caudatum

In a previous study, the apoptotic degeneration of meiotic products outside the paroral region of Paramecium caudatum was indirectly demonstrated by means of “apofluor” staining. In this experiment, conjugating pairs and exconjugants of P. caudatum were stained with either “apofluor” or carbol fuchsin or both to find some direct evidence to demonstrate the apoptotic characteristics of this process. As a result, asynchronous meiotic nuclear degeneration was observed. Furthermore, a number of additional meiotic nuclei were found. Disintegrating/dividing meiotic nuclei outside the paroral region were observed, which might be the origin of these additional meiotic nuclei. Condensed chromatin and disintegrated chromatin attached to the nuclear membrane were also observed in degenerating nuclei, which are the typical morphological characteristics of apoptosis. Comparison of the cells stained by the above two methods indicated that “apofluor”-stained meiotic nuclei could not be detected by carbol fuchsin in some cells, which suggests a time lag between meiotic nuclear DNA degradation and their eventual disappearance. In this study, some direct evidence was found to show that the meiotic nuclear degeneration in P. caudatum is of apoptotic nature, which further confirmed our previous study (Yang et al. 2007) and indicated that morphological apoptotic characteristics discovered in multicellular organisms do exist in unicellular eukaryotic ciliate protozoa.     

禾谷镰孢菌Fusarium graminearum Schwabe分生孢子萌发过程中的核相变化及有丝分裂过程观察

通过Giemsa染色观察禾谷镰孢菌Fusarium graminearum分生孢子萌发过程中的核相变化及有丝分裂过程。观察表明,分生孢子细胞为单核,细胞核在分生孢子细胞内分裂后进入芽管,在芽管内进行多次分裂,使芽管内细胞核数目不断变化。禾谷镰孢菌有丝分裂过程可以分为4个时期,前期染色体逐渐浓缩变短,中期染色体清晰可见,后期染色单体发生分离并向相反的两极移动,末期形成新的子核。有丝分裂过程中染色体的分离同步或不同步,不同步分离中的滞后染色体形成后期桥的现象更为普遍。     

A cytological study of Nematospora coryli pegl

A study is made of nuclear division in Nematospora coryli, a pathogenic yeast. The DNA of cells (grown on a V-8-medium) was stained with leuco-basic fuchsin (Feulgen test) at pH 3.5. After budding has started the rounded nucleus elongates and some differentiation into chromosomes is perceptible. A few slides suggest the number of chromosomes being 4. After some time the nucleus appears to have duplicated. This nucleus migrates towards the isthmus between mother cell and bud. In the isthmus, or just in front of it, the two daughter nuclei proceed to disjoin and move along each other to opposite directions. One daughter nucleus moves into the bud; the other one migrates back into the mother cell.Samples from synchronously growing cultures show that a fraction of the young yeast cells are destined to grow out to asci, in which after about 6 hours the presence of bivalents seems highly probable. The succeeding nuclear divisions take place in the same way as described for the vegetative cells and stop when the majority of the enlarged asci contain 8 nuclei.Problems of haploidy and diploidy are discussed.Small, densely stained bodies are observed in certain vegetative and some meiotic stages. As these bodies contain DNA, their function and possible homology with centrioles is discussed.     

The ultrastructure of mitosis during sporangiogenesis inWoronina pythii (Plasmodiophoromycetes)

Summary Mitotic divisions during sporangiogenous plasmodial cleavage inWoronina pythii were studied with transmission electron microscopy. We conclude that these nuclear divisions (e.g., transitional nuclear division, and sporangial mitoses) share basic similarities with the cruciform nuclear divisions inW. pythii and other plasmo-diophoraceous taxa. The major distinction appeared to be the absence of nucleoli during sporangial mitosis and the presence of nucleoli during cruciform nuclear division. The similarities were especially evident with regard to nuclear envelope breakdown and reformation. The mitotic divisions during formation of sporangia were centric, and closed with polar fenestrae, and characterized by the formation of intranuclear membranous vesicles. During metaphase, anaphase, and telophase, these vesicles appeard to bleb from the inner membrane of the original nuclear envelope and appeared to coalesce on the surface of the separating chromatin masses. By late telophase, the formation of new daughter nuclear envelopes was complete, and original nuclear envelope was fragmented. New observation pertinent to the mechanisms of mitosis in thePlasmodiophoromycetes include a evidence for the incorporation of membrane fragments of the original nuclear envelope into new daughter nuclear envelopes, and b the change in orientation of paired centrioles during sporangial mitosis.     

Staining Mast Cells for Morphometric Evaluation on an Image Analysis System

Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.     

Is actin involved in the nuclear division in Amoeba proteus?

During semi-open mitosis of Amoeba proteus the nuclear envelope is not dispersed and nucleus divides by fission. The presence of actin layer close to nuclear envelope was demonstrated in interphase and telophase nuclei of that amoeba stained with rhodamine labelled phalloidin. In telophase, an accumulation of actin arises in the space between the future daughter nuclei. It appears to be comparable with the contractile ring of dividing cells. This suggests that actin associated with the nuclear envelope of Amoeba proteus may be involved in final separation of the daughter nuclei, forming a constriction ring at the middle of dividing nucleus.     

