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PubMed1999

Pyruvate decarboxylase filaments are associated with the cortical cytoskeleton of asci and spores over the sexual cycle of filamentous ascomycetes.

We show that pyruvate decarboxylase (PDC) 8- to 10-nm-diameter filaments, first described in vegetative cells of Neurospora crassa, are ubiquitously present in filamentous fungi. The cellular arrangement of these structures was examined over the entire sexual cycle of the ascomycetes N. crassa, N. tetraesperma, Podospora anserina, and Sordaria macrospora. PDC-filaments were found associated with the cortical microtubule array of asci and ascospores and absent from the vicinity of spindles and spindle pole bodies. Nocodazole-induced depolymerization of the cortical microtubules results in the loss of PDC-filaments. Moreover, a S. macrospora mutant defective in cortical MT distribution shows abnormal PDC organization. Neurospora asci generated on various metabolic conditions, which modify the presence and relative abundance of PDC-filaments in vegetative cells, have identical patterns of subcellular distribution of these structures. A N. crassa mutant (snowflake) that accumulates giant bundles of PDC-filaments during vegetative growth, shows normal distribution of the filaments during ascogenesis. Thus, the regulation conditioning the presence and supramolecular assembly of PDC-filaments in Neurospora differs between vegetative and sexual cells. Taken together, these results suggest that PDC in filamentous fungi may perform two functions, intervening as an enzyme in vegetative metabolism and as a structural protein associated with the cytoskeleton during sexual development.

Actins↗
PubMedDate not supplied

Internuclear recognition: A possible connection between euascomycetes and homobasidiomycetes.

In the heterothallic Euascomycete Podospora anserina, fertilization is followed by mitotic divisions of parental nuclei, resulting in a plurinucleate stage. Nuclei of opposite mating type then recognize one another and form dikaryons which undergo karyogamy and meioisis. The internuclear recognition is a characteristic feature of the sexual cycle of filamentous Euascomycetes and is controlled by mating-type genes. These genes encode transcription factors which have nucleus-limited expression. It is assumed that this characteristic allows nuclei of different mating type to express a pattern of specific proteins directly involved in internuclear recognition. As the molecular nature of these proteins is unknown, the exact mechanism of internuclear recognition remains elusive. Schuurs et al. (1998) have proposed that internuclear distance affects gene expression through a pheromone/receptor system in the Homobasidiomycete Schizophyllum commune. This model can be applied to internuclear recognition in P. anserina and can account for all data resulting from genetic analyses.

Ascomycota↗
PubMed2000

Programmed ascospore death in the homothallic ascomycete Coniochaeta tetraspora.

Immature asci of Coniochaeta tetraspora originally contain eight uninucleate ascospores. Two ascospore pairs in each ascus survive and mature, and two die and degenerate. Arrangement of the two ascospore types in individual linear asci is what would be expected if death is controlled by a chromosomal gene segregating at the second meiotic division in about 50% of asci. Cultures originating from single homokaryotic ascospores or from single uninucleate conidia are self-fertile, again producing eight-spored asci in which four spores disintegrate, generation after generation. These observations indicate that differentiation of two nuclear types occurs de novo in each sexual generation, that it involves alteration of a specific chromosome locus, and that the change occurs early in the sexual phase. One, and only one, of the two haploid nuclei entering each functional zygote must carry the altered element, which is segregated into two of the four meiotic products and is eliminated when ascospores that contain it disintegrate. Fusion of nuclei cannot be random-a recognition mechanism must exist. More study will be needed to determine whether the change that is responsible for ascospore death is genetic or epigenetic, whether it occurs just before the formation of each ascus or originates only once in the ascogonium prior to proliferation of ascogenous hyphae, and whether it reflects developmentally triggered alteration at a locus other than mating type or the activation of a silent mating-type gene that has pleiotropic effects. Similar considerations apply to species such as Sclerotinia trifoliorum and Chromocrea spinulosa, in which all ascospores survive but half the spores in each ascus are small and self-sterile. Unlike C. tetraspora, another four-spored species, Coniochaetidium savoryi, is pseudohomothallic, with ascus development resembling that of Podospora anserina.

Chromosomes, Fungal↗
PubMed1994

On the identification of group II introns in nucleotide sequence data.

