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The ura5 gene of the ascomycete Sordaria macrospora: molecular cloning, characterization and expression in Escherichia coli.

We cloned the ura5 gene coding for the orotate phosphoribosyl transferase from the ascomycete Sordaria macrospora by heterologous probing of a Sordaria genomic DNA library with the corresponding Podospora anserina sequence. The Sordaria gene was expressed in an Escherichia coli pyrE mutant strain defective for the same enzyme, and expression was shown to be promoted by plasmid sequences. The nucleotide sequence of the 1246-bp DNA fragment encompassing the region of homology with the Podospora gene has been determined. This sequence contains an open reading frame of 699 nucleotides. The deduced amino acid sequence shows 72% similarity with the corresponding Podospora protein.

Amino Acid Sequence↗

Methods for the in vivo and in vitro analysis of [Het-s] prion infectivity.

Prions have been described in mammals and fungi. The [Het-s] infectious genetic element of the filamentous fungus Podospora anserina is the prion form of the HET-s protein. This protein is involved in the control of a cell death reaction termed heterokaryon incompatibility. The infectious form of HET-s corresponds to a self-perpetuating amyloid. The purpose of the present paper is to describe the techniques that can be used to analyse [Het-s] prion propagation in vivo and HET-s amyloid aggregation in vitro. In addition, we report several methods that can be used to infect Podospora with recombinant HET-s amyloid.

Amyloid↗

Peroxide accumulation and cell death in filamentous fungi induced by contact with a contestant.

Podospora anserina and Coprinopsis cinerea (syn. Coprinus cinereus) are endowed with a defence system able to differentiate self vs. non-self and involving the generation of peroxide. Indeed, they produce peroxide when confronted with a filamentous fungus, only in non-self confrontations. Both species are not able to recognize yeasts and show a differential response to bacteria. The accumulation of peroxides in the ascomycete Podospora anserina requires an NADPH oxidase and a MAP kinase cascade, previously shown to be involved in fruit body formation, cell differentiation and cell degeneration. Confrontation is accompanied by the death of the contestant hyphae only in specific combinations of species. As in animals and plants, data suggest that peroxide is likely involved in signalling rather than playing a direct toxic role. Fungi display more complex behaviours than generally acknowledged, i.e. they are able to recognize potential contestants and built up defence reactions involving evolutionary conserved enzymes.

Coprinus↗

Two new fungal inteins.

Until recently the only intein known to be encoded by the nuclear genome of a eukaryote was the VMA intein in the vacuolar ATPase precursor of several species of saccharomycete yeast. This intein has been intensively studied and much information has been gained about its structure, mode of action and evolutionary history. We recently reported a second nuclear intein, Cne PRP8, encoded within the PRP8 gene of the basidiomycete Cryptococcus neoformans. Subsequent studies have found allelic PRP8 inteins in several species of yeast and filamentous ascomycetes. Here we report two further, non-allelic, inteins from ascomycete species. The yeast Debaryomyces hansenii (which also has a VMA intein) has an intein encoded within the sequence of the glutamate synthase gene (GLT1). There are also inteins encoded in the homologous GLT1 genes of the yeast Candida (Pichia) guilliermondii and the filamentous fungus Podospora anserina. These allelic GLT1 inteins occupy exactly the same site in the glutamate synthase and all contain domains that indicate the presence of a homing endonuclease (HEG). Podospora anserina, in addition, contains a second, non-allelic, intein encoded in the chitin synthase gene (CHS2); this intein also contains a HEG domain. We describe the phylogenetic relationships among the four eukaryote nuclear encoded inteins (VMA, PRP8, GLT1 and CHS2). We also consider this phylogeny in the broader context of eubacterial, archaeal and eukaryote viral and organelle inteins.

Amino Acid Sequence↗

A group-I intron in the mitochondrial small subunit ribosomal RNA gene of Sclerotinia sclerotiorum.

A 1,380-bp intervening sequence within the mitochondrial small subunit ribosomal RNA (mt SSU rRNA) gene of the fungus Sclerotinia sclerotiorum has been sequenced and identified as a group-I intron. This is the first report of an intron in the mt SSU rRNA gene. The intron shows close similarity in secondary structure to the subgroup-IC2 introns from Podospora (ND3i1, ND5i2, and COIi5) and Neurospora (ND5i1). The intron has an open reading frame (ORF) that encodes a putative protein of 420 amino acids which contains two copies of the LAGLI-DADG motif. The ORF belongs to a family of ORFs identified in Podospora (ND3i1, ND4Li1, ND4Li2, ND5i2, and COIi5) and Neurospora (ND5i1). The putative 420-aa polypeptide is also similar to a site-specific endonuclease in the chloroplast large subunit ribosomal RNA (LSU rRNA) gene of the green alga Chlamydomonas eugametos. In each clone of S. sclerotiorum examined, including several clones which were sampled over a 3-year period from geographically separated sites, all isolates either had the intron or lacked the intron within the mt SSU rRNA gene. Screening by means of Southern hybridization and PCR amplification detected the intron in the mt SSU rRNA genes of S. minor, S. trifoliorum and Sclerotium cepivorum, but not in other members of the Sclerotiniaceae, such as Botrytis anamorphs of Botryotinia spp., or in other ascomycetous and basidiomycetous fungi.

Amino Acid Sequence↗

Coprophilous fungi of the horse.

A total of 1267 microfungi, including 35 Myxomycetes, were recorded from the fecal samples of the 60 horses; of these 395 were found on 20 saddle-horse feces, 363 on 20 race-horses and 509 on 20 working horses. Eighty two species representing 53 genera were recorded; of these 7 were Zygomycetes, 18 Ascomycetes, 1 Basidiomycetes and 25 Fungi Imperfecti: 2 Myxomycetes. Common coprophilous fungi are in decreasing order Pilobolus kleinii, Saccobolus depauperatus, Mucor hiemalis, Lasiobolus ciliatus, Podospora curvula, Petriella guttulata, M. circinelloides, Coprinus radiatus, Dictyostelium mucoroides, Sordaria fimicola, C. miser, C. stercorariusm, Acremonium sp., Coprotus granuliformis, Graphium putredinis, Iodophanus carneus, Chaetomium murorum, Podospora communis, P. inaequalis, P. setosa, Saccobolus versicolor and Cladosporium cucumerinum. Species of Myrothecium verrucaria, Actinomucor elegans, Kernia nitida, Spiculostilbella dendritica and Mucor parvispora were found exclusively in working-horses feces. Badhamia sp., Anixiopsis stercoraria, Echinobotryum state of D. stemonitis, Geotrichum candidum and Oidiodendron sp. were found only in saddle-horses feces. Chlamidomyces palmarum, Philocopra sp. were found exclusively in race-horses feces. Notes on infrequent or interesting fungi include Thamnostylum piriforme, Phialocephala dimorphospora, Rhopalomyces elegans and Spiculostilbella dendritica.

Animals↗