Transformation to senescence with plasmid like DNA in the ascomycete Podospora anserina

Summary In the ascomycete Podospora anserina senescence through strain aging is under nucleo-cytoplasmic control and inducible in juvenile mycelia by an infective principle transferred after cytoplasmic contact via anastomoses. A specific DNA called plasmid-like (pl) DNA, present exclusively in aging mycelia, was found to be identical with this infective principle, since it was possible to transform juvenile protoplasts to senescence by using purified p1DNA. Therefore a specific function may be attributed to this ccc DNA. Its direct involvement in a genetically programed senescence is confirmed and its development as a vector for transfer of genetic information in eukaryotes can be undertaken.     

Electrophoretic karyotype of the ascomycete Podospora anserina

Summary Fractionation of intact chromosomes of the ascomycete Podospora anserina by contour-clamped homogenous electric field (CHEF) gel electrophoresis resulted in the resolution of five distinct chromosomal bands. Two of these bands migrated as doublets. Using chromosomal standards from Schizosaccharomyces pombe we estimated the size of the P. anserina genome to about 33.4 megabases (Mb). By heterologous hybridization of fractionated chromosomes the rDNA locus was identified on one of the two chromosomes migrating at about 4.9 Mb.     

A two-step protocol for efficient deletion of genes in the filamentous ascomycete Podospora anserina

Deletion of genes in Podospora anserina via conventional methods is an inefficient and time-consuming process since homologous recombination occurs normally only at low frequency (about 1%). To improve the efficiency of replacement, we adopted the two-step protocol developed for Aspergillus nidulans (Chaveroche et al. in Nucleic Acids Res 28:E97, 2000). As a prerequisite, a vector was generated containing a blasticidin resistance cassette for selection in the Escherichia coli host strain KS272 (pKOBEG) and a phleomycin resistance cassette for selection in P. anserina. A derivative of this vector, into which short (∼250 bp) PCR-generated sequences flanking the gene to be deleted have been integrated, is introduced into the E. coli host strain which contains a cosmid with the gene of interest and long 5′ and 3′ flanking sequences. Subsequently, a cosmid is reisolated from E. coli in which the gene of interest is replaced by the resistance cassette. This construct is used to transform P. anserina. The long stretches flanking the resistance cassette facilitate recombination with homologous sequences in the fungal genome and increase the efficiency of gene deletion up to 100%. The procedure is not dependent on the availability of specific auxotrophic mutant strains and may be applicable to other fungi.     

Three mitochondrial unassigned open reading frames of Podospora anserina represent remnants of a viral-type RNA polymerase gene

The mitochondrial DNA of Podospora anserina is complex, consisting of a characteristic set of genes with a large number of introns and a substantial amount of sequence of unknown function and origin. In addition, as indicated by various types of reorganization, this genome is highly flexible. Here we report the identification of three unassigned mitochondrial open reading frames (ORF P', ORF Q', ORF 11) as remnants of a rearranged viral-type RNA polymerase gene. These ORFs are not transcribed and may be derived from the integration of a linear plasmid of the type recently identified in a mutant of P. anserina.     

The mitochondrial plasmid of Podospora anserina: A mobile intron of a mitochondrial gene

Summary In the ascomycete Podospora anserina strain ageing (senescence) is caused by a mitochondrial plasmid. In juvenile mycelia it is an integral part of the mtDNA and becomes liberated during ageing. The nucleotide sequence of this plasmid and of its flanking regions was determined. It consists of 2,539 by and contains an un identified reading frame (URF) originating in the adjacent mtDNA upstream of excision point 1. Within the URF a putative 48 by autonomously replicating sequence (ars) was identified. At both excision sites of the plasmid there are two short nonidentical interrupted palindromes and a few base pairs apart from these palindromes, both upstream and downstream, two short inverted repeats are localised. The experimental data make it evident that the mt plasmid is an intron of the cytochrome c oxidase gene (subunit I) which may be excised at the DNA level and thus become the mobile infective agent causing senescence. The concept of this mobile intron and current hypotheses concerning the relationship between introns and transposons are stressed.     

