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At least 19 recordsLinked to original sources

Presence of a simple tandem repeat in the ITS1 region of the Xylariales.

A Simple Tandem Repeat sequence of 11 nucleotides has been found in the ITS1 region of the rDNA of members of Order Xylariales. The number of repetitions detected ranged from one to six, and they could be found in pure tandem or interspersed. The same core sequences have also been found in DNA from other organisms, although usually not repeated in tandem. These repetitions could have been generated by slipped strand mispairing. The presence of this sequence increases the normal rate of divergence in the ITS1 of the Xylariales. The phylogenetic implications of the presence of this sequence in the molecular taxonomy of Xylariales are also discussed.

Ascomycota↗

Size and sequence heterogeneity in the ITS1 of Xylaria hypoxylon isolates.

During a survey of 375 strains of the Xylariales, one isolate (F127076) was observed to have an ITS1 size of 833 bp. This size exceeds the average ITS1 size in the Xylariales (mean = 209 +/- 57 bp). Comparison of the DNA sequence with GenBank and with a proprietary DNA database revealed low homology with Xylaria hypoxylon ATCC 42768, and with one undescribed Xylaria species. When the ITS2 sequence was compared, these isolates were 96-98% homologous. Sequences of other variable genes confirmed the relatedness among these strains. A closer observation of the ITS sequence of this isolate revealed the presence of three repeated domains of 250 bp plus one truncated domain, showing 52-75% homology. Sequence similarity suggests that the repeated domain is derived from the fusion of the ITS1 with a DNA fragment derived from the gamma-glutamyltranspeptidase gene. This example suggests that the rate of evolution of ITS1 can be independent of the rate of evolution of other genes, even when this variability is not a result of slipped strand misspairing events like in other Xylariales. This observation also indicates that recombination with other nuclear genes could participate in the evolution of the internal transcribed spacer.

Biological Evolution↗

A melanin polyketide synthase (PKS) gene from Nodulisporium sp. that shows homology to the pks1 gene of Colletotrichum lagenarium.

The melanin polyketide synthase (pks) gene of Nodulisporium sp. MF5954 (ATCC74245) was cloned by exploiting its homology to the Colletotrichum lagenarium pks1 gene. Sequence analysis demonstrated that this gene is 70% identical to the C. lagenarium pks1 gene. A gene disruption construct, designed to replace both the ketoacyl synthase and acyl transferase domains with a hygromycin resistance (Hyr) gene, was synthesized, and used to disrupt the Nodulisporium melanin pks1 gene via homologous recombination, resulting in a mel(-) phenotype. Sequence analyses of the gene and of cDNA segments generated by RT-PCR indicate that there are three introns in the 5' half of the gene. The proposed 2159-amino acid product is 72% identical and 78% similar to the 2187-amino acid sequence deduced from the C. lagenarium pks1 gene. This similarity is notable, considering that C. lagenarium is a member of the order Phyllachoales or Sordariales, whereas Nodulisporium is generally believed to be member of the order Xylariales. However, despite the strong resemblance between the amino acid sequences in the acyl transferase domains of the two proteins, only one in five codons are conserved in the DNA sequences that encode this motif. The Nodulisporium sp. pks1 gene sequence and the amino acid sequence deduced from its coding region have been deposited in Genbank under Accession No. AF151533.

Amino Acid Sequence↗

Polyancora globosa gen. sp. nov., an aeroaquatic fungus from Malaysian peat swamp forests.

During an investigation of submerged leaves and twigs sampled from tropical peat swamp forests located in Peninsular Malaysia, an anamorphic fungus not attributable to a described genus was detected and isolated in pure culture. Conidial ontogeny was thoroughly studied and illustrated using both light and SEM, which revealed a unique conidial morphology. Analysis of partial nuLSU rDNA and ITS data revealed a phylogenetic position within the Xylariales (Ascomycota), but family affiliation remained unclear.

Climate↗

Molecular phylogeny of Coniochaetales.

