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In biological systems, reactive oxygen species (ROS) represent 'double edged swords': as signaling molecules they are essential for proper development, as reactive agents they cause molecular damage and adverse effects like degeneration and aging. A well-coordinated control of ROS is therefore of key importance. Superoxide dismutases (SODs) are enzymes active in the detoxification of superoxide. The number of isoforms of these proteins varies among species. Here we report the characterization of the putative protein encoded by Pa_1_10620 that has been previously annotated to code for a mitochondrial ribosomal protein but shares also sequence domains with SODs. We report that the gene is transcribed in P. anserina cultures of all ages and that the encoded protein localizes to mitochondria. In strains overexpressing Pa_1_10620 in a genetic background in which PaSod3, the mitochondrial MnSOD of P. anserina, is deleted, no SOD activity could be identified in isolated mitochondria. However, overexpression of the gene leads to lifespan extension suggesting a pro-survival function of the protein in P. anserina. [ABSTRACT FROM AUTHOR]

Wild-type strains of the ascomycete Podospora anserina are characterized by a limited lifespan. Mitochondria play a central role in this ageing process raising the question of whether apoptosis-like processes, which are also connected to mitochondrial function, are involved in the control of the final stage in the fungal life cycle. While a role of two metacaspases in apoptosis and lifespan control was recently demonstrated in P. anserina, virtually nothing is known about the function of the protein family of apoptosis-inducing factors (AIFs). Here we report data about proteins belonging to this family. We demonstrate that the cytosolic members PaAIF1 and PaAMID1 do not affect lifespan. In contrast, loss of PaAIF2 and PaAMID2, which both were localized to mitochondria, are characterized by a significantly increased ROS tolerance and a prolonged lifespan. In addition, deletion of PaAmid2 severely affects sporogenesis. These data identify components of a caspase-independent molecular pathway to be involved in developmental processes and in the induction of programmed cell death in the senescent stage of P. anserina. [ABSTRACT FROM AUTHOR]

Carotenoids have been identified in the fungus Podospora anserina and a parallel pathway to neurosporene and β-carotene was established. Three genes for the β-carotene branch have been cloned and their function elucidated. They correspond to the al-1, al-2 and al-3 genes from Neurospora crassa. They were individually and in combinations over-expressed in P. anserina in order to modify the carotenoid composition qualitatively and quantitatively. In the resulting transformants, carotenoid synthesis was up to eightfold increased and several intermediates of the pathway together with special cyclic carotenoids, β-zeacarotene and 7,8-dihydro-β-carotene, accumulated. All transformants with an over-expressed al-2 gene (encoding a phytoene synthase and a lycopene cyclase) displayed up to 31% prolonged life span. [ABSTRACT FROM AUTHOR]

In fungi, vegetative incompatibility is a conspecific non-self recognition mechanism that restricts formation of viable heterokaryons when incompatible alleles of specific het loci interact. In Podospora anserina, three non-allelic incompatibility systems have been genetically defined involving interactions between het-c and het-d, het-c and het-e, het-r and het-v. het-d and het-e are paralogues belonging to the HNWD gene family that encode proteins of the STAND class. HET-D and HET-E proteins comprise an N-terminal HET effector domain, a central GTP binding site and a C-terminal WD repeat domain constituted of tandem repeats of highly conserved WD40 repeat units that define the specificity of alleles during incompatibility. The WD40 repeat units of the members of this HNWD family are undergoing concerted evolution. By combining genetic analysis and gain of function experiments, we demonstrate that an additional member of this family, HNWD2, corresponds to the het-r non-allelic incompatibility gene. As for het-d and het-e, allele specificity at the het-r locus is determined by the WD repeat domain. Natural isolates show allelic variation for het-r. [ABSTRACT FROM AUTHOR]

Gene replacement via homologous recombination is a fundamental tool for the analysis of gene function. However, this event is rare in organisms like the filamentous fungus Podospora anserina. We show here that deletion of the PaKu70 gene is an efficient strategy for improving gene manipulation in this organism. By using the Δ PaKu70 strain, it is now possible (1) to produce deletion mutants with an efficiency of 100%, (2) to achieve allelic exchange by introducing a mutated allele associated with a selection cassette at the locus, (3) to introduce a mutation in a gene without co-insertion of a selectable marker and without any modification of the target locus. [ABSTRACT FROM AUTHOR]

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, ). 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. [ABSTRACT FROM AUTHOR]