Subcompartment sugar residues of gastric surface mucous cells studied with labeled lectins.

We examined the intracellular localization of sugar residues of the rat gastric surface mucous cells in relation to the functional polarity of the cell organellae using preembedding method with several lectins. In the surface mucous cells, the nuclear envelope and rough endoplasmic reticulum (rER) and cis cisternae of the Golgi stacks were intensely stained with Maclura pomifera (MPA), which is specific to alpha-Gal and GalNAc residues. In the Golgi apparatus, one or two cis side cisternae were stained with MPA and Dolichos biflorus (DBA) which is specific to terminal alpha-N-acetylgalactosamine residues, while the intermediate lamellae were intensely labeled with Arachis hypogaea (PNA) which is specific to Gal beta 1,3 GalNAc. Cisternae of the trans Golgi region were also stained with MPA, Ricinus communis I (RCA I) which is specific to beta-Gal and Limax flavus (LFA) which is specific to alpha-NeuAc. Immature mucous granules which are contiguous with the trans Golgi lamellae were weakly stained with RCA I, while LFA stained both immature and mature granules. The differences between each lectin's reactivity in the rough endoplasmic reticulum, in each compartment of the Golgi lamellae and in the secretory granules suggest that there are compositional and structural differences between the glycoconjugates in the respective cell organellae, reflecting the various processes of glycosylation in the gastric surface mucous cells.     

Genetic and morphological characterization of a Fusarium verticillioides conidiation mutant

Enteroblastic phialidic conidiation by the corn pathogen Fusarium verticillioides (teleomorph Gibberella moniliformis) produces abundant, mostly single-celled microconidia in distinctive long chains. Because conidia might be critical for establishing in planta associations, we characterized a spontaneous F. verticillioides conidiation mutant in which phialides were incapable of enteroblastic conidiogenesis. Instead of producing a conidium, the phialide apex developed a determinate, slightly undulating, germ tube-like outgrowth, in which nuclei rarely were seen. Electron microscopy showed that the apical outgrowth possessed a thick, rough, highly fibrillar outer wall layer that was continuous with the thinner and smoother outer wall layer of the phialide. Both the inner wall layer and plasma membrane also were continuous between the apical outgrowth and phialide. The apical neck region of mutant phialides lacked both a thickened inner wall layer and a wall-building zone, which were critical for conidium initial formation. No indication of septum formation or separation of the apical outgrowth from mutant phialides was observed. These aberrations suggested the apical outgrowth was not a functional conidium of altered morphology. The mutation did not prevent perithecium development and ascosporogenesis. Genetic analyses indicated that a single locus, designated FPH1 (frustrated phialide), was responsible for the mutation. The conidiogenesis mutants were recovered only during certain sexual crosses involving wild-type conidiating parents, and then only in some perithecia, suggesting that mutation of FPH1 might be meiotically induced, perhaps due to mispairing between homologous chromosomes and deletion of the gene from a chromosome.     

Ultrastructural Cytochemistry of Secretory Granules of Esophageal Glands of Meloidogyne incognita

Ultrastructural cytochemical tests for several enzymes, proteins, carbohydrates, and nucleic acids were conducted on secretory granules o£ dorsal and subventral esophageal glands of preparasitic second-stage juveniles and the dorsal gland of adult females of Meloidogyne incognita. Secretory granules in the subventral glands of juveniles stained positive for acid phosphatase. Peroxidase, DNase, RNase, cellulase, and nucleic acids were not detected in these granules. Secretory granules in the dorsal gland of adult females stained positive for peroxidase (pH 7.6) in < 50% of the tests, Acid phosphatase, β-glucuronidase, DNase, RNase, polyphenoloxidase, cellulase, and carbohydrates were not detected in dorsal gland granules in adult females. Positive staining with cobalt thiocyanate, a stain for amino groups of basic proteins, occurred in secretory granules in the dorsal gland, ribosomes, and chromatin in adult females. Ribosomes, nuclei, and secretory granules of the dorsal gland of adult females intensely stained when incubated in three reagents specific for nucleic acid.     

Vegetative nuclear structures and their mode of division inCyathus species

Vegetative nuclear division in the homokaryotic and dikaryotic hyphae ofCyathus olla Brodie,C. setosus Brodie andC. bulleri Brodie was investigated. In the homokaryotic hyphae a nucleolus develops within a globular condensed nucleus consisting of a folded up filament. As the nucleolus increases in size, the nucleus unfolds and can assume a ring, horseshoe or filament configuration. The filament duplicates and (usually when unwound from the nucleolus) divides longitudinally. Occasionally, strand separation occurs while the filament is wrapped in the form of a ring around the nucleolus. The daughter nuclei may condense before the next division. In the dikaryotic hyphae the same nuclear cycle occurs as in the homokaryons except that an extra nuclear condensation to the globular form can occur in both the clamp and tube nuclei. The division of these two nuclei is not always synchronous and, moreover, the stage of karyokinesis of the clamp nucleus is not closely synchronized with the formation of the clamp connection. A deeply stained granule is associated with the nucleus. Some granules can be observed to be connected to the nucleus by a faintly Feulgen positive thread-like structure but other granules are sessile. The granule or centriole-like body is thought to direct the nuclear unfolding process. It may divide prior to, or after nuclear division.     