Although the taxonomy of ascomycetes has changed dramatically, generic delimitation within the recently proposed order Coniochaetales has not been resolved. In order to clarify the phylogenetic relationships of genera in the Coniochaetaceae, we performed a molecular study based on the analyses of the sequences of the partial SSU and of the variable domains of the LSU rDNA genes. The phylogenetic trees obtained do not support the monophyly of the genera Coniochaeta, Coniochaetidium, Ephemeroascus, and Poroconiochaeta. A morphological study confirmed that there were not enough differences to distinguish these genera, and the latter three are treated as synonyms of Coniochaeta. The phialidic anamorph proved to be an informative phylogenetic character in Coniochaetales, while that the type of ascomata (cleistothecial or perithecial) and the ornamentation of the ascospore walls were of little taxonomic value at the generic level. The circumscription of the genus Coniochaeta is revised. The genera Coniocessia and Coniolariella are proposed as new within the order Xylariales to accommodate Coniochaeta nodulisporioides, and C. gamsii, respectively. The taxonomic position of Synaptospora and Wallrothiella subiculosa are also discussed.

Ascomycota↗

Molecular systematics of the Amphisphaeriaceae based on cladistic analyses of partial LSU rDNA gene sequences.

The Amphisphaeriaceae is an important family of ascomycetes within the Xylariales. There has been, however, disagreement regarding the taxonomic placement of many genera within this family and whether it should be confined to ascomycetes producing Pestalotiopsis-like anamorphs. In this study, phylogenetic relationships among members of the Amphisphaeriaceae are investigated using partial sequences of the 28S rDNA. Molecular data provided further evidence to support the association of several coelomycetous genera with the ascomycetous Amphisphaeriaceae. Phylogenetic analyses also show that all ascomycetous genera possessing Pestalotiopsis-like anamorphs are monophyletic and confirm the anamorphic-teleomorphic connections of some. There is, however, insufficient evidence to support the restriction of Amphisphaeriaceae to genera, which produce Pestalotiopsis-like anamorphs, because the phylogenetic placement of Amphisphaeria umbrina is not fully resolved and its affinities with other members received low bootstrap support. The results also indicate that Iodosphaeria and Arecophila should be excluded from the Amphisphaeriaceae. The placement of Lanceispora in the Amphisphaeriaceae is doubtful. A broad concept of the family Amphisphaeriaceae is advocated until further data are available.

Ascomycota↗

Screening of basidiomycetes for antimicrobial activities.

As a part of a screening programme developed to evaluate the antimicrobial activity of basidiomycetes, 317 isolates representing 204 species collected in Spain were screened against a range of human clinical pathogens and laboratory controls. Extracts from 45% of the isolates, representing 109 species, showed antimicrobial activity. Antibacterial activity was more pronounced than antifungal activity. The proportion of extracts from basidiomycetes showing antimicrobial activity was similar to or above that obtained for representative orders of Ascomycetes, such as Pezizales and Xylariales, but lower than that produced by members of the orders Diaporthales, Eurotiales, Hypocreales, Leotiales and Sordariales. Suprageneric taxa (orders and families) did not show pronounced differences in their antimicrobial activities though such differences were observed at the genus level, suggesting that the ability to produce these bioactive compounds is not homogenously distributed amongst the basidiomycetes. Isolates from some species showed large differences in their ability to produce metabolites with antimicrobial activity, possibly reflecting genetic differences at the infraspecific level.

Anti-Bacterial Agents↗

Molecular diversity of bamboo-associated fungi isolated from Japan.

Bamboos are common and useful plants in Japan but little information is available about their endophytes. In this study, 257 fungal strains were isolated from bamboo tissues, and 71 representative strains were characterized by 18S rRNA gene and internal transcriber spacer region sequence analysis. Phylogenetic analysis showed that the fungal isolates were located in Sordariomycetes and Dothideomycetes. Xylariales was the dominant group within bamboos. Several rRNA gene sequences were not similar to any current sequence in the database and might be a novel species or genera that could represent sources of novel biological compounds. These findings reveal that bamboos are a huge reservoir of microorganisms that should be extensively investigated.

Bambusa↗

L-696,474, a novel cytochalasin as an inhibitor of HIV-1 protease. I. The producing organism and its fermentation.

A novel cytochalasin, L-696,474, (18-dehydroxy cytochalasin H) that inhibits HIV-1 protease was discovered in fermentations of a bark-inhabiting Ascomycete, Hypoxylon fragiforme. The product was first identified from extracts of an agar medium. Fermentation studies on a number of media indicated that the product can be made on several solid and liquid media. Optimum production was obtained from growth in a complex medium composed of glycerol, glucose, citrate, Ardamine, soybean meal, tomato paste, and inorganic salts. Other Hypoxylon spp., related species of Xylariales, and other fungi known to produce cytochalasins, were also surveyed for their ability to make L-696,474. Only one other Hypoxylon fragiforme isolate was found to make this novel cytochalasin; none of the other cultures surveyed made L-696,474 or any other compounds which inhibit HIV-1 protease.