Senescence in Podospora anserina is always correlated with extrachromosomal mitochondrial DNA amplification (senDNA). Here we report a quantitative kinetic analysis of the molecular events that occur in the mitochondrial DNA of several wild-type cultures during aging. For each culture, the amplification of senDNA molecules and the modifications of the mitochondrial chromosome are analyzed at different ages and in relation with two age-related parameters: growth rate and fertility. We find senDNAs exponentially amplified from the germination state and particular regions of the mitochondrial chromosome preferentially lost only in the pre-senescent state, i.e. a few centimeters before the growth arrest of the mycelium. This late loss of information, concomitant with the first phenotypic expression of senescence (loss of fertility), begins in the regions from which the senDNAs originate and culminates with the growth arrest of the mycelium. [ABSTRACT FROM AUTHOR]

The het-c locus controls heterokaryon formation in Neurospora crassa. It is subject to balancing selection operating to maintain polymorphism at that locus in natural populations. We have isolated hch, the het-c homolog from the related species Podospora anserina (hch for het-c homolog), in order to determine if this gene also functions as a het gene in that species. The het-c and hch sequences are highly similar but differ in the region defining allele specificity in N. crassahet-c. Analysis of hch variability in 11 natural P. anserina isolates with different het genotypes revealed no polymorphism. This suggested that hch does not function as a het gene. However, heterologous expression of the N. crassa het-cPA allele in P. anserina triggers a growth defect reminiscent of the het-c incompatibility reaction. [ABSTRACT FROM AUTHOR]

Rearrangements of the mitochondrial DNA (mtDNA) are a hallmark of senescence in wild-type strains of the ascomycete P. anserina. These rearrangements include the systematic amplification of the first intron (pl-intron) of the cytochrome oxidase subunit-I gene (CoI) as a circular DNA molecule (plDNA). In addition, deletions and amplifications of other regions of the mtDNA occur. The molecular basis of the underlying processes is not understood in detail. A comparative analysis of the wild-type strain and of the long-lived mutant grisea, affected in the uptake of copper, revealed that mtDNA instabilities are dependent on the availability of cellular copper. In the mutant, the first steps in the corresponding pathway, including the transcription of the CoI gene, the splicing of the pl-intron and the transposition of this mobile element, are not impaired. In contrast, recombination processes between short direct repeats, as well as rearrangements between two tandem intron copies leading to the formation of plDNA, appear to be affected. Additional copper in the growth medium rescues this molecular phenotype. We suggest that copper is a cofactor of a component of the molecular machinery leading to the characteristic age-related mtDNA rearrangements. [ABSTRACT FROM AUTHOR]

A differential protein display screen resulted in the identification of a 27-kDa protein which strongly accumulates during the senescence of Podospora anserina cultures grown under standard conditions. After partial determination of the amino-acid sequence by mass-spectrometry analysis of trypsin-generated fragments, pairs of degenerated primers were deduced and used to amplify parts of the sequence coding for the protein. These PCR products were utilized to select specific cDNA and genomic clones from DNA libraries of P. anserina. A subsequent DNA-sequence analysis revealed that the 27-kDa protein is encoded by a discontinuous gene, PaMth1, capable of coding for 240 amino acids. The first three amino-terminal residues appear to be removed post-translationally. The deduced amino-acid sequence shows significant homology to S-adenosylmethionine (SAM)-dependent methyltransferases. We hypothesize that the 27-kDa protein, PaMTH1, is involved in age-related methylation reactions protecting aging cultures against increasing oxidative stress. [ABSTRACT FROM AUTHOR]

Degenerative processes in the filamentous fungus Podospora anserina are strongly correlated with the instability of the mitochondrial genome. Among the sources of instability is the mobile group-II intron COX1-i1, also called intron α, which encodes a protein with a reverse transcriptase activity. In this paper we characterize, through PCR experiments, mitochondrial recombinant DNA molecules joining the 5′ end of intron α to the 3′ end of tRNA sequences including the CCA motif. The structure of these junctions led us to propose that they were most probably initiated by a RNA template switching of the reverse transcriptase encoded in COX1-i1. This activity might be involved in a number of mitochondrial rearrangements occurring in degenerative syndromes and in some long-lived mutants. [ABSTRACT FROM AUTHOR]

The unavoidable arrest of vegetative growth in Podospora anserina (senescence process) is always correlated with rearrangements of the mitochondrial chromosome, mainly consisting in the amplification of particular regions as tandemly repeated circular molecules (senDNAs). One sequence systematically amplified in senescent cultures corresponds precisely to the first intron (intron α) of the cox1 gene; nevertheless, other regions (called β and γ) are also frequently amplified. The experiments presented in this paper show that cellular death is in some cases associated with the sole presence of large amounts of senDNA β. In addition, we provide evidence that senDNA β and senDNA α accumulate by different mechanisms, as previously proposed. This suggests that β senDNAs have a lethal effect on the mycelium on their own and most likely have replicative properties independent of the presence of sequence α. These data do not fit well with the current opinion that gives an essential role to intron α in the senescence of P. anserina. [ABSTRACT FROM AUTHOR]

