Heterokaryon formation in the basidiomycete Schizophyllum commune by electrofusion of protoplasts

Summary Conditions for high frequency electrofusion of protoplasts from the basidiomycete Schizophyllum commune are described. Visual inspection revealed up to 30% of the protoplasts engaged in fusion. Using complementing nutritional mutations, nearly 7% of the regenerated protoplasts could be recovered as heterokaryotic mycelia. The method is probably equally applicable to other basidiomycetes such as Agaricus bisporus, permitting the recovery of fusion products in the absence of selection markers.     

Direct studies of dikaryotization in Schizophyllum commune

Compatible matings of Schizophyllum commune were performed on glucose-peptone-yeast extract medium appended with gelatin (18%) and studied by phase contrast microscopy during nuclear migration. Three categories of nuclear migration were observed. Type I involved a pulsatile jerking of the entire cytoplasmic contents of the hypha, changed direction periodically, and, during periods of cytoplasmic tranquility, the nucleus continued to migrate. Type II A migration of nuclei occurred in the absence of visible cytoplasmic flow. Both Type I and Type II A nuclear movements exceeded the hyphal growth rate by 10--20-fold. Type II B nuclear migration also occurred in the absence of visible cytoplasmic flow and the velocity was within the range of the hyphal growth rate. No specific organelles that were detected either directed or facilitated Type II A or Type II B nuclear movements. The nucleolus could either lead or trail relative to the direction of nuclear movement. Nuclear migration can be attributed to both cytoplasmic flow and self motility, depending upon the particular regions of the migration hypha in which it occurs.     

Mitosis in hyphae ofSchizophyllum commune Fr.

Summary In hyphae of the basidiomyceteSchizophyllum commune Fr. mitosis occurs through a longitudinal division of a strand of chromatin, which is followed by a parallel separation of the daughter strands.     

Insoluble hydrophobin complexes in the walls of Schizophyllum commune and other filamentous fungi

Two closely related cysteine-rich hydrophobic proteins, Sc3p and Sc4p, of the basidiomycete Schizophyllum commune are developmentally regulated and associated with the walls of aerial hyphae and fruit-body hyphae. They are present in the walls as hot-SDS-insoluble complexes which can be extracted with formic acid. The hydrophobins can then be dissociated by oxidation with performic acid. However, extraction of the walls with trifluoroacetic acid results in both solubilization and dissociation of the hydrophobin complexes into monomers. This suggests that non-covalent interactions are responsible for formation of these insoluble complexes. Carboxymethylation with iodoacetic acid only occurred after reduction with DTT indicating all cysteines in the monomeric hydrophobins involved in intramolecular disulfide bridges. Abundant proteins with similar properties were found in walls from all other filamentous fungi tested, including the basidiomycetes Pleurotus ostreatus, Coprinus cinereus, Agaricus bisporus, and Phanerochaete chrysosporium, the ascomycetes Aspergillus nidulans, Neurospora crassa, and Penicillium chrysogenum, and the zygomycete Mucor mucedo.     

Intergeneric hybridization between Pleurotus ostreatus and Schizophyllum commune by PEG-induced protoplast fusion

Intergeneric hybridization between Pleurotus ostreatus and Schizophyllum commune was studied using PEG-induced fusion. The fusion of protoplasts from auxotrophic mutant strains resulted in the formation of fusion hybrids in the frequencies of 3.6 to 7.3×10^–5. Most of these fusion hybrids were monokaryotic and sterile and no heterokaryosis occurred. Most fusants showed a significantly higher nuclear DNA content when compared to parental strains and no diploids (parent 1 genome plus parent 2 genome) were found. Some fusion hybrids revealed both parental fragments in nuclear and mitochondrial rDNA PCR profiles. AP-PCR (Arbitrarily-primed Polymerase Chain Reaction) fingerprints also indicated that most of the fusion products were recombinant hybrids.     

Enzyme activities associated with arabitol and mannitol biosynthesis and catabolism in Schizophyllum commune

Enzymes of polyol metabolism were studied in basidiospore germination of Schizophyllum commune during periods of in vivo arabitol and mannitol pool depletion (growth on glucose-asparagine) and during their subsequent synthesis (growth on acetate-NH ₄ ⁺ ). Optimal conditions for assays were established and specific activities of enzymes employing d-arabitol, d-mannitol, d-ribulose, d-fructose and d-xylulose as substrates were traced. Inquiries into the products formed during these reactions showed that d-ribulose generated arabitol while d-fructose produced mannitol with d-xylulose giving rise to xylitol. The dehydrogenase reactions were further investigated using polyacrylamide disc gel electrophoresis. Here was revealed the existence of at least two separate enzymatic activities pertaining to the catabolism of arabitol and mannitol. Also noted were the electrophoretic patterns when d-sorbitol, ribitol, xylitol and ethanol were used as substrates.     

