Cloning and comparison of Aα mating-type alleles of the basidiomycete Schizophyllum commune

Summary An A mating-type allele (A4) was isolated by walking the chromosome from the closely linked PAB1 gene. A cosmid clone containing the A1 allele isolated from the walk was used as a probe to recover the A1 allele from another cosmid library. Cosmids encoding mating-type activity were identified by transforming Schizophyllum cells and screening for activation of A-regulated development. Putative mating-type transformants were confirmed in mating tests and genetic analyses of progeny. The identity of the specific alleles isolated was demonstrated by showing that their effectiveness in transforming for mating type is limited to recipient strains possessing an A allele different from the one encoded by the cloned sequences. Transforming DNA is active in trans, suggesting that A encodes a diffusible product. Restriction mapping shows that A1 and A4 are coded in the same physical region of the genome, but within a subregion that contains extensive sequence divergence. In addition, Southern analyses show that there is only one copy of A1 or A4 per haploid genome, and that they do not cross-hybridize to one another or to any of the other A alleles. A1 and A4 were subcloned as 2.8 and 1.2 kb fragments, respectively, retaining in transformation all the mating-type activity demonstrated of the original cosmids.     

Transformation of the basidiomycete, Schizophyllum commune

Summary Protoplasts of aSchizophyllum commune tryptophan auxotroph (trp1), deficient in indole-3-glycerol phosphate synthetase (IGPS), were transformed to trp⁺ with plasmid DNA containing the SchizophyllumTRP1 sequence. Efficiencies up to 30 transformants per microgram of plasmid DNA were obtained. Southern blots reveal that the transforming DNA is integrated in chromosomal DNA. The trp⁺ phenotype of transformants is stable in meiosis and mitosis. Transformants possess IGPS activity comparable to wild-type cells.     

Molecular characterization of ura1, a mutant allele for orotidine-5'-monophosphate decarboxylase in Schizophyllum commune

Abstract The basis of the auxotrophic ural phenotype in Schizophyllum commune has been investigated. Two point mutations causing changes in conserved amino acid positions 62 (from lysine to glutamate) and 79 (from leucine to phenylalanine) most likely are the cause for the observed phenotype, whereas the overall gene structure was unchanged. Since reversion rates in this locus are extremely low, a single point mutation could not be expected to be the cause for the mutation. Besides the two point mutations expected to be induced by UV mutagenesis, the two alleles investigated from independently isolated strains differ by approximately 7% in nucleic acid sequence and about 3% in amino acid sequence, indicating a distant relationship between the strains used.     

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.     

Enhanced glucan formation of filamentous fungi by effective mixing,oxygen limitation and fed-batch processing

Summary Glucan formation ofSchizophyllum commune andSclerotium glucanicum were investigated. Process data obtained during batch cultivation are presented. Glucan release can be improved by oxygen limitation. Thus, growth and glucan release are influenced by oxygen in opposite ways. Possible pathways of this oxygen-dependent regulation are discussed. A draft-tube/propeller system, rushtonturbine-, fan- and helicon-ribbon-impeller as well as a fundaspi and intermig agitator were tested. The 4-bladed fan impeller withd ^*=0.64 yielded the best results, since effective bulk mixing is much more important than bubble break up (micromixing) with regard to this system. Fed-batch cultivation always resulted in higher rates of glucan formation than the batch process.     

Direct studies of dikaryotization in Schizophyllum commune

The growth, duplication and fate of multikaryotic hyphae bearing true clamp connections, as derived from compatible matings of Schizophyllum commune, were studied by phase contrast microscopy. The nuclei (N) of multikaryotic apices maintained a near central position during hyphal growth. True clamp connection formation occurred with near synchronous mitosis followed by septal synthesis across the clamp neck and main hyphal axis. Nuclear progeny after mitosis in a hexakaryon included 6 N in the apex, 1 N in the clamp and 5 N in the penultimate cell; the solitary nucleus in the clamp later entered the penultimate cell. Similar events occurred for clamp connection formation and mitosis in the trikaryon, quadrikaryon or pentakaryon, whether in the apex or primary branches. Nuclear content of the multikaryotic apex (2 N through 10 N) had no apparent effect on the rate of individual hyphal growth. Reduction of the nuclear number in a trikaryon occurred by long-term entrappment of a solitary nucleus in the clamp and subsequent outgrowth of the dikaryotic penultimate cell. Occasionally, more than one nucleus became entrapped in the clamp cell. The ephemeral nature of the multikaryon was indicated by the fact that older cultures appeared to be exclusively dikaryotic hyphae at the colony periphery.     

Purification and Some Properties of β-Xylosidase from Emericella nidulans

β-Xylosidase was purified 662 fold from a culture filtrate by ammonium sulfate fractionation, gel filtration on Biogel P-100, DEAE-Sephadex chromatography, and gel filtration on Sephadex G-200. With isoelectric focusing, the purified β-xylosidase found to be homogeneous on SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis. The molecular weight was estimated by gel filtration to be 240,000, and 116,000 by SDS polyacrylamide gel electrophoresis. The purified β-xylosidase had an isoelectric point at pH 3.25, and contained 4% carbohydrate residue. The optimum pH was found to be in the range of 4.5 ~ 5, and the optimum temperature was 55°C. The enzyme activity was inhibited by Hg^{2 +}, SDS, and N-bromosuccinimide at a concentration of 1 × 10^?3 m, and also p-chloromercuribenzoate at a concentration of 1 × 10^?4m. The purified enzyme hydrolyzed phenyl β-d-xyloside (k_o = 302.6 sec^?1),β-nitrophenyl β-d-xyloside (k_o = 438.9 sec^?1), o-nitrophenyl β-d-xyloside (k_o = 431.0 sec^?1), p-chlorophenyl β-d-xyloside (k_o = 207.9 sec^?1), o-chlorophenyl β-d-xyloside (k_o = 211.8 sec^?1), β-methylphenyl β-d-xyloside k_o = 96.5 sec^?1), o-methylphenyl β-d-xyloside (k_o = 83.1 sec^?1), p-methoxyphenyl β-d-xyloside (k_o = 99.3 sec^?1), o-methoxyphenyl β-d-xyloside (k_o= 100.0 sec^?1), xylobiose (k_o = 992A sec^?1), xylotriose (k_o = 1321.9 sec^?1), xylotetraose (k_o = 7S9.1 sec^?1) and xylopentaose (k_o = 508.0 sec^?1). On enzymic hydrolysis of phenyl β-d-xyloside, the reaction product was found to be β-d-xylose with retention of the configuration. The purified β-xylosidase was practically free of a-xylosidase and β-glucosidase activities.     

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.     

高相对分子质量裂褶多糖的制备与结构鉴定

以裂褶菌（Schizophyllum commune）发酵生产的培养物为研究对象,经过活性炭脱色、Sevag法脱杂蛋白、乙醇沉淀得粗多糖,再经Sephadex G-200柱层析分离纯化得裂褶多糖纯品.通过Sephadex G-200凝胶层析法测得其平均分子质量为436kDa;由气相色谱、红外光谱、高碘酸氧化、Smith降解、^13C核磁共振等方法表征其化学结构,推测其结构为具有（1→6）分支的β-〔1-3）-D-葡聚糖：其组成重复单元应该含有3个主链糖基和一个单糖分支,主链的取代发生在C-1和C-3之间,即为1→3糖苷键;分支发生在C-1和C-6之间,即为1→6糖苷键,其中分支点在主链糖基的C-6上.