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PubMed1993

The mat- allele of Podospora anserina contains three regulatory genes required for the development of fertilized female organs.

In the filamentous fungus Podospora anserina, mating type is specified by a single locus with two alternate alleles, termed mat- and mat+. A previous study has shown that the mat+ sequence consists of 3.7 kb and contains a single gene relevant to the sexual cycle. This gene, called FPR1, encodes a protein with a HMG DNA-binding domain and is required for fertilization and for the development of the fertilized fruiting body. The mat- sequence, which is 4.7 kb in length, displays a more complex structure. We present here the characterization of two genes, called SMR1 and SMR2, which are present in the mat- allele along with the FMR1 gene. FMR1, whose role in the sexual cycle has been already partially described, encodes a protein with an alpha 1-domain and was shown to control fertilization. We demonstrate that these three genes are required for the developmental events that occur in the female organ after fertilization. The additional role of FMR1 requires a region of unknown function that is distinct from the alpha 1-domain. SMR1 encodes a protein with a putative acidic/hydrophobic alpha-helix, which has been proposed to be a feature common to transcriptional activators. The protein sequence deduced from SMR2 contains an HMG motif suggesting that it is a transcription factor.

Alleles↗
PubMed1991

Two allelic genes responsible for vegetative incompatibility in the fungus Podospora anserina are not essential for cell viability.

Vegetative incompatibility is a lethal reaction that destroys the heterokaryotic cells formed by the fusion of hyphae of non-isogenic strains in many fungi. That incompatibility is genetically determined is well known but the function of the genes triggering this rapid cell death is not. The two allelic incompatibility genes, s and S, of the fungus Podospora anserina were characterized. Both encode 30 kDa polypeptides, which differ by 14 amino acids between the two genes. These two proteins are responsible for the incompatibility reaction that results when cells containing s and S genes fuse. Inactivation of the s or S gene by disruption suppresses incompatibility but does not affect the growth or the sexual cycle of the mutant strains. This suggests that these incompatibility genes have no essential function in the life cycle of the fungus.

Alleles↗
PubMed1993

Deletion of the mating-type sequences in Podospora anserina abolishes mating without affecting vegetative functions and sexual differentiation.

The mating-type locus of Podospora anserina controls fusion of sexual cells as well as subsequent stages of development of the fruiting bodies. The two alleles at the locus are defined by specific DNA regions comprising 3.8 kb for mat+ and 4.7 kb for mat-, which have identical flanking sequences. Here we present the characterization of several mutants that have lost mat(+)-specific sequences. One mutant was obtained fortuitously and the other two were constructed by gene replacement. The mutants are deficient in mating with strains of either mat genotype but are still able to differentiate sexual reproductive structures. The loss of the mating type does not lead to any discernible phenotype during vegetative growth: in particular it does not change the life span of the strain. The mutants can recover mating ability if they are transformed with DNA containing the complete mat+ or mat- information. The transformants behave in crosses as do the reference mat+ or mat- strains, thus indicating that the transgenic mat+ and mat- are fully functional even when they have integrated at ectopic sites.

Alleles↗
PubMed1995

A novel family of linear plasmids with homology to plasmid pAL2-1 of Podospora anserina.

Three recently isolated wild-type strains of the ascomycete Podospora anserina were analyzed for the presence of linear mitochondrial plasmids. In one of these strains, designated Wat, at least 12 distinct plasmid-like elements were identified. From molecular analyses a minimum number of 78 individual linear molecules with proteins bound to their 5' ends was estimated. In addition, the different members of this family of typical linear plasmids were shown to possess a common central region and terminal sequences which differ from one plasmid to another due to the presence of different numbers of a 2.4 kb sequence module. Finally, the pWa6 plasmids share a high degree of sequence similarity with pAL2-1, a linear plasmid previously identified in mitochondria of a long-lived mutant of P.anserina. A mechanism is proposed which explains the generation of these distinct, closely related extra-chromosomal genetic traits.

Ascomycota↗
PubMed1994

Evidence for a life span-prolonging effect of a linear plasmid in a longevity mutant of Podospora anserina.

The linear mitochondrial plasmid pAL2-1 of the long-lived mutant AL2 of Podospora anserina was demonstrated to be able to integrate into the high molecular weight mitochondrial DNA (mtDNA). Hybridization analysis and densitometric evaluation of the mitochondrial genome isolated from cultures of different ages revealed that the mtDNA is highly stable during the whole life span of the mutant. In addition, and in sharp contrast to the situation in certain senescence-prone Neurospora strains, the mutated P. anserina mtDNA molecules containing integrated plasmid copies are not suppressive to wild-type genomes. As demonstrated by hybridization and polymerase chain reaction (PCR) analysis, the proportion of mtDNA molecules affected by the integration of pAL2-1 fluctuates between 10% and 50%. Comparative sequence analysis of free and integrated plasmid copies revealed four differences within the terminal inverted repeats (TIRs). These point mutations are not caused by the integration event since they occur subsequent to integration and at various ages. Interestingly, both repeats contain identical sequences indicating that the mechanism involved in the maintenance of perfect TIRs is active on both free and integrated plasmid copies. Finally, in reciprocal crosses between AL2 and the wild-type strain A, some abnormal progeny were obtained. One group of strains did not contain detectable amounts of plasmid pAL2-1, although the mtDNA was clearly of the type found in the long-lived mutant AL2. These strains exhibited a short-lived phenotype. In contrast, one strain was selected that was found to contain wild-type A-specific mitochondrial genomes and traces of pAL2-1. This strain was characterized by an increased life span. Altogether these data suggest that the linear plasmid pAL2-1 is involved in the expression of longevity in mutant AL2.