Podospora anserina mutations inhibiting several developmental alternatives and growth renewal

Summary A mutation (modG) was selected on the basis of the suppression of a defect resulting from a mutation of modD gene. The modG mutation shows the same developmental consequences as modD mutations: the absence of protoperithecia and aerial hyphae, the non-renewal of growth from stationary cells and the non-germination of ascospores. Investigations of heterokaryotic mycelia showed that the action of modD and modG mutations on the formation of protoperithecia and aerial hyphae is autonomous. Furthermore, taking advantage of the thermosensitivity of a modD mutation, it was shown that the mutations have no incidence on cell differentiation prior to the achievement of stationary physiology. These findings lead to suggest the existence, in Podospora anserina, of a stage of cell totipotency, which we call the switch stage, that derives from the stationary state through the action of modD and mode genes. Accordingly, the switch stage would be the intermediate in the process of growth renewal from stationary cells and the basic stage for the selection of the developmental alternatives (aerial hyphae, protoperithecia ...) of Podospora mycelia.     

The linear mitochondrial plasmid pAL2-1 of a long-lived Podospora anserina mutant is an invertron encoding a DNA and RNA polymerase

The molecular characterization of an additional DNA species (pAL2-1) which was identified previously in a long-lived extrachromosomal mutant (AL2) of Podospora anserina revealed that this element is a mitochondrial linear plasmid. pAL2-1 is absent from the corresponding wild-type strain, has a size of 8395 bp and contains perfect long terminal inverted repeats (TIRs) of 975 bp. Exonuclease digestion experiments indicated that proteins are covalently bound at the 5 termini of the plasmid. Two long, non-overlapping open reading frames, ORF1 (3,594 bp) and ORF2 (2847 bp), have been identified, which are located on opposite strands and potentially encode a DNA and an RNA polymerase, respectively. The ORF1-encoded polypeptide contains three conserved regions which may be responsible for a 3–5 exonuclease activity and the typical consensus sequences for DNA polymerases of the D type. In addition, an amino-acid sequence motif (YSRLRT), recently shown to be conserved in terminal proteins from various bacteriophages, has been identified in the amino-terminal part of the putative protein. According to these properties, this first linear plasmid identified in P. anserina shares all characteristics with invertrons, a group of linear mobile genetic elements.     

Transformation of Podospora anserina with a dominant resistance gene

Summary The Podospora anserina immortal mutant incoloris, vivax was transformed to benomyl resistance with a -tubuline gene from a resistant Neurospora crassa strain. The transforming plasmid was integrated into the genome of the transformants, and subsequent Southern analysis and retransformation experiments provided no evidence for autonomous replication. Non-homologous integration was demonstrated in some of the transformants. Resistance to benomyl varied widely among the transformants and was conserved after the transformants were grown on non-selective medium.     

Induction of longevity by cytoplasmic transfer of a linear plasmid inPodospora anserina

InPodospora anserina the longevity inducing linear plasmid pAL2-1 was transferred from the extrachromosomal long-lived mutant AL2 to the shor-tlived wild-type strain A. The resulting strain, AL2-IV, exhibited the long-lived phenotype. In the short-lived progeny of crosses between this strain and wild-type strain A, the plasmid was absent. In contrast, all long-lived progeny contained both the autonomous plasmid as well as copies of it integrated in the mitochondrial DNA (mtDNA). Molecular analysis revealed that the integrated plasmid copies most likely resulted from ade novo integration of the autonomous element and the generation of AT-linker sequences at the integration site. We conclude that once the plasmid is present in mitochondria of a particular genetic background, it is able to integrate into the mtDNA and to induce longevity.     

Genetics of translational fidelity in Podospora anserina: are all the genes involved in this ribosomal function identified?

