Oxylipins and ascospore morphology in the ascomycetous yeast genus Dipodascus

Immunofluorescence microscopy was used to assess members of the yeast genus Dipodascus for the presence of 3-hydroxy oxylipins. Fluorescence was associated with the aggregating ascospores in all species tested, thus suggesting the association of 3-hydroxy oxylipins with these cells, especially the surrounding slime sheaths. An ultrastructural study of the ascospores revealed sheaths with indentations, probably caused by the close packing of the ascospores to form clusters. In addition, an increase in the neutral and glycolipid fractions as well as a decrease in the phospholipid fraction during ascosporogenesis in D. ambrosiae was found. This revised version was published online in June 2006 with corrections to the Cover Date.     

Bioprospecting for novel oxylipins in fungi: The presence of 3-hydroxy oxylipins in Pilobolus

As previously found in various members of the Mucorales, 3-hydroxy oxylipins in Mucor genevensis are associated with the sporangia, i.e. mainly the columella structure and between aggregating sporangiospores. To determine if this phenomenon is also true in distantly related members, the mucoralean fungus Pilobolus was examined. This fungus is characterized by relatively large sub sporangial-columella structures which actively eject sporangia in a sticky liquid for attachment onto herbage surrounding its growth medium – in this case horse dung. Strikingly, this fungus produced a novel oxylipin i.e. a 3-hydroxy monounsaturated fatty acid, possibly a nonenoic acid, which is mainly associated with the sub sporangial-columella structure and aggregating sporangiospores. The specificity of the antibody against 3-hydroxy oxylipins used in immunofluorescence mapping of the mucoralean fungi, was further confirmed in the yeast, Saccharomycopsis malanga which produces 3-hydroxy palmitate in crystal form. These crystals occur between aggregating yeast cells. On the basis of the available data, we hypothesize that 3-hydroxy oxylipins probably function as adhesives, attaching fungal cells to each other or to other surfaces through entropic based hydrophobic forces and/or hydrogen bonds.     

Mapping 3-hydroxy oxylipins on ascospores of <Emphasis Type="Italic">Eremothecium sinecaudum</Emphasis>

3-Hydroxy oxylipins were uncovered on ascospores of Eremothecium sinecaudum using immunofluorescence microscopy. This was confirmed by gas chromatography mass spectrometry. These oxylipins were observed only on ascospore parts characterised by nano-scale surface ornamentations simulating a corkscrew as demonstrated by scanning electron microscopy. Conventional ascospore staining further confirms its hydrophobic nature. Using confocal laser scanning microscopy we found that the corkscrew part with spiky tip of needle-shaped ascospores may play a role in rupturing the ascus in order to affect its release. Through oxylipin inhibition studies we hypothesise a possible role for 3-hydroxy oxylipins in facilitating the rupturing process.     

The cytoskeleton in the unique cell reproduction by conidiogenesis of the long-neck yeast Fellomyces (Sterigmatomyces) fuzhouensis

Summary. The morphology of conidiogenesis and associated changes in microtubules, actin distribution and ultrastructure were studied in the basidiomycetous yeast Fellomyces fuzhouensis by phase-contrast, fluorescence, and electron microscopy. The interphase cell showed a central nucleus with randomly distributed bundles of microtubules and actin, and actin patches in the cortex. The conidiogenous mother cell developed a slender projection, or stalk, that contained cytoplasmic microtubules and actin cables stretched parallel to the longitudinal axis and actin patches accumulated in the tip. The conidium was produced on this stalk. It contained dispersed cytoplasmic microtubules, actin cables, and patches concentrated in the cortex. Before mitosis, the nucleus migrated through the stalk into the conidium and cytoplasmic microtubules were replaced by a spindle. Mitosis started in the conidium, and one daughter nucleus then returned to the mother via an eccentrically elongated spindle. The cytoplasmic microtubules reappeared after mitosis. A strong fluorescence indicating accumulated actin appeared at the base of the conidium, where the cytoplasm cleaved eccentrically. Actin patches then moved from the stalk together with the retracting cytoplasm to the mother and conidium. No septum was detected in the long neck by electron microscopy, only a small amount of fine “wall material” between the conidium and mother cell. Both cells developed a new wall layer, separating them from the empty neck. The mature conidium disconnected from the empty neck at the end-break, which remained on the mother as a tubular outgrowth. Asexual reproduction by conidiogenesis in the long-neck yeast F. fuzhouensis has unique features distinguishing it from known asexual forms of reproduction in the budding and fission yeasts. Fellomyces fuzhouensis develops a unique long and narrow neck during conidiogenesis, through which the nucleus must migrate into the conidium for eccentric mitosis. This is followed by eccentric cytokinesis. We found neither an actin cytokinetic ring nor a septum in the long neck, from which cytoplasm retracted back to mother cell after cytokinesis. Both the conidium and mother were separated from the empty neck by the development of a new lateral wall (initiated as a wall plug). The cytoskeleton is clearly involved in all these processes. Correspondence and reprints: Department of Biology, Faculty of Medicine, Masaryk University, Tomešova 12, 602 00 Brno, Czech Republic.     