Intraspecific hybridization between Coprinus cinereus and Schizophyllum commune by PEG-induced protoplast fusion and electrofusion

Two irreversible inhibitors, iodoacetamide and diethylpyrocarbonate, were used to select intraspecific fusion products of two mushroom species, Coprinus cinereus and Schizophyllum commune. Iodoacetamide was the more suitable inhibitor because it gave a low breakage frequency and low survival rate of the cells in the inactivation experiments. Fusion-induced by polyethylene glycol and electro-fusion were compared and, under optimal conditions, gave fusion frequencies of 16.7% to 50.0% and 6.9% to 8.4%, respectively. All fusion progeny were heterokaryons (dikaryons) and had clamp connections. There were no differences in the morphology and fruiting ability of the fusion progeny and those of the heterokaryons generated from mating.     

Chimeric pheromone receptors in the basidiomycete Schizophyllum commune

The pheromone receptor system of the basidiomycete Schizophyllum commune is capable of ligand discrimination to confer mating specificity. The pheromone receptors of the B alpha locus were investigated for ligand discrimination in a strategy of domain swapping experiments. Several altered phenotypes of chimeric receptors have been found. These include constitutive pheromone receptors which need no ligand for activation of the downstream cascade of events. In addition, receptors still dependent on ligand were identified that had altered pheromone activation profiles, including promiscuous receptors that are activated by pheromones of all nine specificities, including the former self. In addition, highly discriminative receptors were created which are activated by only two of the eight non-self-specificities. The chimeric receptors identify the last third of the receptor as the determinant for B alpha 1 specificity, whereas B alpha 2 specificity resides in noncontiguous domains covering the first and middle parts of the receptor molecule.     

Isolation of a new potent cytotoxic pigment along with indigotin from the pathogenic basidiomycetous fungus Schizophyllum commune

An indole derivative, schizocommunin, was isolated along with indigotin (indigo), indirubin, isatin, and tryptanthrin, from the liquid culture medium in which a culture of Schizophyllum commune, isolated from the bronchus of a human patient with allergic bronchopulmonary mycosis, had been grown. The structure of schizocommunin was established by spectroscopic investigation. Schizocommunin showed the strong cytotoxicity against murine lymphoma cells. The assignments of the ¹H- and ¹³C-NMR signals of indigotin were also listed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Recovery of degenerate schizostatin production by screening single-basidiospore isolates inSchizophyllum commune

To recover degenerate schizostatin production in aSchizophyllum commune isolate, SANK 17785, we examined schizostatin production of 30 single-basidiospore isolates obtained from a basidiocarp of SANK 17785. One of the isolates showed high productivity (136–154 μg/ml) and maintained its high productivity for 26 mo. To explore other isolates with high schizostatin production, 76 single-basidiospore isolates from 7 wild basidiocarps were obtained and their schizostatin production was quantified. Four of the isolates produced more schizostatin than SANK 17785 did originally.     

Two morphological markers indicating dikaryosis in Schizophyllum commune

Summary In the wood destroying basidiomycete Schizophyllum commune a method is described to recognize the onset of dikaryosis rapidly in using recessive genetic markers. The gene ai ⁺/ai causes in its mutant recessive allele (ai) the production of dark coloured fruit bodies. This can be made use of to evaluate macroscopically the formation of a dikaryon. Another useful marker is the gene rd ⁺/rd. The recessive allele (rd) causes phenotypically the formation of a round looking mycelium instead of the fringed looking mycelium, the wild type. This genetic marker which is closely linked to the A-incompatibility factor is therefore also qualified to detect the onset of dikaryosis without much effort.     

裂褶菌及裂褶菌多糖研究进展

裂褶菌是一种十分重要的食药兼用真菌,含有丰富的生理活性物质,裂褶菌多糖作为一种极有开发前景的生物活性物质已得到国内外的普遍重视。综述了裂褶菌的生物学特性、营养成分、药用价值、栽培现状以及裂褶菌多糖的化学分析和药理作用的研究进展,并讨论了裂褶菌和裂褶菌多糖的研究前景。     

Degradation of ferulic acid by a white rot fungus <Emphasis Type="Italic">Schizophyllum commune</Emphasis>

Summary A ubiquitous white rot fungus Schizophyllum commune was used for the first time to study the degradation of ferulic acid. Vanillic acid was observed as one of the major products of ferulic acid catabolism, with vanillin formed as an intermediate. Almost 99.9% ferulic acid with a initial concentration of 5 mM was consumed by this fungus after 16 days of incubation at 37 °C.     