Four different consensus sequences (GTI, group II identifiers) have been derived from domains V of known group II introns and are used as query input sequences for sensitive database screenings with the FASTA and LFASTA programs. The set of four GTI sequences can identify all domains V of the 96 known group II introns in the completely sequenced chloroplast genomes of Marchantia polymorpha, Epifagus virginiana, Oryza sativa, Nicotiana tabacum and the completely sequenced mitochondrial genomes of Saccharomyces cerevisiae, Podospora anserina, Schizosaccharomyces pombe and Marchantia polymorpha. Seven moderately high-scoring hits can easily be rejected as false-positives since they do not fulfil secondary structure requirements. Large FASTA outputs obtained after screening the entire nucleotide sequence database are evaluated in a second step by a program (D5SCAN) that allows the assignment of variable selection criteria for potential domain V secondary structures. Database searches with these routines yield evidence for several group II intron sequences previously unrecognized. These include novel intron structures in the cyanobacterium Synechocystis and in the mitochondrial genomes of Marchantia, soybean, pea, broad bean, sugar beet and a heterobasidiomycete. Potential intron remnants are found contributing to the secondary structure of rRNAs in several trypanosome species. At a given sensitivity of 95% positively identified true domains V, the search routine produces one false positive hit per 10,000 kb.

Amino Acid Sequence↗
PubMed1994

Transposable group II introns in fission and budding yeast. Site-specific genomic instabilities and formation of group II IVS plDNAs.

The recent report on RNA-mediated group II intron (IVS, intervening sequence) transposition in mitochondria (mt) of Saccharomyces cerevisiae and Podospora anserina and the demonstration of reverse transcriptase (RT) activity encoded by the mobile S. cerevisiae intron cox1-aI1 suggests that group II introns constitute a new class of site-specific retro-like (retroid) elements. This is supported by the finding that the mitochondrial cob1-bI1 intron from the fission yeast Schizosaccharomyces pombe, encoding an RT-like open reading frame, is transposed in mtDNA populations. In agreement with the involvement of an RNA-intermediate in IVS transposition: First, the insertion sites were preceded by at least an IBS1-like (intron binding site) motif, which corresponds to the upstream exon and suffices to form the IBS1/EBS1 (EBS: exon binding site) base-pairing interactions. Second, intron transposition was conservative with respect to sequences flanking the insertion sites. We formulated the hypothesis that transient IVS insertion at non-allelic sites followed by recombination can be viewed as a general molecular mechanism, applicable equally well to site-specific genomic instabilities involving splice-site borders of group II introns and to the formation of extra-genomic IVS plasmid DNAs (plDNAs). We used polymerase chain reaction (PCR) techniques to detect infrequent rearrangements in mtDNA and report here on duplicative IVS transposition, twintron formation (e.g. bI1 insertion into another bI1 intron), and IVS insertions at canonical 5' exon-intron borders in S. pombe (cob1-bI1) and in S. cerevisiae (cox1-aI1). These data substantiate the concept that group II intron homing, IVS transposition and circular IVS plDNA formation involve a common RNA-mediated mechanism. Finally, the findings suggest that extra-genomic group II IVS copies are not restricted to senescence mycelia of P. anserina, but constitute natural components of group II IVS-containing genomes.

Apoproteins↗
PubMed1992

The wheat mitochondrial genome contains an ORF showing sequence homology to the gene encoding the subunit 6 of the NADH-ubiquinone oxidoreductase.

A region of the mitochondrial (mt) DNA of wheat was studied because of its homology with other plant mtDNAs. Sequence analysis revealed an open reading frame encoding a polypeptide of 247 amino acids. Comparison of the sequence of the putative polypeptide with the protein sequence data of the Swiss-Prot library reveals homology with subunit 6 of the NADH-ubiquinone complex of mitochondria from Marchantia polymorpha, Podospora anserina, Chlamydomonas reinhardtii and of chloroplasts from M. polymorpha and Oryza sativa. No similarity was detected when compared with the subunit 6 of animal mitochondria, probably due to the rapid evolution of the sequence. A single 1.2 kb transcript appears in northern RNA blots. We found 15 edited sites of which only 13 give amino acid changes. This is the first report of a mt nad6 gene in higher plants.

Amino Acid Sequence↗
PubMed1995

Genetic transformation of the pathogenic fungus Wangiella dermatitidis.