The histological localization of DNA in rabbit medullary neurons

The nuclei of unfixed isolated rabbit neurons cleared on incubation with DNAse (10 mg/ml), but not RNAse (10 mg/ml). The nuclei stained for DNA with eight chromosomal or nuclear stains more intensely than the cytoplasm, and less intensely after treatment with DNAse (10 mg/ml). On the other hand, when the whole tissue was embedded and sectioned, DNA did not appear to be stained in the nucleus; the nucleolus and the cytoplasm were more heavily stained than the nucleoplasm. Possible explanations for this apparent anomaly are considered. It was concluded that DNA diffused out of the nucleus during embedding and sectioning, and that the colouration of the nucleolus and cytoplasm with the eight staining systems used was due to other nucleotides present.     

Characterization of an Aspergillus nidulans mutant with abnormal distribution of nuclei in hyphae, metulae, phialides and conidia

The V10 deteriorated variant of Aspergillus nidulans has hyphae, metulae, phialides and conidia with abnormal nuclear distributions. The alterations observed were: increase in the number of nuclei in hyphae, metulae and phialides, presence of anucleate, uninucleate and multinucleate conidia, abnormal vegetative growth and defective conidiation. When 0.5 M NaCl was added to the medium, an increase in the number of conidia was observed but their morphology and number of nuclei were not modified. The gene responsible for these alterations was named anuA1. The anuA1 gene is located on linkage group VII and is possibly involved in nuclear migration to hyphae, metulae, phialides and conidia.     

Development of the Mycetozoan Echinosteliopsis oligospora*

SYNOPSIS. The mycetozoan genus Echinosteliopsis, resembling the myxomycete Echinostelium in some of its features, is described. The single species, E. oligospora Reinhardt & Olive, forms small sporocarps which consist of a basal disk, stalk and a sporangium with only 1–8 spores. Spores form progressively, not simultaneously, by segmentation. The spores germinate to release non-flagellate amebae which, in liquid, assume a characteristic broad, fan shape. Each ameba has one or more nuclei. The nucleus is distinctive because of refractile, globular to elongate peripheral bodies which cytochemical tests indicate to be primarily RNA. At the time of nuclear division the characteristic RNA bodies disappear and, as observed with the phase microscope and in stained preparations, optically dense material accumulates in the middle area of the nucleus. Threads, either a spindle or actual chromatin, can be seen attached to the nuclear membrane. The threads separate to opposite poles as the nucleus elongates. During this division process the nuclear membrane apparently remains intact. Synchronous binucleate divisions, as well as a tripolar nuclear division, have been observed. Uninucleate and synchronous binucleate divisions may or may not be followed by cytokinesis. The absence of cell division after nuclear division leads to the production of cells with varying numbers of nuclei. Nuclear divisions in early sporangial stages and in spores have not been observed. The spores are uni- to multinucleate. In 8-spored sporangia and in most 4-spored sporangia there is a characteristic small “stalk spore” at the apex of the stalk. The stalk spore germinates slowly, if at all, but the larger spores germinate readily. No evidence of a sexual process has been found.     

Neuronal types and their percent distribution within the magnocellular nuclei of the human basal forebrain

Three neuronal types constituting the magnocellular nuclei of the human basal forebrain have been differentiated with the aid of preparations stained for both Nissl material and pigment deposits: type I = large multipolar neurons contain loosely packed and faintly stained lipofuscin granules occupying a large portion of the cell body; type II = large spindle-shaped neurons reveal a densely packed accumulation of coarse and intensely stained lipofuscin granules, and type III = small nerve cells, scattered among these large neuronal components, with only a small number of faintly stained lipofuscin granules. The determination of the projection areas of the somata of the three neuronal types has led to a distribution pattern with three peaks. The ratio of the nerve cell types has been evaluated: 73.6% type I; 8.6% type II, and 17.8% type III neurons.     

Formation of a micronuclei-like structure induced by colchicine treatment in Saccharomyces cerevisiae

Minute nuclei named “smaller nuclei” were generated when the cells of Saccharomyces cerevisiae were treated with colchicine. The formation of “smaller nuclei” seemed to be related to nuclear division because those nuclei were only produced under conditions suitable for nuclear division. The fact that the average DNA content of “smaller nuclei” was almost one tenth of that of the isolated normal diploid nuclei showed that the “smaller nuclei” are not condensed nuclei but aneuploid nuclei like micronuclei in animal cells. It appeared therefore likely that a micronuclei-like structure could be produced by colchicine treatment in S. cerevisiae.