In Podospora 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 a de 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In the extrachromosomal mutant AL2 of the ascomycete Podospora anserina longevity is correlated with the presence of the linear mitochondrial plasmid pAL2-1. In addition to this autonomous genetic element, two types of closely related pAL2-1-homologous molecules were detected in the high-molecular-weight mitochondrial DNA (mtDNA). One of these molecules is of linear and the other of circular structure. Both molecules contain pAL2-1 sequences which appear to be integrated at the same site in the mtDNA. Sequence analysis of a DNA fragment cloned from one of these molecules revealed that it contains an almost full-length copy of pAL2-1. At the site of plasmid integration a 15-nucleotide AT-spacer and long inverted mtDNA sequences were identified. Finally, two giant linear plasmid-like DNAs of about 50 kbp and 70 kbp were detected in pulsed-field gels of mutant AL2. These molecules are composed of mtDNA and pAL2-1-specific sequences and may result from the integration of mtDNA sequences into linear plasmid pAL2-1. [ABSTRACT FROM AUTHOR]

A shuttle cosmid vector, pANsCos1, has been constructed for Escherichia coli and filamentous fungi. This vector contains two cos sequences separated by a single XbaI restriction site. pANsCos1 allows the efficient construction of representative genomic libraries from as little as 15-20 μg of genomic DNA. Due to the presence of a functional hygromycin B phosphotransferase gene ( hph) transformation of fungal protoplasts with pANsCos1, or derivatives of it, results in the formation of hygromycin B-resistant transformants. The T7 and T3 RNA polymerase promoter sequences flanking the cloning site, in combination with two adjacent NotI sites facilitate genomic walking and the rapid construction of restriction maps of cloned inserts. [ABSTRACT FROM AUTHOR]

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 (α, β, γ and ε) of the mitochondrial chromosome. The β region gives rise to senDNAs with variable sizes, but sharing a 1-kb common sequence. Here, we present a molecular analysis of five β 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 β senDNAs could be generated via a reverse transcription step. We have furthermore shown that the β sen DNAs produce specific transcripts which undergo normal processing of their introns. We propose that a transcription start site, located in the β common region, is involved in mitochondrial replication allowing the amplification of the β senDNAs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

An electrophoretic karyotype of the filamentous fungus Podospora anserina has been obtained using contour-clamped homogenous electric field gel electrophoresis. Six chromosomal bands were separated with one migrating as a doublet. The size of the chromosomes was estimated to be between 3.8 and 6.0 megabase pairs (mb) using the chromosomes of Schizosaccharomyces pombe as size standards, giving a total genome size of about 34 mb for the P. anserina genome. Homologous probes were used to assign five of the seven linkage groups (LGs) to chromosomal bands on the gel. Analysis of reciprocal translocation strains allowed us to complete the karyotype. In decreasing size order, the P. anserina chromosomes are LG I (6.0 mb); LG II (5.5 mb); LG V (5.1 mb); LG III (4.9 mb); LGs VI and VII (4.3 mb) and LG IV (3.8 mb). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The Podospora anserina immortal mutant incoloris, vivax was transformed to benomyl resistance with a β-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. [ABSTRACT FROM AUTHOR]

We started partial overlapping hybridization on chromosome V from the centromere-linked gene SU8 towards centromere 5. Transformation of Podospora anserina with the cloned sequences and subsequent genetic analysis reveal that cosmids issuing from a 60 kb region never integrate near centromere 5, whereas cosmids from either side return mostly to this locus. Molecular analysis shows that heterologous integrations of cosmids spanning the 60 kb region occurs in a specific sequence of 11 kb common to all the inserts. We propose that this sequence contains a gene or a structure which is lethal in a duplicated form. Straightforward interpretation of genetic data suggests that this structure does not correspond to centromere 5. Its function is still unknown. [ABSTRACT FROM AUTHOR]