Summary Su12-1 and su12-2 are two ribosomal suppressor mutations previously described in the fungus Podospora anserina. Revertants were isolated on the criteria of either improved growth at 27 °C (for su12-1) or suppression of the paromomycin hypersensitivity (for su12-2). Among 45 mutations lying outside the su12 locus, only one was found which defines a new antisuppressor locus, AS9. About 3/4 of these mutations are antisuppressor mutations localized in the previously identified AS6 and AS7 genes. While the AS6 mutations harbour diverse phenotypes, all the mutations lying in the AS7 gene lead to the same phenotypic alterations. In addition, two new su3 mutations were obtained and shown to display an antisuppressor effect on su12-1.     

Reversion of a long-living,undifferentiated mutant of Podospora anserina by copper

The Podospora anserina nuclear mutant grisea displays an undifferentiated growth phenotype (diminished production of aerial hyphae), is female sterile (lack of perithecia), has a prolonged life-span compared to the wild-type strain, and lacks detectable phenoloxidase (laccase and tyrosinase) activity. Reversion of all of these characteristics to those of the wild-type phenotype was accomplished by supplementing the growth medium with extra amounts of copper salts. These results indicate that the primary defect of the grisea strain is in its copper uptake and/or distribution in the cells.     

Sequence diversity and unusual variability at the het-c locus involved in vegetative incompatibility in the fungus Podospora anserina

The het-c locus of the filamentous fungus Podospora anserina controls heterokaryon formation through genetic interaction with alleles of the unlinked loci het-e and het-d. We have isolated four wild-type and two mutant alleles of the het-c locus. A comparison of the predicted proteins encoded by the different wild-type alleles revealed an unusual high level of amino-acid replacements compared to silent polymorphisms but only one amino-acid difference is sufficient to modify the specificity of het-c alleles. Chimeric genes constructed in vitro may exhibit a new specificity different from that of any known wild-type allele.     

Despite mtDNA polymorphism the mobile intron (plDNA) of the COI gene is present in ten different races of Podospora anserina

Summary Polymorphism of mitochondrial DNA (mtDNA) from ten different geographical races of Podospora anserina was characterized by means of restriction and hybridization analysis. Our data reveals that the mobile intron (plDNA), first characterized by DNA sequencing in strain s (Osiewacz and Esser 1984), is present in all of the races investigated. Therefore, we conclude that the mobile intron is not an optional intron of the COI gene in wild type strains. In addition, the detailed physical and genetical mapping of mtDNA from three different races with different lifespans leads us to propose that length polymorphism can result due to the lack or presence of intron sequences of structural mitochondrial genes. In view of our results we discuss the function of the mobile intron during senescence in P. anserina.     

Sequence of the nuclear ATP synthase subunit 9 gene of Podospora anserina: lack of similarity to the mitochondrial genome

Summary The nuclear gene coding for the mitochondrial subunit 9 of the F₀F₁-ATP synthase complex was isolated from a genomic library of Podospora anserina. Nucleotide sequencing revealed an open reading frame capable to code for 144 amino acids including an amino-terminal pre-sequence of 63 amino acid residues for mitochondrial import of the pre-proteolipid. The P. anserina proteolipid shows extensive sequence identity with the corresponding gene products of the related filamentous fungi Neurospora crassa, Aspergillus nidulans and Aspergillus niger. In contrast to the situation in Saccharomyces cerevisiae, N. crassa and A. nidulans, no sequence similarity of the ATP synthase subunit 9 gene to the mitochondrial genome of P. anserina could be detected. Thus, in P. anserina this gene appears to be exclusively encoded by the nuclear genome.     

Autonomously replicating sequences in young and senescent mitochondrial DNA from Podospora anserina

Summary Senescence in the filamentous fungus Podospora anserina is characterized by the accumulation of multimeric circular mitochondrial DNA molecules, known as senDNAs. These tandemly repeated DNA sequences, which originate from broadly dispersed regions of the young mitochondrial genome, behave as independently replicating molecules. In this study, the yeast transformation system was used to assay senDNAs and their young mtDNA counterparts for the presence of autonomously replicating sequences. P. anserina mtDNA fragments were cloned into the yeast vector YIp5 and the hybrid YPM plasmids were used to transform yeast. All of the senDNAs and their homologous young mtDNAs promoted high frequency transformation and extrachromosomal maintenance of YPM plasmids. The putative origin of replication for the P. anserina mitochondrial genome was also cloned into YIp5 and shown to confer autonomously replicating properties.     