Genetic transformation of Wangiella dermatitidis was studied using three plasmid vectors (pAN7-1, pWU44, and pKK5) and both electroporation and polyethyleneglycol-mediated methods. pAN7-1 contains the E. coli hygromycin B (HmB) phosphotransferase (hph) gene. Expression of the hph gene confers resistance to antibiotic HmB. Selection for resistance, indicative of transformation, resulted in 10 203 HmB-resistant colonies/micrograms pAN7-1 on medium containing 100 micrograms HmB/ml. Strains of W. dermatitidis used in this study have innate sensitivity to HmB at a critical inhibitory concentration of 20-40 micrograms/ml. Vectors pWU44 and pKK5 contain a URA5 gene from Podospora anserina. A ura5 auxotroph of W. dermatitidis was transformed to prototrophy with pWU44 or pKK5 by complementation. Transformation frequencies for these two plasmids were between 17-50 transformants/micrograms vector DNA. Southern blotting analysis and polymerase chain reaction detection of DNA from putative transformants confirmed transformation.

Base Sequence↗
PubMed1990

Development of a genetic transformation system for Histoplasma capsulatum: complementation of uracil auxotrophy.

We have developed a simple and efficient transformation system for the dimorphic fungus Histoplasma capsulatum. Mutants of H. capsulatum defective in orotidine-5'-monophosphate pyrophosphorylase were transformed to prototrophy by the cloned URA5 gene of the filamentous fungus Podospora anserina. Abortive and mitotically stable transformants were obtained. The stable transformants had integrated copies of the plasmid, some in tandem head-to-tail orientation. Free plasmid identical to the transforming plasmid was present in some of the transformants. We obtained a transformation efficiency of up to 30 transformants/micrograms DNA for plasmid pPAura5-1 (9.2 kb). pPW2001, a smaller plasmid (4.7 kb) derived from pPAura5-1, transformed H. capsulatum more efficiently (up to 155 transformants/micrograms DNA).

DNA Replication↗
PubMed1993

Cotranscriptional expression of mitochondrial genes for subunits of NADH dehydrogenase, nad5, nad4, nad2, in Marchantia polymorpha.

Three genes for the subunits of the NADH dehydrogenase (nad5, nad4, and nad2) are tandemly clustered on the liverwort mitochondrial genome. Their gene products showed high levels of amino acid sequence identity with the corresponding subunits from higher plant mitochondria (82.8-84.4%), and significant levels of identity with those from liverwort chloroplast (32.0-33.5%). Podospora anserina mitochondria (21.4-45.9%), and human mitochondria (18.4-27.9%). In addition, these three subunits from liverwort mitochondria have conserved amino acid residues in their central regions. The gene nad5 is interrupted by a 672 bp group I intron, while genes nad4 and nad2 are interrupted by group II introns of 899 bp and 1418 bp, respectively. Northern blot analysis using exon-intron specific probes indicated that these three genes are transcribed as a single precursor mRNA of 9.6 kb in length and are processed into mature mRNA molecules in liverwort mitochondria. Several regions of this nad gene cluster are repeated in the liverwort mitochondrial genome.

Amino Acid Sequence↗
PubMed1993

Cloning and analysis of the mating type genes from Cochliobolus heterostrophus.

Cochliobolus heterostrophus, a heterothallic Ascomycete, has a single mating type locus with two alternate forms called MAT-1 and MAT-2. MAT-1 was cloned by complementing a MAT-2 strain using a cosmid library from a MAT-1 strain and screening for a homothallic transformant. The cosmid recovered from this transformant was able to re-transform a MAT-2 strain to homothallism and MAT identity was proven by restriction fragment length polymorphism and conventional genetic mapping. All homothallic transformants could mate with either MAT-1 or MAT-2 strains, although the number of ascospores produced by self matings or crosses to MAT-2 strains was low. Progeny of selfed homothallic transformants were themselves homothallic. MAT-2 was cloned by probing a cosmid library from a MAT-2 strain with a fragment of insert DNA from a MAT-1 cosmid. A 1.5 kb subclone of either MAT-containing cosmid was sufficient to confer mating function in transformants. Examination of the DNA sequence of these subclones revealed that MAT-1 and MAT-2 contain 1297 bp and 1171 bp, respectively, of completely dissimilar DNA flanked by DNA common to both mating types. Putative introns were found (one in each MAT gene) which, when spliced out, would yield open reading frames (ORFs) that occupied approximately 90% of the dissimilar DNA sequences. Translation of the MAT-1 ORF revealed similarity to the Neurospora crassa MATA, Podospora anserina mat-, and Saccharomyces cerevisiae MAT alpha 1 proteins; translation of the MAT-2 ORF revealed similarity to the N. crassa MATa, P. anserina mat+, and Schizosaccharomyces pombe mat-Mc proteins. These gene products are all proven or proposed DNA binding proteins. Those with similarity to MAT-2 are members of the high mobility group.

