Papers by Robert Boulianne
Journal of Basic Microbiology, May 1, 2002
Microbiology, Aug 1, 2000
Fruiting body formation in the basidiomycete Coprinus cinereus is a developmental process that oc... more Fruiting body formation in the basidiomycete Coprinus cinereus is a developmental process that occurs as a response of the mycelium to external stimuli. First, localized, highly branched hyphal structures (knots) are formed as a reaction to nutritional depletion. Hyphal-knot formation is repressed by light ; however, light signals are essential for the development of the hyphal knot into an embryonic fruiting body (primordium) as well as karyogamy, meiosis and fruiting body maturation. The role of the different environmental signals in the initial phases of fruiting body development was analysed. It was observed that two fungal galectins, Cgl1 and Cgl2, are differentially regulated during fruiting body formation. cgl2 expression initiated in early stages of fruiting body development (hyphal knot formation) and was maintained until maturation of the fruiting body, whereas cgl1 was specifically expressed in primordia and mature fruiting bodies. Immunofluorescence and immunoelectron microscopy studies detected galectins within specific fruiting body tissues. They localized in the extracellular matrix and the cell wall but also in membrane-bound bodies in the cytoplasm. Heterologous expression of Cgl2 in Saccharomyces cerevisiae indicated that secretion of this protein occurred independently of the classical secretory pathway.
Biochemistry and Cell Biology, May 1, 1992
We have used high resolution two-dimensional gel electrophoresis to identify and characterize pro... more We have used high resolution two-dimensional gel electrophoresis to identify and characterize proteins that may represent products of genes involved in establishing positional information along the proximal–distal axis of the regenerating forelimb of the newt Notophthalmus viridescens. At least 24 proteins have been found whose synthesis and (or) abundance is increased in proximal (midstylopodial) regenerates relative to midzeugopodial (distal) regenerates at either of two regeneration stages, the early dedifferentiation and moderate bud stages. Four of these same proteins show an axial asymmetry at both stages. Ten distal-specific proteins were also identified, although only one was common to both stages. More significantly, 6 of these 34 proteins (molecular masses of 73, 73, 51.5, 44.0, 19.5, and 16.5 kilodaltons and isoelectric points of 6.70, 6.74, 6.0, 6.05, 5.9, and 6.98, respectively) are regulated to proximal levels by treatment of distal regenerates with retinoic acid (RA) at both stages. An additional five are proximalized by RA at only one regeneration stage. Since the effect of RA is to proximalize positional information in blastema cells, these 11 proteins represent gene products that could be involved in a biochemical cascade leading to the establishment of positional information in the regenerating limb along this axis.Key words: positional information, retinoic acid, regeneration, blastema, pattern formation, newt.
Surface fibrils (fimbriae) have been observed on fungi from every major group. Fimbriae are thoug... more Surface fibrils (fimbriae) have been observed on fungi from every major group. Fimbriae are thought to be involved in the following cell to cell interactions: conjugation, flocculation and adhesion. Several higher fungi exibit two other types of interactions: hyphal fusion (anastomosis) and clamp connection formation. As a prelude to examining the role of fimbriae in these processes, the fimbriae of two fungi that undergo these fusion events were examined. Electron microscopy studies revealed that Coprinus cinereus and Schizophyllum commune are fimbriated. C. cinereus fimbriae were 5 nm in diameter and 0.5 to 20 11m in length. Fimbriae of C. cinereus oidia were more numerous and longer than those of the hyphal stage. S. commune fimbriae were also 5 nm in diameter, but were only 0.5 to 2 11m in length. There was an unequal distribution of fimbriae on the hyphal surfaces of S. commune. Fimbriae were sparsely distributed over the entire hyphal surface, with higher densities of fibrils present on the side growths of the hyphae found in the older sections of the mycelium. Antiserum raised against Ustilago violacea fimbrial protein (AU) crossreacted strongly with 37 and 39 kd C. cinereus mycelial proteins. In contrast, AU bound very weakly to 89 and 92 kd S. commune mycelial proteins. Since AU cross-reacted poorly with S. commune fimbrial proteins, it was impossible to further characterize the fimbriae of this specIes. The 37 and 39 kd C. cinereus proteins, were isolated by electroelution and were shown to be able to form fibrils the same diameter as oidial fimbriae. The 37 kd protein was shown to be composed of several proteins with isoelectric points ranging from pH 6.1 to 7.63. Furthermore, 10. Immunoblots of U. violacea proteins 11. Inhibition of conjugation in U. violacea
Gene, Dec 1, 1992
Cloning and differential expression during the sexual cycle of a meiotic endonuclease-encoding ge... more Cloning and differential expression during the sexual cycle of a meiotic endonuclease-encoding gene from the basidiomycete Coprinus cinereus
Canadian Journal of Microbiology, Nov 1, 1992
Fimbriae from the fungus Ustilago violacea were shown to consist of 74-kDa protein subunits. The ... more Fimbriae from the fungus Ustilago violacea were shown to consist of 74-kDa protein subunits. The 74-kDa protein was strongly recognized on immunoblots reacted with a polyclonal antiserum against U. violacea fimbrial protein. Staining of the subunits with periodic acid – Schiff reagent indicated that the proteins were glycosylated. Approximately 10% of the glycoprotein was estimated to be carbohydrate, since treatment of defimbriated cells with tunicamycin or removal of the carbohydrate portion of the subunit by trifluoromethanesulfonic acid treatment resulted in a 67-kDa polypeptide. Mannose was identified as the predominant sugar. Separation of fimbrial protein by two-dimensional gel electrophoresis resolved it into a least six isoelectric forms. All isoelectric forms were recognized on immunoblots by the antiserum. Examination of fimbrial protein samples from three independent isolates indicated that the production of these isoelectric forms and posttranslational glycosylation of fimbrial protein were not dependent on mating type or environmental conditions. Key words: fimbriae, glycoprotein, oligosaccharides, Ustilago violacea, fungus.