In senescing strains of the filamentous ascomycete Podospora anserina, the first intron (il) of the mitochondrial gene for cytochrome-c-oxidase subunit I ( CO I), a group II intron, accumulates as a circular mitochondrial plasmid (plDNA). In both juvenile and senescent wild type strain s two highly abundant transcripts were detected homologous to il and plDNA. In this report we show that these RNAs are identical molecules having different conformations: the first is a lariat, the second a corresponding linear molecule probably resulting from breakage of the branched circular form. Our findings suggest that the transcripts arise from processing of CO I pre-mRNA, including il, rather than from transcription of the excised plasmid. The significance of the lariat RNA concerning plDNA amplification via a postulated reverse transcription mechanism is discussed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Mapping and transcription studies have revealed that in Podospora anserina the causative agent of senescence, a mitochondrial plasmid (p1DNA), is identical with intronl of the discontinuous gene for cytochrome-c-oxidase subunit 1 (COI), which is 2 kpb from the discontinuous gene for cytochrome b (Cytb). A mitochondrial mutant ( ex1) devoid of the COI, but not of the Cytb gene provides longevity. A molecular model for the onset of senescence is presented. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The nucleotide sequence in three regions of the mitochondrial plasmid (p1DNA) of Podospora anserina has been determined for almost 1,000 by of the altogether 2.4 kb. The first region beginning around excision point E1 comprises 652 by and is characterized by an AT content of about 58%. A long open reading frame (URF) (555 bp) can be attributed to this region coding for an unidentified polypeptide. No reading frame could be attributed to the second region (247 bp), since in all 6 possible phases numerous termination codons were detected. Its AT content was found to be 71%. The third region (87 bp) which ends with excision point E2 is too small for further analysis. From these data it follows that the p1DNA consists of two regions with different functions at least. However, only the larger one beginning near excision point E1 appears to code for a protein. [ABSTRACT FROM AUTHOR]

We have examined the structure of the rRNA genes from the mitochondrial genome of Podospora anserina. Using R-loop analysis, nuclease protection experiments, and Southern blot hybridization analysis we have observed two intervening sequences (IVS) in the large rRNA gene, and none in the small rRNA gene. the IVS sequences are 1.65 kbp and 2.73 kbp long, and the larger of the two is in the position of the conserved IVS found in the mitochondrial genomes of other fungi. We have detected precursor transcripts for the large rRNA, and these data support the observation of two IVS in this gene. We also note that the large and small rRNA genes are separated by approximately 6 kbp of DNA. [ABSTRACT FROM AUTHOR]

In developing a system for molecular cloning with the Podospora anserina plasmid (p1DNA) it is necessary to find recipient strains which are resistant to p1DNA mediated senescence. Three long lived double mutants which fail to exhibit spontaneous aging were genetically and biochemically analysed. All mutants were infected with p1DNA. The mutant ca viv became irreversibly senescent and therefore was not further tested. The second mutant, gr viv showed some symptoms of aging but never died. The third strain i viv remained resistant to aging from p1DNA infection and has thus proven to be the best host strain available for molecular cloning in this system. A DNA analysis of the latter two strains revealed: These experimental results are interpreted as follows: [ABSTRACT FROM AUTHOR]

Emetine resistant mutants have been isolated in the fungus Podospora anserina. Genetic analysis of 75 mutants has shown that resistance is controlled by at least two genes, EmR1 and EmR2. In vitro studies showed that resistance to emetine in EmR1 and EMR2 mutants was due to alterations in the protein synthesis machinery. Reconstitution experiments with ribosomal subunits prepared from wild-type and resistant strains indicated that emetine resistance is associated with the 40S subunit. The strains resistant to emetine are also hypersensitive to anisomycin in vivo. In poly-U directed polyphenylalanine synthesis or run off of polysomes, it was shown that mutant ribosomes do not differ from those of wild-type with regard to anisomycin sensitivity, pointing out that initiation would be lso an anisomycin target. The ribosomal proteins isolated from resistant mutants were analyzed by two dimensional gel electrophoresis and compared with the proteins of wild-type ribosomes. The EmR40 mutant was found to have an altered S14 protein. In EmR69, three proteins of the small subunit S13, S15 and S17 were altered, nevertheless only altered S13 and S17 proteins segregate with the resistance character. The comparison of proteolytic digestion of S13 and S17 on SDS gel allowed us to conclude that S13 and S17 would be homologous proteins. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In order to develop an eukaryotic vector with the Podospora plasmid, further characterization is required of the mitochondrial DNA into which this plasmid is integrated, a physical map (restriction sites) of the Podospora chondriome (size 95 kb) has been completed. As prerequisite for the establishment of a genetic (functional) map, 70% of the chondriome was cloned in E. coli vectors. Using mitochondrial genes from Saccharomyces cerevisiae, six structural genes were located on the Podospora chondriome by cross hybridization experiments. There is strong evidence that the plasmid is inserted into the cytochrome b gene. A comparison of the genetic map of the Podospora chondriome with those of Neurospora crassa and Aspergillus nidulans exhibits a rather good accordance with respect to the sequence of genes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The nuclear gene coding for the mitochondrial subunit 9 of the FF-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]