Amino Acid Sequence↗
PubMed1992

The PYR1 gene of the plant pathogenic fungus Colletotrichum graminicola: selection by intraspecific complementation and sequence analysis.

A spontaneous uridine-requiring auxotroph of Colletotrichum graminicola was recovered by selection for resistance to 5-fluoro-orotic acid. The auxotroph lacked orotate phosphoribosyl transferase (OPRTase) and was complemented with a clone from a cosmid library of C. graminicola DNA. A 3.1 kb HindIII-SalI fragment was subcloned from the cosmid and it could efficiently transform the auxotrophic strain to uridine prototrophy and integrate by site-specific recombination. This DNA fragment contains an open reading frame that is similar to OPRTase genes of the fungi Sordaria macrospora, Trichoderma reesei, Podospora anserina, and Saccharomyces cerevisiae. Based on the sequence similarities and the ability to restore uridine prototrophy, we conclude that the fragment contains the C. graminicola gene for OPRTase, which we have named PYR1. Our results demonstrate that cloning by complementation is feasible in C. graminicola, that the gene for OPRTase from C. graminicola can be useful as a selectable marker in transformation of the fungus, and that the OPRTase gene product is similar to OPRTase from other fungi.

Amino Acid Sequence↗
PubMed1994

The Claviceps purpurea glyceraldehyde-3-phosphate dehydrogenase gene: cloning, characterization, and use for the improvement of a dominant selection system.

The GPD 1 gene of Claviceps purpurea coding for glyceraldehyde-3-phosphate dehydrogenase was cloned and sequenced, including 1,800 bp of its 5' upstream region. This gene shows an identical structure to the gpd gene of Podospora anserina and Cryphonectria parasitica (one intron at an identical position) with high homology at both the DNA and amino-acid levels. Two fragments of the promoter spanning from the ATG to -500 bp and to -1,400 bp were fused to the phleomycin-resistance gene. Both constructs transformed C. purpurea at a high rate. The enhanced expression of the long vector construct indicates the presence of additional elements between -500 bp and -1,400 bp upstream of the initiation codon.

Amino Acid Sequence↗
PubMed1994

Linear plasmids, pLm9 and pLm10, can be isolated from the phytopathogenic ascomycete Leptosphaeria maculans by pulsed-field gel electrophoresis.

Two linear DNA plasmids (pLm9 and pLm10, sized 9 and 10 kb respectively) were isolated from the phytopathogenic ascomycete Leptosphaeria maculans, using pulsed-field gel electrophoresis. pLm9 and pLm10 are found only in aggressive isolates of L. maculans but, because aggressive and non-aggressive strains appear to be different species, these plasmids are probably not involved in pathogenicity. pLm9 and pLm10 copurify with a mitochondrially-enriched cell fraction, and do not hybridise to chromosomal or mitochondrial DNA, or to each other. Exonuclease digestions suggest that both these molecules contain covalently-bound proteins at their 5' termini. pLm9 hybridises to the RNA polymerase of a linear plasmid from the ascomycete Podospora anserina, and pLm10 hybridises to the DNA polymerase from the same P. anserina plasmid, suggesting that pLm9 and pLm10 encode their own replication and transcription enzymes.

Ascomycota↗
PubMed1993

Isolation and transformation of uracil auxotrophs of the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Uracil auxotrophs of Phanerochaete chrysosporium were isolated using 5-fluoroorotate resistance as a selection scheme. The ura3 auxotrophs deficient in orotidylate decarboxylase and ura5 auxotrophs deficient in orotate phosphoribosyl transferase were characterized by enzyme assays and complementation tests. The ura5 auxotrophs were transformed to prototrophy with the ura5 gene from the ascomycete Podospora anserina. The ura3 auxotrophs were transformed to prototrophy with the ura3 gene from the basidiomycete Schizophyllum commune. The P. chrysosporium ura3 gene was isolated from a lambda EMBL3 genomic library using the S. commune ura3 gene as a probe. A 6.6-kb fragment incorporating the ura3 gene was subcloned into Bluescript SK+(pURA3.1) and used to transform P. chrysosporium ura3 auxotrophic strains. The pURA3.1 insert was mapped for restriction sites and the approximate location of the ura3 gene within the insert was determined. Double auxotrophic strains were transformed with either of two marker genes and the resulting single auxotrophic strains were crossed to demonstrate genetic recombination between two nuclei of identical genetic background.