Ascomycota↗

Self-splicing of the mobile group II intron of the filamentous fungus Podospora anserina (COI I1) in vitro.

The first intron of the mitochondrial gene coding for cytochrome oxidase subunit I (COI I1) of Podospora anserina can undergo self-splicing in vitro at high concentrations of NH4Cl or KCl. Under these conditions cleavage at the 5' splice junction takes place without branch formation probably via hydrolysis by water or OH- and the intron is released in a linear form. In vitro transcripts that contain mutated introns with large deletions in nonconserved domain IV comprising greater than 50% of the intronic sequence display a more efficient splicing reaction and, surprisingly, 5' cleavage via transesterification and lariat formation is re-established to a low degree under NH4Cl. In contrast to the self-splicing group II introns aI5 gamma and bI1 from yeast mitochondria cleavage at the 3' splice site of the Podospora intron is reduced and cleavage by hydrolysis in trans (i.e. exon reopening) is almost completely suppressed. Both observations could be interpreted as a result of unfavourable spatial conformations of the intron that (i) lead to a steric hindrance of the 5' exon to attack the 3' splice site in cis and (ii) block intron-dependent cleavage reaction of the ligated exons in trans. Alternatively, the possibility that a weak overall interaction of the postulated exon- with the corresponding intron-binding sites (EBS-IBS pairings) is responsible for the remarkable differences to the self-splicing reaction of other group II introns is discussed.

Ascomycota↗

Virus-like genetic organization and expression strategy for a double-stranded RNA genetic element associated with biological control of chestnut blight.

The complete nucleotide sequence of the largest double-stranded (ds) RNA present in hypovirulent strain EP713 of the chestnut blight pathogen, Cryphonectria parasitica, was determined and the predicted genetic organization was confirmed by translational mapping analysis. The deduced RNA sequence was 12 712 bp in length, excluding the terminal poly(A):poly(U) homopolymer domain. The strand terminating with 3'-poly(A) contained two contiguous large open reading frames (ORF A and ORF B) beginning at nucleotide residues 496-498 and extending to nucleotide positions 11 859-11 861. The junction between ORF A and ORF B consisted of the sequence 5'-UAAUG-3', where UAA served as the termination codon for ORF A and AUG was the 5'-proximal initiation codon within ORF B. ORF A (622 codons in length, excluding the termination codon) was recently shown to encode two polypeptides, p29 and p40, which were generated from a nascent polyprotein by an autocatalytic event mediated by p29 (Choi et al., 1991). A similar autocatalytic event was observed during in vitro translation of ORF B (3165 codons in length) resulting in the release of a 48 kd polypeptide from the amino-terminal portion of the ORF B-encoded polyprotein. These results are discussed in terms of the opportunities they provide for elucidating the molecular basis of transmissible hypovirulence and possible origins of hypovirulence-associated dsRNAs.

Amino Acid Sequence↗

The contribution of defective RNAs to the complexity of viral-encoded double-stranded RNA populations present in hypovirulent strains of the chestnut blight fungus Cryphonectria parasitica.

Hypovirulent strain EP713 of the chestnut blight fungus Cryphonectria (Endothia) parasitica harbors a family of viral encoded double-stranded (ds) RNAs thought to be responsible for the hypovirulence phenotype. These include L-dsRNA, described in the accompanying paper (Shapira et al., 1991); several prominent species in the estimated size range of 8 to 10 kb, referred to here as M-dsRNAs; and several smaller species designated S-dsRNAs which range in size from approximately 0.6 to 1.7 kb. The characterization of the M- and S-dsRNA species is the subject of this report. Results from polymerase chain reaction mapping and molecular hybridization analysis indicate that the M- and S-dsRNA species are internally deleted forms of L-dsRNA. Three different S-dsRNA species were cloned and sequenced. Each species contained a single deletion breakpoint and retained either 149, 155 or 156 bp of the terminus corresponding to the 5'-end of the coding strand and 440, 447 or 449 bp of the other terminus. Two of the S-dsRNA species contained, within the boundaries of the breakpoint, additional sequence information consisting of 42 bp or 95 bp that appeared to be unrelated to the L-dsRNA sequence. These results demonstrate that defective RNAs contribute significantly to the complexity of dsRNA populations found in hypovirulent strains of C. parasitica and provide a first approximation of the location of cis-acting signals involved in their replication.