Sequence analysis of the gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Podospora anserina: use of homologous regulatory sequences to improve transformation efficiency

Summary The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene of Podospora anserina has been isolated from a genomic library by heterologous hybridization with the corresponding gene of Curvularia lunata. The coding region consists of 1014 nucleotides and is interrupted by a single intron. The amino-acid sequence encoded by the gpd gene shows a high degree of sequence identity with the corresponding gene products of various fungi. Multiple alignments of all fungal GPD sequences so far available resulted in the construction of a phylogenetic tree. The evolutionary relationships of the various fungi belonging to different taxa will be discussed on the basis of these data. Sequence analysis of 1.9 kbp of the 5 non-coding region revealed the presence of typical fungal promoter elements. Utilizing different parts of the 5 regulatory sequence of the Podospora gpd gene, expression vectors containing a dominant selectable marker gene (hygromycin B phosphotransferase) have been constructed for the transformation of P. anserina protoplasts. The use of these homologous gpd regulatory sequences resulted in a significant increase in transformation efficiencies compared to those obtained with vectors in which the selectable marker gene is under the control of the corresponding heterologous promoter of Aspergillus nidulans.     

Positive screening and transformation of ura5 mutants in the fungus Podospora anserina: characterization of the transformants

Summary To develop a transformation system in the filamentous fungus Podospora anserina we have selected ura5 mutants deficient in orotidylic acid pyrophosphorylase using a positive screening. These mutants could be transformed to prototrophy by an hybrid vector carrying the ura5 gene of this organism. The properties of the transformants have been analysed. In most cases integration of the transforming vector occurred outside the ura5 locus and frequently repeated tandem copies of the vector were found. Reversion of the transformants could also be selected and we found that it can occur by exact or only partial excision of the integrated vector.Abbreviations bp base pairs - kb 1,000 bp - EtBr ethidium bromide - PEG polyethyleneglycol     

Electrophoretical and immunological comparison of the ribosomal proteins of the fungus Podospora anserina and the yeast Saccharomyces cerevisiae

Summary The ribosomal proteins of two ascomycetes Podospora anserina and Saccharomyces cerevisiae, were compared by two dimensional electrophoresis in two different gel systems and we found only five pairs of proteins which have kept homologous physico-chemical properties under these conditions. An immunological analysis was performed by radioimmunodetection of proteins blotted on nitrocellulose sheet after separation by electrophoresis, with four sera directed against the r-proteins of each subunit of these fungi. So, we pointed out many common antigenic sites present on proteins which do not co-migrate except for yeast L3 and L1 of P. anserina which have the same properties.     

Genome analysis of filamentous fungi: identification and characterization of an unusual GT-rich minisatellite in the ascomycete Podospora anserina

A genomic DNA fragment, PaGT7-5, of Podospora anserina was cloned that contains three different repetitive sequence motifs including a minisatellite with an unusual structure. This element, PaMin1, consists of ten copies of a 16 bp repeat unit with five GT dinucleotide repeats. Adjacent to PaMin1, a short poly(GT) stretch and four repeats of a 12 bp sequence were identified. Six alleles which differ in the number of the minisatellite unit were demonstrated in 18 different P. anserina strains. The flanking sequences of PaMin1 did not display any sequence alterations. A PCR analysis of total DNA from cultures of different age revealed no sequence changes, indicating that this repetitive DNA remains stable during aging. Southern-blot hybridization experiments of DNA from different strains detected only minor fragment length polymorphisms in the immediate vicinity of PaMin1. In contrast, major polymorphisms were observed at a greater distance from the locus indicating that this locus can be used as an informative probe to discriminate between different P. anserina strains. Received: 12 March / 24 May 1998