Tadanori Aimi

Identification and linkage mapping of the genes for the putative homeodomain protein ( hox1 ) and the putative pheromone receptor protein homologue ( rcb1 ) in a bipolar basidiomycete, Pholiota nameko

In the current studies, we sequenced and characterized the gene for the homeodomain protein (hox1) in a bipolar mushroom, Pholiota nameko, which is a putative homologue of A mating type genes in the tetrapolar basidiomycete, Coprinopsis cinerea. We also sequenced and characterized the gene for the pheromone receptor (rcb1) in P. nameko, which is a putative homologue of the B mating type genes in C. cinerea. Restriction fragment length polymorphism (RFLP) and linkage analyses indicated that the both genes are present as a single locus on the different chromosome. Moreover, in P. nameko, the hox1 gene was mapped to the A mating type locus in linkage group I. However, rcb1 was not linked to the A mating type locus and was mapped to the other linkage group. These results strongly suggest that hox1 regulates with incompatibility in the bipolar mushroom, and that rcb1 may not affect the mating function in P. nameko. This is the first report regarding the structure of the mating type genes in bipolar mushrooms.

Rapid Species Identification of Cooked Poisonous Mushrooms by Using Real-Time PCR

ABSTRACT Species-specific identification of the major cooked and fresh poisonous mushrooms in Japan was performed using a real-time PCR system. Specific fluorescence signals were detected, and no nonspecific signals were detected. Therefore, we succeeded in developing a species-specific test for the identification of poisonous mushrooms within 1.5 h.

An archaeal protein homologous to mammalian SRP54 and bacterial Ffh recognizes a highly conserved region of SRP RNA

The gene encoding the 54 kDa protein of signal recognition particle (SRP54) in the hyperthermophilic archaeon Pyrococcus furiosus has been cloned and sequenced. Recombinant P. furiosus SRP54 (pf‐SRP54) and the N‐terminal G‐domain and C‐terminal M‐domain (pf‐SRP54M) of pf‐SRP54 with an amino‐terminal addition of six histidine residues were expressed in Escherichia coli and subjected to binding experiments for SRP RNA, non‐conserved 213‐nucleotide RNA (helices 1, 2, 3, 4 and 5) and conserved 107‐nucleotide RNA (helices 6 and 8) from SRP RNA. The RNA binding properties of the purified protein were determined by filter binding assays. The histidine‐tagged pf‐SRP54M bound specifically to the conserved 107‐nucleotide RNA in the absence of pf‐SRP19, unlike the eukaryotic homologue, with an apparent binding constant (K) of 18 nM.

Genomic structure of the A mating-type locus in a bipolar basidiomycete, Pholiota nameko

In the bipolar basidiomycete, Pholiota nameko, the homeodomain protein, A4-hox1, located at the A mating-type locus, is known to regulate mating compatibility. In the present study, we investigated the genomic structure of the P. nameko A mating-type locus and its flanking region. A second homeodomain gene (A4-hox2) was discovered upstream of A4-hox1; this together with the conserved gene order around the A mating-type locus and their similar transcription direction were found in P. nameko, another bipolar mushroom, Coprinellus disseminatus, and two tetrapolar mushrooms, Coprinopsis cinerea and Laccaria bicolor. Analysis of the deduced protein sequences of the homeodomain protein genes from two strains of P. nameko show that the putative functional domains differ from those of the homeodomain proteins of the tetrapolar mushrooms, C. cinerea and L. bicolor.

Complete mitochondrial DNA sequence of the Japanese flying fish Cypselurus hiraii

In this study, to develop a technique that enables authentication of processed seafood, the complete nucleotide sequence of the mitochondrial genome for the Japanese flying fish Cypselurus hiraii was determined. Three segments spanning the entire genome were amplified using polymerase chain reaction, and products were subsequently used as templates for direct sequencing with 60 primers. The genome (16 528 base pairs) was found to contain the same 37 genes (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) as those found in other vertebrate mitochondrial genomes, with the gene order being identical to that typical of vertebrates. A major noncoding region between the tRNAPro and tRNAPhe genes (868 base pairs) appears to be the control (D-loop) region, as it has several conserved blocks characteristic of control regions.

Transcriptional regulation of two cellobiohydrolase encoding genes (cel1 and cel2) from the wood-degrading basidiomycete Polyporus arcularius

In the current studies, we sequenced and characterized the genomic and complementary deoxyribonucleic acid clones encoding the cellobiohydrolase encoding genes cel1 and cel2 of Polyporus arcularius. The predicted amino acid sequences of Cel1 and Cel2 are similar to glycosyl hydrolase family 7 and 6 proteins, respectively. The expression of cel1 and cel2 was induced by microcrystalline cellulose (Avicel) and cellopentaose but repressed by glucose, cellobiose, cellotriose, and cellotetraose. There was a very low level of cel1 and cel2 transcription regardless of the carbon source. These results suggest that P. arcularius cells constitutively express a very low level of cellulase that can degrade insoluble crystalline cellulose and that the transcription of cel1 and cel2 in the cells is induced by products produced by these endoglucanases such as cellooligosaccharides.