Base Sequence↗

A viral gene confers hypovirulence-associated traits to the chestnut blight fungus.

A viral double-stranded (ds)RNA associated with reduced virulence (hypovirulence) and the accompanying biological control of the chestnut blight fungus, Cryphonectria parasitica, was shown recently to contain two contiguous coding domains designated ORF A and ORF B. We report here that transformation of an isogenic virulent, dsRNA-free C. parasitica strain with a cDNA copy of ORF A conferred traits similar to those exhibited by the dsRNA-containing hypovirulent strain: characteristics included reduced pigmentation, reduced laccase accumulation and suppressed conidiation. However virulence was not reduced, indicating an apparent uncoupling of associated traits from hypovirulence. These results establish a direct cause and effect relationship between a viral dsRNA genetic element present in a hypovirulent C. parasitica strain and specific phenotypic traits. They demonstrate further that these traits are not the result of a general reaction of the fungus to the presence of the replicating viral RNA, but are caused by a specific viral coding domain.

Genes, Viral↗

1H and 13C NMR assignments for two new xanthones from the endophytic fungus Xylaria sp. FRR 5657.

The complete 1H and 13C NMR assignments are reported for the new natural products, 7-hydroxy-3-(hydroxymethyl)-1-methoxy-9H-xanthen-9-one (1) and 2,5-dihydroxy-8-methoxy-6-methyl-9-oxo-9H-xanthene -1-carboxylic acid (2). Both of these secondary metabolites were isolated from the fermentation culture of the endophytic fungus Xylaria sp. FRR 5657. 1D and 2D NMR experiments that included 1H, gCOSY, gHSQC and gHMBC were used for the determination of the structure and assignment of these xanthones.

Carbon Isotopes↗

Sequential oxygenation of linoleic acid in the fungus Gaeumannomyces graminis: stereochemistry of dioxygenase and hydroperoxide isomerase reactions.

Linoleic acid is sequentially oxygenated to (7S,8S)-dihydroxylinoleic acid by dioxygenase and hydroperoxide isomerase activities present in the fungus Gaeumannomyces graminis (Brodowsky, I. D., Hamberg, M., and Oliw, E. H., J. Biol. Chem. 267, 14738-14745 (1992)). Linoleic acids stereospecifically deuterated at C-7 and C-8 were prepared by biological desaturation of the corresponding stearates and used to determine the stereochemistry of the hydrogen abstractions occurring in the dioxygenase- and hydroperoxide isomerase-catalyzed reactions. The dioxygenase reaction was found to involve stereospecific abstraction of the pro-S hydrogen from C-8 followed by antarafacial insertion of dioxygen to produce (8R)-hydroperoxylinoleic acid. The hydroperoxide isomerase reaction consisted of conversion of (8R)-hydroperoxylinoleic acid into (7S,8S)-dihydroxylinoleic acid by stereospecific elimination of the pro-S hydrogen from C-7 and intramolecular suprafacial insertion of oxygen at C-7. Accordingly, during the conversion of linoleic acid into (8R)-hydroperoxylinoleic acid, the absolute configuration of C-8 was inverted, while the conversion of (8R)-hydroperoxylinoleic acid into (7S,8S)-dihydroxylinoleic acid occurred with retention of absolute configuration at C-7.

Deuterium↗

Efficient synthesis of a 72-kDa mitochondrial polypeptide using the yeast Ty expression system.

Using the Ty system from yeast we report the efficient expression of a heterologous eukaryotic gene encoding a 72 kDa mitochondrial polypeptide. The pFM2IIBgIII expression vector was initially modified for this purpose by inserting the factor X(a) protease cleavage site. The TyA gene, which encodes the structural component of the yeast virus-like particles (VLPs), and the eukaryotic yst1 gene, encoding a 72 kDa mitochondrial tyrosyl-tRNA synthetase from the filamentous fungus Podospora anserina, were subsequently fused to the factor X(a) cleavage site. The resulting chimeric gene, in which the two polypeptide coding sequences are separated by the factor X(a) cleavage site, was expressed in yeast. High yield expression of this foreign protein, which was isolated from yeast transformants as hybrid TyVLPs, was verified after factor X(a) treatment by SDS polyacrylamide gel electrophoresis and antibody detection. The strategy presented here should be useful for expressing a wide variety of eukaryotic genes.

Base Sequence↗