Ascospore aggregation and oxylipin distribution in the yeast Dipodascopsis tothii

Upon cultivation of the yeast Dipodascopsis tothii in its sexual stage, small ascospores are released individually from the ascus tip, which then assemble in sheathed cluster balls. In contrast to Dipodascopsis uninucleata, this yeast produced smooth bean shaped ascospores with sheath-like appendages that assemble in a disordered sheathed ball of ascospores outside the ascus. Strikingly, upon release, the ascus tip contained 3-hydroxy oxylipins, while the released ascospore clusters contained little or no 3-hydroxy oxylipins as indicated by immunofluorescence microscopy. In D. uninucleata, these oxylipins are concentrated on the spore surface and interspore matrix, but not on the ascus tip.     

Variation in functional ascospore parts in the ascomycetous yeast Dipodascopsis uninucleata

A variation in functional ascospore morphology was detected using electron microscopy (EM) in two varieties of the yeast Dipodascopsis uninucleata, i.e., D. uninucleata var. uninucleata and D. uninucleata var. wickerhamii. It was found that the latter produces ascospores characterized by the absence of small surface hooks which have been implicated in the release and re-assembly of ascospores in D. uninucleata var. uninucleata. These varieties are closely related on the basis of their mode of sexual reproduction, ascospore morphology as observed under the light microscope, physiological characteristics as well as the extent of divergence in the variable D1/D2 domain of the large subunit 26S ribosomal DNA.     

Bioprospecting for novel hydroxyoxylipins in fungi: presence of 3-hydroxy palmitic acid in Saccharomycopsis malanga

Electron microscopy studies indicated that the major oxylipin 3-hydroxy palmitic acid (16:0) was associated with aggregating vegetative cells and formed a web-like structure around these cells. Cross sections through this structure showed a hydrophilic outer layer and a more hydrophobic inner layer suggesting that the web-like structure is in fact tube-like micelles. This information sheds more light on the role of these hydroxyoxylipins in fungi.     

Acetylsalicylic acid as antifungal in <Emphasis Type="Italic">Eremothecium</Emphasis> and other yeasts

Interesting distribution patterns of acetylsalicylic acid (ASA, aspirin) sensitive 3-hydroxy (OH) oxylipins were previously reported in some representatives of the yeast genus Eremothecium—an important group of plant pathogens. Using immunofluorescence microscopy and 3-OH oxylipin specific antibodies in this study, we were able to map the presence of these compounds also in other Eremothecium species. In Eremothecium cymbalariae, these oxylipins were found to cover mostly the spiky tips of narrowly triangular ascospores while in Eremothecium gossypii, oxylipins covered the whole spindle-shaped ascospore with terminal appendages. The presence of these oxylipins was confirmed by chemical analysis. When ASA, a 3-OH oxylipin inhibitor, was added to these yeasts in increasing concentrations, the sexual stage was found to be the most sensitive. Our results suggest that 3-OH oxylipins, produced by mitochondria through incomplete β-oxidation, are associated with the development of the sexual stages in both yeasts. Strikingly, preliminary studies on yeast growth suggest that yeasts, characterized by mainly an aerobic respiration rather than a fermentative pathway, are more sensitive to ASA than yeasts characterized by both pathways. These data further support the role of mitochondria in sexual as well as asexual reproduction of yeasts and its role to serve as a target for ASA antifungal action.     

