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At least 19 recordsLinked to original sources
PubMed1992

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↗
PubMed1990

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1992

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↗
PubMed1994

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↗
PubMed1996

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↗
PubMed1995

Phylogenetic ordinal placement based on rDNA sequences of the freshwater genera Ophioceras and Pseudohalonectria.

The ordinal placement of two closely related freshwater genera, Ophioceras and Pseudohalonectria, was assessed by using phylogenetic analysis of morphological characters, partial sequences of the large subunit ribosomal DNA and restriction site variations in the internal transcribed spacer (ITS). The two genera have some morphological features that are used to define taxa in both the Sordariales and Diaporthales, and, hence, their phylogenetic relationships are unclear. Equally weighted analyses of thirty-eight morphological characters produced unresolved phylogenetic trees and unequivocal conclusions could not be drawn based on the morphological data. The polymerase chain reaction-amplified ITS region was variable in length between the two genera and restriction sites in the ITS region were determined. Analysis of variation in restriction sites in the ITS region placed Ophioceras and Pseudohalonectria in one clade with taxa sampled from Sordariales. About 350 basepairs of DNA sequence from the 5' end of the large subunit rDNA were also determined. In phylogenetic analysis of the sequence data with Hypocrea lutea and Nectria cinnabarina as outgroups, Ophioceras and Pseudohalonectria showed a closer relationship to Neurospora crassa, Schizothecium sp., and Sordaria fimicola of the Sordariales than to Cryphonectria parasitica and Endothia gyrosa of the Diaporthales.

Ascomycota↗
PubMed1995

Thermodynamic mapping of the inhibitor site of the aspartic protease endothiapepsin.

The discovery that the protease from the human immunodeficiency virus (HIV) belongs to the aspartic protease family has generated renewed interest in this class of proteins. In this paper, the interactions of endothiapepsin, an aspartic proteinase from the fungus Endothia parasitica, with the inhibitor pepstatin A have been studied by high-sensitivity calorimetric techniques. These experiments have permitted a complete characterization of the temperature and pH-dependence of the binding energetics. The binding reaction is characterized by negative intrinsic binding enthalpy and negative heat capacity changes. The association constant is maximal at low pH (2 x 10(9) M-1 at pH 3) but decreases upon increasing pH (8.1 x 10(6) M-1 at pH 7). The binding of the inhibitor is coupled to the protonation of one of the aspartic moieties in the Asp dyad of the catalytic site of the protein. This phenomenon is responsible for the decrease in the apparent affinity of the inhibitor for the enzyme upon increasing pH. The experimental results presented here indicate that the binding of the inhibitor is favored both enthalpically and entropically. While the favorable enthalpic contribution is intuitively expected, the favorable entropic contribution is due to the large gain in solvent-related entropy associated with the burial of a large hydrophobic surface, that overcompensates the loss in conformational and translational/rotational degrees of freedom upon complex formation. The characteristics of the molecular recognition process have been evaluated by means of structure-based thermodynamic analysis. Three regions in the protein contribute significantly to the free energy of binding: the residues surrounding the Asp dyad (Asp32 in the N-terminal lobe and Asp215 in the C-terminal domain) and the flap region (Ile73 to Asp77). In addition, the rearrangement of residues that are not in immediate contact with the inhibitor provides close to 40% of the protease contribution to the binding free energy. On the other hand, the two statine residues provide more than half of the inhibitor contributions to the total free energy of binding. It is demonstrated that a previously developed empirical structural parametrization of the thermodynamic parameters that define the Gibbs energy, accurately accounts for the binding energetics and its temperature and pH-dependence.

Aspartic Acid Endopeptidases↗
PubMed1994

A viral dsRNA element of the chestnut blight fungus with a distinct genetic organization.

We have sequenced overlapping complementary DNA clones representing the viral double-stranded (ds) RNA from hypovirulent strain NB58 of the chestnut blight fungus Cryphonectria parasitica. Cryphonectria hypovirus 2-NB58 (CHV2-NB58) dsRNA contains 12,507 base pairs, excluding the poly(A) tail at the 3' end of the plus strand, and is organizationally similar to the largest dsRNA from the virus of strain EP713 (CHV1-713; identical to HAV; Shapira et al., (1991), EMBO J. 10, 731-739). CHV2-NB58 and CHV1-713 dsRNAs share approximately 60% nucleotide sequence identity. On the poly(A)-containing strand of CHV2-NB58, a 487-residue nontranslated region precedes two open reading frames, designated ORF A (438 codons) and ORF B (3291 codons). The connecting pentanucleotide sequence UAAUG (1802-1806) terminates ORF A and initiates ORF B. In contrast to the 69-kDa ORF A product of CHV1-713, the 50-kDa CHV2-NB58 ORF A product did not undergo autoproteolysis under the conditions tested, nor were motifs associated with cysteine proteases present in the CHV2-NB58 ORF A sequence. CHV2-NB58 ORF B products appear to be homologous with CHV1-713 ORF B products, and the motifs involved in autoproteolysis of the N-terminal 48 kDa of CHV1-713 ORF B were identified in the CHV2-NB58 ORF B product. Motifs associated with RNA polymerase and helicase activities were highly conserved between CHV2-NB58 and CHV1-713 and were found at similar genomic positions in the C-terminal half of ORF B.