Canadian Journal of Microbiology, May 1, 1996
Fimbriae of the anther smut fungus, Microbotryum violaceum are polymers of six 74-kDa glycoprotei... more Fimbriae of the anther smut fungus, Microbotryum violaceum are polymers of six 74-kDa glycoprotein isoforms. Digestion of fimbrial monomers with α-mannosidase yielded two polypeptides with masses of 70 and 48 kDa. The 70-kDa polypeptide is probably a product of incomplete digestion and the 48-kDa polypeptide is the aglycone. Thus, most of the carbohydrate component of fimbrial protein is mannose. Previous observations have suggested that fimbriae are necessary for mating in M. violaceum. Further evidence for this role was obtained in the present study by showing that mating is inhibited by an anti-fimbrial protein antiserum, by mannose and related sugars glucose and arabinose, and by the lectin concanavalin A. Since inhibition was not complete, however, two mechanisms for adhesion between compatible cells were proposed, one fimbrial dependent and one independent. Lastly, fimbrial protein from a1 but not a2 mating types bound to a mannose–agarose column, suggesting a lectin-like capability. The fimbrial dependent mechanism of cell-to-cell adhesion may involve binding of the mannose residues of the fimbriae of a2 cells by the fimbriae of a1 cells.Key words: mating, Microbotryum violaceum, lectin, fimbriae.
Biochemistry and Cell Biology, 1993
Biochemistry and Cell Biology, 1993
Morphogenetic effects of retinoic acid (RA) on the urodele amphibian limb regenerate pattern have... more Morphogenetic effects of retinoic acid (RA) on the urodele amphibian limb regenerate pattern have been well documented, but little is known regarding the mechanism of this action of RA at the molecular level. Since exogenous RA, at concentrations sufficient to cause proximalization, represents a significant stress to newts and has been shown previously to elicit increased synthesis of heat shock proteins (HSPs) in mouse embryo limb buds, we investigated the effects of this putative morphogen on the synthesis of members of the 70-kilodalton (70-kDa) stress protein family in amputated forelimbs of the newt Notophthalmus viridescens. Injection (i.p.) of RA in dimethyl sulfoxide (DMSO), at a dose sufficient to cause significant proximal–distal reduplication of the pattern in 50% of animals treated, resulted in increased synthesis and accumulation of a 73-kDa protein with a pi of approximately 6.75. The synthesis of this same protein is increased in limb tissues as a result of a brief 35...
Surface fibrils (fimbriae) have been observed on fungi from every major group. Fimbriae are thoug... more Surface fibrils (fimbriae) have been observed on fungi from every major group. Fimbriae are thought to be involved in the following cell to cell interactions: conjugation, flocculation and adhesion. Several higher fungi exibit two other types of interactions: hyphal fusion (anastomosis) and clamp connection formation. As a prelude to examining the role of fimbriae in these processes, the fimbriae of two fungi that undergo these fusion events were examined. Electron microscopy studies revealed that Coprinus cinereus and Schizophyllum commune are fimbriated. C. cinereus fimbriae were 5 nm in diameter and 0.5 to 20 11m in length. Fimbriae of C. cinereus oidia were more numerous and longer than those of the hyphal stage. S. commune fimbriae were also 5 nm in diameter, but were only 0.5 to 2 11m in length. There was an unequal distribution of fimbriae on the hyphal surfaces of S . commune . Fimbriae were sparsely distributed over the entire hyphal surface, with higher densities of fibril...
Canadian Journal of Microbiology, 1996
Fimbriae of the anther smut fungus, Microbotryum violaceum are polymers of six 74-kDa glycoprotei... more Fimbriae of the anther smut fungus, Microbotryum violaceum are polymers of six 74-kDa glycoprotein isoforms. Digestion of fimbrial monomers with a-mannosidase yielded two polypeptides with masses of 70 and 48 kDa. The 70-kDa polypeptide is probably a product of incomplete digestion and the 48-kDa polypeptide is the aglycone. Thus, most of the carbohydrate component of fimbrial protein is mannose. Previous observations have suggested that fimbriae are necessary for mating in M. violaceum. Further evidence for this role was obtained in the present study by showing that mating is inhibited by an anti-fimbrial protein antiserum, by mannose and related sugars glucose and arabinose, and by the lectin concanavalin A. Since inhibition was not complete, however, two mechanisms for adhesion between compatible cells were proposed, one fimbrial dependent and one independent. Lastly, fimbrial protein from a1 but not a2 mating types bound to a mannose-agarose column, suggesting a lectin-like capability. The fimbrial dependent mechanism of cell-to-cell adhesion may involve binding of the mannose residues of the fimbriae of a2 cells by the fimbriae of a1 cells.