Ascomycota↗
PubMed1990

Cloning and sequencing of URA10, a second gene encoding orotate phosphoribosyl transferase in Saccharomyces cerevisiae.

Orotate phosphoribosyl transferase (OP-RTase) catalyses the transformation of orotate to OMP in the pyrimidine pathway. In the yeast Saccharomyces cerevisiae, the URA5 gene is known to encode this enzyme activity. In this paper we present the cloning and sequencing of a yeast gene, named URA10, encoding a second OPRTase enzyme. Comparison of the predicted amino acid sequences between URA5 and URA10 genes shows more than 75% similarity. These sequences have also been compared to those of Escherichia coli, Podospora anserina, Sordaria macrospora and Dictyostelium discoideum. Remarkable similarities in the primary structure of these proteins have been found. Gene disruption experiments revealed that URA10 gene expression is responsible for the leaky phenotype of a ura5 mutant. Assays of OPRTase activity in extracts from ura5 and ura10 mutants indicate that the URA10 product contributes only 20% of the total activity found in wild type cells.

Amino Acid Sequence↗
PubMedDate not supplied

Cloning of an Aspergillus niger invertase gene by expression in Trichoderma reesei.

The filamentous fungus Aspergillus niger produces two glycosylated forms of the sucrose-hydrolysing enzyme, invertase. In contrast, some Trichoderma species lack invertase and are unable to utilise sucrose as a sole carbon source. Using an A. niger genomic library constructed in a cosmid vector containing the ura5 gene of Podospora anserina as a selectable marker, and the T. reesei ura5- strain as a sucrose-minus recipient strain, an A. niger invertase gene (suc1) has been cloned by a sib selection procedure. PAGE and enzyme analysis confirmed that transformants had acquired invertase activity. The cloned gene contained DNA sequences which were complementary to the amino-acid sequences of tryptic peptides found in invertase purified from A. niger. The suc1 invertase gene can be used as a dominant selectable marker for the transformation of Trichoderma strains.

Aspergillus niger↗
PubMed1994

Senescence-specific mitochondrial DNA molecules in P. anserina: evidence for transcription and normal processing of the RNA.

In Podospora anserina the phenomenon of senescence was previously shown to be correlated with the presence of senescence-specific circular DNAs (senDNAs), resulting from the amplification of distinct regions (alpha, beta, gamma and epsilon) of the mitochondrial chromosome. The beta region gives rise to senDNAs with variable sizes, but sharing a 1-kb common sequence. Here, we present a molecular analysis of five beta senDNAs. We have determined the nucleotide sequence around the circularization site of each senDNA monomer. In two cases, the presence of a tRNA gene, very close to the 3' end of the monomer, has been observed. This suggests that some beta senDNAs could be generated via a reverse transcription step. We have furthermore shown that the beta senDNAs produce specific transcripts which undergo normal processing of their introns. We propose that a transcription start site, located in the beta common region, is involved in mitochondrial replication allowing the amplification of the beta senDNAs.

Base Sequence↗
PubMed1989

Structure and expression of the URA5 gene of Saccharomyces cerevisiae.

The URA5 gene of Saccharomyces cerevisiae encodes orotate phosphoribosyl transferase (EC 2.4.2.10; OPRTase) which catalyses the transformation of orotate to OMP in the pyrimidine pathway. We present in this paper the cloning and the sequencing of this gene, the last in the yeast pyrimidine pathway to be cloned. We have deduced the protein sequence of the OPRTase of S. cerevisiae from the DNA sequence and compared it to that of Escherichia coli, Podospora anserina and Dictyostelium discoideum. Some important similarities in the structure of these four proteins have been found. Finally, we have quantified the transcription of the URA5 gene in different physiological conditions and confirmed that it was not under the control of UTP or any intermediary product of the pathway.

Amino Acid Sequence↗