Amino Acid Sequence↗
PubMed1994

cDNA-derived hypovirus RNA in transformed chestnut blight fungus is spliced and trimmed of vector nucleotides.

Unencapsidated double-stranded viral RNAs belonging to the genus Hypovirus attenuate virulence of the chestnut blight fungus, Cryphonectria parasitica. A full-length cDNA clone of hypovirus CHV1-713 double-stranded RNA was recently shown to be infectious when introduced into the C. parasitica genome by DNA-mediated transformation. In this study, we show that the viral RNA derived from the chromosomally integrated cDNA copy is trimmed of extraneous vector nucleotide sequences. The cDNA-derived viral RNA was also found to contain a 73-bp deletion located within the 5'-noncoding leader sequence as a result of a pre-mRNA splicing event. Implications of these results are discussed in terms of hypovirus RNA replication and anticipated field studies involving engineered hypovirulent C. parasitica strains.

Base Sequence↗
PubMed1977

X-ray analysis and circular dichroism of the acid protease from Endothia parasitica and chymosin.

The structure of an acid proteinase from Endothia parasitica has been solved by x-ray diffraction using multiple isomorphous replacement. A 3 A resolution map was interpreted in terms of a bilobal structure with a long 25 A cleft. The secondary structure is mostly distorted beta-sheet. The circular dichroism was measured and model curves for different secondary structures were fitted by least squares indicating a large component of beta-structure. The structure was seen to be homologous with that of the acid proteinase from R. Chinensis and hence with pepsin and chymosin. A rotation function against diffraction data from chymosin crystals confirm confirm this and suggested an approach to the solution of this structure.

Ascomycota↗
PubMed1994

DNA markers linked to Malus floribunda 821 scab resistance.

Breeding resistant apple plants is an alternative way to control fungal pathogens reducing the environmental impact due to the use of pesticides. The breeding of apple cultivars resistant to Venturia inaequalis could be much improved by marker-assisted selection. A molecular marker closely linked to the resistance locus called Vf could replace selection based on infection studies. To find such molecular markers, DNA of progenies from crossings of a resistant and a susceptible apple tree was subject to bulked segregant analysis. Two markers were found with a genetic distance of 10.6% and 19.7% recombination frequency to the Vf locus.

DNA Primers↗
PubMed1980

A new mutant form of the ribosomal protein L21 in the fungus Podospora anserina: identification of the structural gene for this protein.

A muation in the cyR1 gene of the fungus Podospora anserina confers resistance to cycloheximide and leads to an alteration of the 60S ribosomal protein L21 (Bégueret et al. 1977). Nine revertants of this mutant were isolated and the properties of these strains were analyzed. It was found that one revertant strain contains a new mutant form of L21. It is proposed that cyR1 gene is the structural gene for protein L21 and that the alteration of this protein is responsible for the resistance to cycloheximide in vivo.

Ascomycota↗
PubMed1981

Mutations affecting translational fidelity in the eucaryote Podospora anserina: characterization of two ribosomal restrictive mutations.

Fifty-nine mutations that restrict suppressor efficiency were selected in the fungus Podospora anserina using four different screening methods. Previous genetic analysis has shown that these antisuppressors lie in six loci and that they could be similar to ribosomal restrictive mutations known in Escherichia coli. The present study deals with the response of two of them, AS1-1 and AS6-1, to paromomycin and low temperature both in vivo and in vitro. The data demonstrate that ribosomes of the mutant and double-mutant strains are equally resistant to the ambiguity effect of paromomycin. These data are the first demonstration of mutations that increase translational fidelity in eucaryotic organism.

Ascomycota↗
PubMed1990

Detection of a protein encoded by a class II mitochondrial intron of Podospora anserina.

In the filamentous fungus Podospora anserina, the amplification as circular DNA molecules of the first intron (intron alpha) of the CO1 mitochondrial gene, encoding the cytochrome oxidase subunit 1, is known to be strongly associated with aging of strains. In this study we have attempted to detect the protein potentially encoded by the open reading frame (ORF) contained in this intron. This was done by the Western blot technique using specific antisera raised against three polypeptides encoded by three non-overlapping fragments of this ORF adapted to the universal code and overexpressed in Escherichia coli. We examined about thirty independent subclones of Podospora derived from two different geographic races (A, s), using wild-type and mutant strains, young and senescent cultures. A 100 kDa polypeptide, encoded by the class II intron alpha, was detected in five senescent subclones which all showed strong amplification of the intronic alpha sequence (Sen DNA alpha).

Cloning, Molecular↗