Vegetative incompatibility in the ascomycete Rosellinia necatrix studied by fluorescence microscopy

We describe our examination of the cytological characteristics of the vegetative incompatibility reaction in a filamentous ascomycetes, Rosellinia necatrix, by analyzing the fluorescence emitted by ethidium bromide and acridine orange stained nuclei. Hyphal anastomosis between incompatible strains, which were field and single ascospore isolates, were observed with cell death showing fused hyphae, and nuclei debris which were intensified by staining with ethidium bromide. In contrast, the nuclei in a living cell were not intensified by staining with ethidium bromide but were intensified by staining with acridine orange. A strain was found which did not form a barrier reaction, but which could be shown to undergo cell death and therefore showed a positive vegetative incompatibility reaction. We also examined the vegetative incompatibility among five single ascospore isolates and the putative parent strain from the same perithecium; all strains were incompatible. These results strongly suggest that vegetative incompatibility in R. necatrix is regulated by many loci.

Cytological Analysis of Anastomoses and Vegetative Incompatibility Reactions in Helicobasidium monpa

Our examination of the cytological characteristics of the vegetative incompatibility reaction in a filamentous basidiomycete, Helicobasidium monpa, by analyzing the fluorescence emitted by ethidium bromide and acridine orange stained nuclei is described. Hyphal anastomoses between strains belonging to different mycelium compatibility groups (MCG) were observed with cell death in fused hyphae, whose nuclei were intensified by ethidium bromide. In contrast, the nuclei in a living cell were not intensified by staining with ethidium bromide, but were intensified by staining with acridine orange. These results indicate that in H. monpa, ethidium bromide staining is a useful method for detecting dead cells. We also examined the relationships between the alternation of ploidy and hyphal anastomosis formation using the newly developed method on filamentous fungi. The tetraploid monokaryon strain derived from the original dikaryon strain by continuous subculture could not be fused to any wild type strains, but the original dikaryon strain could be fused without cell death to only the same MCG strain. In contrast, the haploid dikaryon strain derived from the original monokaryon strain fuses to several strains belonging to different MCGs without cell death. These results suggested that the cellular ploidy of this fungus is closely related to its mating system and, H. monpa may be a self-fertilizing fungus.

Enhancement of the viscometric endocellulase activity of Polyporus arcularius CMCase IIIa by cellobiose and cellooligosaccharides

Purification and viscometric characterization of three CMCases from Polyporus arcularius were carried out. The three CMCases, I, II, and IIIa, were estimated to have molecular masses of 39.1 kDa, 36.3 kDa, and 24.3 kDa, respectively. The addition of cellobiose and cellooligosaccharides to the reaction mixtures of CMCase I and II inhibited viscometric endocellulase activity. Following the addition of 20 mM cellobiose, CMCase I and II activities fell to about 30%–36% of their activity in the absence of cellobiose. CMCase IIIa activity, on the other hand, increased in proportion to the increase in cellobiose or cellooligo-saccharide concentration. Maximal enhancement of CMCase IIIa activity was observed following the addition of cellobiose, whereas less enhancement was observed with cellooligosaccharides spanning more than two glucoside units. The addition of 20 mM cellobiose resulted in an increase greater than 500% in CMCase IIIa activity. Inhibition of CMCase I and II by cellobiose and cellooligosaccharides may be the result of competition between the substrate and the reaction products. One of the reaction products of CMCase IIIa may bind to a site other than the active site of the enzyme, thus enhancing CMCase IIIa activity.

Molecular characterization of a xylanase-producing thermophilic fungus isolated from Japanese soil.

We describe the molecular characteristics of Scytalidium thermophilum isolated from Japanese soil. The S. thermophilum isolates produced higher xylanase activity than Humicola lanuginosa isolated from Japanese soil. A G/11 family xylanase-encoding gene was detected in the S. thermophilum genome by using the polymerase chain reaction technique. The S. thermophilum AF101-3 strain, which was one of the isolates in this study, grew well at 37°C and 50°C, and contained the maximum xylanase activity detected among the isolates. Phylogenetic analysis revealed that the S. thermophilum strains isolated from Japanese soil were clustered in a different group from the S. thermophilum strains reported by Lyons et al. [Mycol Res 104:1431, 2000], suggesting that the S. thermophilum strains isolated in this study are genetically new isolates. Therefore, the genetic diversity of S. thermophilum might be higher than that of H. lanuginosa. Moreover, this is the first report about detection of a xylanase-encoding gene in S. thermophilum. RID=”” ID=”” Correspondence to: T. Morinaga; email: tmorina@bio.hiroshima-pu.ac.jp