普通裂褶菌真菌学及实验室研究进展

普通裂褶菌是一种条件致病菌,可侵犯人体多个器官多种组织,导致各种表现形式的疾病,如脑脓肿、脑膜炎、鼻窦炎、肺部真菌球、蜂窝肺、肺结节、甲真菌病等.由于普通裂褶菌感染引起的真菌病病例少见报道,临床医生对其认识不足,再加上某些实验室不具备鉴定条件,所以感染该菌的病例常被漏诊、误诊.现将近年来临床分离的普通裂褶菌株真菌学及实...     

Ultrastructural differences between wall apices of growing and non-growing hyphae ofSchizophyllum commune

Summary Newly synthesized chitin at the hyphal apex ofSchizophyllum commune was shown to be highly susceptible to chitinase degradation and solubilization by dilute mineral acid. With time this chitin became gradually more resistant to these treatments. With a combination of the shadow-cast technique and electron microscopic autoradiography it could be shown that this process occurred as the newly synthesized chitin moved into subapical parts of growing hyphae but also in non-growing apices which had ceased growth after incorporation of theN-acetyl[6-³H]glucosamine. These results are in agreement with a model which explains apical morphogenesis by assuming that the newly synthesized wall material at the apex is plastic due to the presence of individual polymer chains but becomes rigidified because of subsequent physical and chemical changes involving these polymers.Dedicated to Dr. A.Quispel, Professor of Botany at the University of Leiden, on occasion of his retirement.     

裂褶菌营养菌丝蛋白质成分的分析

对裂褶菌菌丝中蛋白质的氨基酸组成及含量进行了测定。结果表明 ,裂褶菌中所含粗蛋白的质量分数为 2 8.76 % ,所测定的 17种氨基酸总质量分数为 12 0 .13g·kg- 1 。其中 7种必需氨基酸的质量分数为 4 5 .36g·kg- 1 ,占氨基酸总量的 37.76 % ;10种非必需氨基酸的质量分数为 79.5 0g·kg- 1 ,占氨基酸总量的 6 2 .2 4 %。     

Direct studies of nuclear movements in Schizophyllum commune

Summary Measurements of nuclear positions in apical cells of homokaryotic mycelia and dikaryotic mycelium of Schizophyllum commune showed that nuclei occupied a near central position in most cases. Forward nuclear movements observed in living hyphal apices occurred at rates within the range of hyphal growth and could account for the maintenance of centrally located nuclei. Opposed nuclear movements followed mitosis and greatly exceeded the rate of hyphal growth. Septum disruption and rapid nuclear movements characterized an A _xB_mut homokaryon. Neither cytoplasmic streaming nor actively participating granules or filaments could account for any of these nuclear movements.     

裂褶菌培养基形态学观察和DNA序列分析

目的通过观察裂褶菌在5种培养基上的生长状态、扫描电镜及DNA序列分析,了解该菌形态学及分子生物学等方面的特征。方法菌落转种于沙氏培养基(SDA),麦芽浸膏琼脂(MEA),马铃薯葡萄糖琼脂(PDA),玉米粉琼脂(CMA)和察氏琼脂(CZA)平皿培养基,27℃和37℃培养2周,观察菌落生长情况,进行扫描电镜检测及DNA序列分析。结果菌落在SDA,MEA和PDA上生长状态较好,呈蓬松白色羊毛状;尿素酶试验阳性,放线菌酮耐受试验阴性。光镜下见分支分隔菌丝、侧生的钉状突起及类水母体变异子实体。扫描电镜见菌丝分隔处闭锁联合、侧生钉状突起和泪滴状球形分泌物。经26S rDNAD1/D2区序列分析证实该菌株为裂褶菌。结论裂褶菌只有丝状型一种菌落形态;分支分隔菌丝及分隔处闭锁联合,侧生钉状突起和泪滴状球形分泌为其形态学特征;孢子由类水母状子实体产生。     

Cell wall metabolism during fruiting of the basidiomycete Schizophyllum commune

During the development of fruit bodies of the basidiomycete Schizophyllum commune, the alkali-insoluble (R glucan) and alkali-soluble (S glucan) cell wall fractions are synthesized during the entire course of morphogenesis. The water soluble glucan (WSG) is not synthesized after an early stage. There is also a relative increase in the proportion of S glucan during development which appears related to a change in the proportion of the components synthesized. Data are also presented to show that several fruiting mutants also have specific cell wall differences, and that there is a significant contribution to cell wall structure by genes which do not cause a macroscopically observable change in phenotype.