Prevalence and distribution of introns in non-ribosomal protein genes of yeast

Relatively few genes in the yeast Saccharornyces cerevisiae are known to contain intervening sequences. As a group, yeast ribosomal protein genes exhibit a higher prevalence of introns when compared to non-ribosomal protein genes. In an effort to quantify this bias we have estimated the prevalence of intron sequences among non-ribosomal protein genes by assessing the number of prp2-sensitive mRNAs in an in vitro translation assay. These results, combined with an updated survey of the GenBank DNA database, support an estimate of 2.5% for intron-containing non-ribosomal protein genes. Furthermore, our observations reveal an intriguing distinction between the distributions of ribosomal protein and non-ribosomal protein intron lengths, suggestive of distinct, gene class-specific evolutionary pressures.     

Oxylipin covered ascospores of Eremothecium coryli

Eremothecium coryli is known to produce intriguing spindle-shaped ascospores with long and thin whip-like appendages. Here, ultra structural studies using scanning electron microscopy, indicate that these appendages serve to coil around themselves and around ascospores causing spore aggregation. Furthermore, using immunofluorescence confocal laser scanning microscopy it was found that hydrophobic 3-hydroxy oxylipins cover the surfaces of these ascospores. Using gas chromatography–mass spectrometry, only the oxylipin 3-hydroxy 9:1 (a monounsaturated fatty acid containing a hydroxyl group on carbon 3) could be identified. Sequential digital imaging suggests that oxylipin-coated spindle-shaped ascospores are released from enclosed asci probably by protruding through an already disintegrating ascus wall.     

Manganese accumulation in yeast cells

Summary Manganese accumulation was studied by room-temperature electron spin resonance (ESR) spectroscopy inSaccharomyces cerevisiae grown in the presence of increasing amounts of MnSO₄. Mn²⁺ retention was nearly linear in intact cells for fractions related to both low-molecular-mass and macromolecular complexes (free and bound Mn²⁺, respectively). A deviation from linearity was observed in cell extracts between the control value and 0.1 mM Mn²⁺, indicating more efficient accumulation at low Mn²⁺ concentrations. The difference in slopes between the two straight lines describing Mn²⁺ retention at concentrations lower and higher than 0.1 mM, respectively, was quite large for the free Mn²⁺ fraction. Furthermore it was unaffected by subsequent dialyses of the extracts, showing stable retention in the form of low-molecular-mass complexes. In contrast, the slope of the line describing retention of bound Mn²⁺ at concentrations higher than 0.1 mM became less steep after subsequent dialyses of the cell extracts. This result indicates that the macromolecule-bound Mn²⁺ was essentially associated with particulate structures. In contrast to Cu²⁺, Mn²⁺ had no effect on the major enzyme activities involved in oxygen metabolism except for a slight increase of cyanide-resistant Mn-superoxide dismutase activity, due to dialyzable Mn²⁺ complexes.     

Splenic white pulp as a thymus-independent area in the African clawed toad,Xenopus laevis

Summary An indirect immunofluorescence study of the frozen sections of the spleen of an anuran amphibian, Xenopus laevis, showed that lymphocytes bearing a small amount of immunoglobulin (Ig) were localized mostly in the white pulp of non-immunized toads. There were fewer fluorescent cells in the red pulp. In the toads hyperimmunized with human gamma globulin (HGG), cells with strong cytoplasmic fluorescence increased significantly in the outer part of the white pulp. Electron microscopy of spleens from these toads showed that plasma cells at different stages of maturation were abundant in the white pulp, whereas in the red pulp, a smaller number of maturer plasma cells were observed. These results indicate that, in contrast with its mammalian counterpart, the splenic white pulp of this anuran is the site where thymusin-dependent lymphocytes commence blast formation and transformation into plasma cells.     