Base Sequence↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1995

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.

Alleles↗
PubMed1994

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.

Cell Nucleus↗
PubMed1991

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

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.

Amino Acid Sequence↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1984

Immunological comparison of individual ribosomal proteins in six species of the genus Podospora.

The ribosomal proteins of six species belonging to the genus Podospora have been compared by electrophoretical and immunological methods. The important variability in the electrophoretical properties is similar to other lower eukaryotes but we could specify the extent of the variability of the r-proteins by the analysis of immunological relationships between individual proteins. Radioimmuno-detection of ribosomal proteins blotted on nitrocellulose sheets after separation on two-dimensional gels, was used to determine common antigenic sites on the r-proteins of the different species. These sites may be present on proteins that do or do not have identical electrophoretical mobilities. Furthermore, the proteins which co-migrate do not necessarily cross-react.

Antigen-Antibody Complex↗
PubMed1985

Cloning and characterization of the rDNA repeat unit of Podospora anserina.

DNA coding for ribosomal RNA in Podospora anserina has been cloned and was found as a tandemly repeated 8.3 kb sequence. The cloned rDNA was characterized by restriction endonuclease mapping. The location of 5.8S, 18S and 28S rRNA coding regions was established by DNA-RNA hybridization and S1 nuclease mapping. The organization of P. anserina rRNA genes is similar to that of Neurospora crassa and Aspergillus nidulans. The rDNA unit does not contain the sequence coding for 5S RNA.

Ascomycota↗
PubMed1980

Characterization and cloning of plasmid like DNA of the ascomycete Podospora anserina.

The previously reported existence of plasmid-like (pl) DNA in senescent mycelia of Podospora anserina was confirmed using new methodology. Detailed anaysis of bulk DNA has further shown a possible relationship between pl DNA and mt DNA. According to biophysical and electron microscopic experiments the pl DNA was found to consist of oligomeres having a basic unit with a contour length of 0.75 micrometer corresponding to 2.4 kb. To overcome the handicap that pl DNA is only produced in rather small amounts in the aging mycelia, this DNA was cloned in E. coli after insertion into a bacterial plasmid vector, pBR 322. It was possible to isolate a stable hybrid plasmid consisting of the vector and only one integrated monomere of pl DNA. The composition of this hybrid plasmid was confirmed by restriction endonuclease analysis and heteroduplex formation. A restriction map of the pl DNA is presented and its insertion site onto pBR 322 indicated.

Ascomycota↗
PubMed1993

Assignment of linkage groups to the electrophoretically-separated chromosomes of the fungus Podospora anserina.

An electrophoretic karyotype of the filamentous fungus Podospora anserina has been obtained using contour-clamped homogeneous 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).

Ascomycota↗
PubMed1995

Evidence for giant linear plasmids in the ascomycete Podospora anserina.

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.

Base Sequence↗
PubMed1988

DNA sequence and organization of the mitochondrial ND1 gene from Podospora anserina: analysis of alternate splice sites.

Earlier, we reported that the ND1 mitochondrial gene of Podospora anserina is mosaic, containing at least three class I introns. We have now completed the sequence of the ND1 gene and have determined that it contains four class I introns of 1,820, 2,631, 2,256 and 2,597 bp with the entire gene complex containing 10,505 bp, only 1,101 of which are exon sequences. Introns 1 and 3 appear to be related in that their open reading frames (ORFS) exhibit extensive amino acid sequence similarity and like the URFN sequence from Neurospora crassa have multiple sequence repetitions. Introns 2 and 4 are similar in that both appear to be mosaic introns. Where intron 2 has many short ORFS, intron 4 has two, 391 and 262 aa respectively. The first ORF has some patch work sequence similarity with one of the intron 2 ORFs but the second ORF is strikingly similar to the single intron ORF in the ND1 gene of N. crassa. Just upstream of the sequences necessary to form the central core of the P. anserina intron 4 secondary structure, there is a 17 bp sequence which is an exact replica of the exon sequence abutting the 5' flank of the 1,118 bp N. crassa ND1 intron. Secondary structure analysis suggests that the 2,597 bp intron 4 can fold as an entity but a similar structure can be constructed just from an 1,130 bp portion by utilizing the 17 bp element as an alternate splice site. Detailed structural analysis suggests that intron 4 (as well as the single ND1 intron from N. crassa) can utilize helical configurations which bring the downstream open reading frame into juxtaposition with the exon sequences.

Amino Acid Sequence↗
PubMed1986

An additional class II intron with homology to reverse transcriptase in rapidly senescing Podospora anserina.

Senescence in Podospora anserina is maternally inherited and the parameters of senescence are race specific. We have compared the restriction enzyme fragment maps of race A, the most rapidly senescing race, with race s and have found three inserts in race A which are not present in race s mitochondrial DNA. Fragment A was mapped and found to be located downstream of the so-called alpha senDNA, a class II intron, near the 5' end of the COI gene, separated from alpha senDNA by two class I introns. DNA sequence analysis showed that fragment A is also a class II intron, but with only 10% DNA sequence homology to alpha senDNA. Like alpha senDNA, intron A contains significant amino acid homology with known reverse transcriptases. The importance of this additional class II intron in the mitochondrial genome with the relative rate of senescence in race A is discussed.

Amino Acid Sequence↗