Microbiology (Reading, England), 2000
Fruiting body formation in the basidiomycete Coprinus cinereus is a developmental process that oc... more Fruiting body formation in the basidiomycete Coprinus cinereus is a developmental process that occurs as a response of the mycelium to external stimuli. First, localized, highly branched hyphal structures (knots) are formed as a reaction to nutritional depletion. Hyphal-knot formation is repressed by light; however, light signals are essential for the development of the hyphal knot into an embryonic fruiting body (primordium) as well as karyogamy, meiosis and fruiting body maturation. The role of the different environmental signals in the initial phases of fruiting body development was analysed. It was observed that two fungal galectins, Cgl1 and Cgl2, are differentially regulated during fruiting body formation. cgl2 expression initiated in early stages of fruiting body development (hyphal knot formation) and was maintained until maturation of the fruiting body, whereas cgl1 was specifically expressed in primordia and mature fruiting bodies. Immunofluorescence and immuno-electron mi...
Molecular Biology of Fungal Development, 2002
... and Georg-August-Universität Gottingen, Gottingen, Germany Eline Polak,* Alan PF Bottoli,✝ Ma... more ... and Georg-August-Universität Gottingen, Gottingen, Germany Eline Polak,* Alan PF Bottoli,✝ Marcel Hollenstein,☨ Piers J. Walser, Robert P. Boulianne ... Upon meiosis, the exogenously formed sexual spores (basidiospores) bud off the basidium [5; this volume, Chapters 14 by ...
Molecular and General Genetics MGG, 1998
Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium.... more Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium. Some strains also produce large, inflated single cells (chlamydospores) at the agar/air interface, and hyphal aggregates (hyphal knots) that can develop into sclerotia. Monokaryons show various reactions upon transformation with heterologous A mating type genes. Production of oidia in such A-activated transformants is repressed in the dark and induced by blue light. Five of six monokaryons tested following transformation with A genes showed induced production of hyphal knots and sclerotia in the dark, and at least three strains showed enhanced chlamydospore production in the dark. Continuous incubation under blue light inhibited formation of hyphal knots, sclerotia and chlamydospores in both competent monokaryons and in A-activated transformants. On artificial medium and on a 12 h light/12 h dark regime, A-activated transformants of one distinct monokaryon (218) formed fruit-body primordia that were arrested in development before karyogamy. Our studies show that A mating type genes control all major differentiation processes in Coprinus, but whether developmental processes can proceed depends on the genetic background of the strain.
Journal of Microbiological Methods, 1999
In this study we present an indexed genomic library of homokaryon AmutBmut constructed within a n... more In this study we present an indexed genomic library of homokaryon AmutBmut constructed within a novel cosmid carrying pub1 + as a selectable Coprinus marker. The average insert size per cosmid comprises 41 kb. We screened the library and detected copies of known (al-2, P-tub, cgll, ras, trp I) and of new Coprinus genes (cat, lacl, lac2, lac3). Screening was performed either by Southern blot hybridisation or more efficiently by non-radioactive PCR amplification. We successfully applied PCR with specific and with degenerate primers, multiplex PCR and colony PCR in library screening. Our results suggest a new, more efficient pooling strategy for future high throughput screenings to be used in PCR with pooled cosmid DNAs, or in a less laborious approach using pooled Escherichia coli colonies for PCR.
Journal of Biological Chemistry, 1997
Galectins are members of a genetically related family of -galactoside-binding lectins. At least ... more Galectins are members of a genetically related family of -galactoside-binding lectins. At least eight distinct mammalian galectins have been identified. More distantly related, but still conserving amino acid residues critical for carbohydrate-binding, are galectins in chicken, eel, frog, nematode, and sponge. Here we report that galectins are also expressed in a species of fungus, the inky cap mushroom, Coprinus cinereus. Two dimeric galectins are expressed during fruiting body formation which are 83% identical to each other in amino acid sequence and conserve all key residues shared by members of the galectin family. Unlike most galectins, these have no N-terminal post-translational modification and no cysteine residues. We expressed one of these as a recombinant protein and studied its carbohydrate-binding specificity using a novel nonradioactive assay. Binding specificity has been well studied for a number of other galectins, and like many of these, the recombinant C. cinereus galectin shows particular affinity for blood group A structures. These results demonstrate not only that the galectin gene family is evolutionarily much older than previously realized but also that fine specificity for complex saccharide structures has been conserved. Such conservation implies that galectins evolved to perform very basic cellular functions, presumably by interaction with glycoconjugates bearing complex lactoside carbohydrates resembling blood group A.
Uploads
Papers by Robert Boulianne