Microfilaments and microtubules: the news from yeast

New evidence that cortical actin patches and the endocytic machinery share components supports the idea that actin patches are in fact transient membrane coats at the initial stage of endocytosis. Recent studies of actin cables have identified formins as the core of a novel actin-filament-assembling machine. Meanwhile, microtubule-binding proteins have been found in the kinetochore, and factors affecting microtubule dynamic instability have been identified.     

Calcitonin-like cells in the pharynx of the ascidian Styela clava

Summary A small group of granulated endocrine cells have been described in the endostylar region of the pharynx in Styela. These cells are argyrophilic and exhibit calcitonin-like immunofluorescence. Tests with antisera to other peptides all proved negative. Cells from animals exposed to elevated calcium levels showed a degree of degranulation. The possibility that these cells may represent ancestral ultimobranchial C cells is discussed.The authors are very grateful to Julia Polak and Susan Van Noorden, Royal Postgraduate Medical School, London, for the kind gift of all antisera used. Animals were collected by courtesy of Mr. David Houghton and his staff of the Admiralty Marine Trials Station, Portsmouth. This research was carried out during the tenure of SRC grant no. B/RG 82919 to one of us (M.C.T.) — The localization of polypeptide hormones in the pharynx and gut of protochordates     

Development of quasi-multicellular bodies of Treponema denticola

The formation of quasi-multicellular bodies of Treponema denticola was analysed using different electron microscopical methods. These bacteria could develop four different conformations: (i) normal helical forms; (ii) twisted spirochetes, forming plaits; (iii) twisted spirochetes, forming club-like structures; (iv) spherical bodies in different size. Treponemes within spherical bodies, plaits, and clubs proved to be enclosed in a common outer sheath in which the normal arrangement of their axial flagella was lost. The development of the quasi-multicellular bodies starting from the monoforme spirochetes was elucidated and this morphogenetic process is illustrated by a schematic drawing. Factors which might be involved in the induction of the structures are discussed and their possible pathogenetic importance is considered.     

Repair of 8-methoxypsoralen photoinduced cross-links in yeast

Summary The repair of interstrand cross-links induced by 8-methoxypsoralen plus UVA (365 nm) radiation DNA was analyzed in diploid strains of the yeast Saccharomyces cerevisiae. The strains employed were the wild-type D₇ and derivatives homozygous for the rad18-1 or the rad3-12 mutation. Alkaline step-elution and electron microscopy were performed to follow the process of induction and removal of photoinduced crosslinks. In accordance with previous reports, the D₇ rad3-12 strain failed to remove the induced lesions and could not incise cross-links. The strain D₇ rad18-1 was nearly as efficient in the removal of 8-MOP photoadducts after 2 h of post-treatment incubation as the D₇ RAD⁺ wild-type strain. However, as demonstrated by alkaline step-elution and electron microscopic analysis, the first incision step at DNA cross-links was three times more effective in D₇ rad18-1 than in D₇ RAD⁺. This is consistent with the hypothesis that the RAD18 gene product is involved in the filling of gaps resulting from persistent non-informational DNA lesions generated by the endonucleolytic processing of DNA cross-links. Absence of this gene product may lead to extensive strand breakage and decreased recognition of such lesions by structural repair systems.     

Tubulin conformation in microtubule-free cells ofVigna sinensis

Summary The primary leaf, epicotyl, and root cells ofVigna sinensis seedlings grown continuously in a 0.08% colchicine solution, become microtubule-free and polyploid. In meristematic root cells a tubulin transformation is detected 1–3 h after the treatment had begun. Tubulin strands are organized at the positions of the pre-existing microtubules. Frequently, the strands converge on or are organized in the cortical cytoplasmic zone where in normal cells the preprophase microtubule band (PMB) is assembled. In meristematic root cells subjected to a 6–12 h colchicine treatment, the tubulin strands become perinuclear, entering the cortical cytoplasm at regions close to the nucleus. One day after the onset of the treatment, tubulin generally forms a continuous reticulum of interconnected strands in all the organs examined. In most cells this reticulum surrounds the nucleus partly or totally or lies close to it, exhibiting variable configurations in different cells. After prolonged treatments, the organization of the tubulin reticulum changes further. Now this consists of crystal-like structures interconnected by thin strands.On thin sections of fixed tissue the tubulin strands consist of paracrystalline material. The distribution of this material in the affected cells coincides with that of tubulin reticulum visualized by immunofluorescence. In transverse planes each strand exhibits circular subunits arranged close to one another in a hexagonal pattern but in longitudinal ones variable images were observed. The paracrystalline material persists in root cells subjected to an 8-day continuous colchicine treatment. The immunolabeled strands seem to be composed of tubulin-colchicine complexes and not pure tubulin.     

Recombinant human fibrinogen expressed in the yeast Pichia pastoris was assembled and biologically active

Fibrinogen is a large plasma glycoprotein with a molecular mass of 340 kDa that plays a critical role in the final stage of blood coagulation. Human plasma fibrinogen is a dimeric molecule comprising two sets of three different polypeptides (Aα, 66 kDa; Bβ, 55 kDa; γ, 48 kDa). To express recombinant human fibrinogen in the methylotrophic yeast Pichia pastoris, we constructed an expression vector containing three individual fibrinogen chain cDNAs under the control of the mutated AOX2 (mAOX2) promoter. First, P. pastoris GTS115 was transformed with the vector, but the expressed recombinant fibrinogen suffered severe degradation by yeast-derived proteases under conventional nutrient culture conditions. Fibrinogen degradation was prevented by using the protease A-deficient strain SMD1168 as a host strain and regulating the pH of the culture to between 5.5 and 7.0. Western blot analysis revealed that the Aα, Bβ and γ chains of recombinant fibrinogen were assembled and secreted as a complete molecule. The Bβ chain of the recombinant fibrinogen was N-glycosylated but the Aα chain, as in plasma fibrinogen, was not. The γ chains however were heterologous, one being N-glycosylated and the other not. The recombinant fibrinogen was capable of forming a thrombin-induced clot in the presence of factor XIIIa and both the glycosylated and the non-glycosylated γ chains were involved in the formation of cross-linking fibrin. The present study indicates that the recombinant fibrinogen expressed in P. pastoris, although different from plasma fibrinogen in post-translational modification, is correctly assembled and biologically active.     

Mapping the distribution of 3-hydroxylipins in the Mucorales using immunofluorescence microscopy

The distribution of endogenous 3-hydroxylipins (3-OH oxylipins) in representatives of the Mucorales was mapped using immunofluorescence microscopy. Strains of each of the following genera were examined: Absidia, Actinomucor, Cunninghamella, Mortierella (subgenus Micromucor), Mortierella (subgenus Mortierella), Mucor and Rhizomucor. Immunofluorescence microscopy was carried out using an antibody that was raised against 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (3R-HETE), which cross-reacts with other 3-OH oxylipins. Subsequently, the occurrence and distribution of the antibody on the various reproductive stages of each fungus was noted. In Absidia, Actinomucor, Mortierella (subgenus Micromucor), Mucor and Rhizomucor, 3-OH oxylipins were found to be associated with the columellae and/or wall of the sporangium. In the representative of Cunninghamella, the 3-OH oxylipins were associated with the single-spored sporangiola. No 3-OH oxylipins were detected in the strains representing Mortierella (subgenus Mortierella).     

Genetic control of translational fidelity in yeast: molecular cloning and analysis of the allosuppressor gene SAL3

Summary The fidelity of translation in the yeast Saccharomyces cerevisiae is controlled by a number of gene products. We have begun a molecular analysis of such genes and here describe the cloning and analysis of one of these genes, SAL3. Mutations at this locus, and at least four other unlinked loci (designated SAL1-SAL5), increase the efficiency of the tRNA ochre suppressor SUQ5, and are thus termed allosuppressors. We have cloned the SAL3 gene from a yeast genomic library by complementation of a sal3 mutation. Integration of the cloned sequence into the yeast chromosome was used to confirm that the SAL3 gene had been cloned. SAL3 gene is present in a single copy in the yeast genome, is transcribed into a 2.3-kb polyadenylated mRNA and encodes a protein of M_r 80 000. The size of the SAL3 gene product strongly suggests that it is not a ribosomal protein.