Filamentous Fungus Podospora Anserina Research Articles

Efficient Synthesis of a 72-kDa Mitochondrial Polypeptide Using the Yeast Ty Expression System

Using the Ty system from yeast we report the efficient expression of a heterologous eukaryotic gene encoding a 72 kDa mitochondrial polypeptide. The pFM2IIBglII expression vector was initially modified for this purpose by inserting the factor Xaprotease cleavage site. TheTyAgene, which encodes the structural component of the yeast virus-like particles (VLPs), and the eukaryoticyst1 gene, encoding a 72 kDa mitochondrial tyrosyl-tRNA synthetase from the filamentous fungusPodospora anserina, were subsequently fused to the factor Xacleavage site. The resulting chimeric gene, in which the two polypeptide coding sequences are separated by the factor Xacleavage site, was expressed in yeast. High yield expression of this foreign protein, which was isolated from yeast transformants as hybrid TyVLPs, was verified after factor Xatreatment by SDS polyacrylamide gel electrophoresis and antibody detection. The strategy presented here should be useful for expressing a wide variety of eukaryotic genes.

A nonmammalian homolog of the PAF7 gene(Zellweger syndrome) discovered as a gene involved in caryogamy in the fungus Podospora anserina

The car1 gene of the filamentous fungus Podospora anserina was cloned by complementation of a mutant defective for caryogamy (nuclear fusion), a process required for sexual sporulation. This gene encodes a protein that shows similarity to the mammalian PAF1 protein (Zellweger syndrome). Besides sequence similarity, the two proteins share a transmembrane domain and the same type of zinc finger motif. A combination of molecular, physiological, genetical, and ultrastructural approaches gave evidence that the P. anserina car1 protein is actually a peroxisomal protein. This study shows that peroxisomes are required at a specific stage of sexual development, at least in P. anserina, and that a functional homolog of the PAF1 gene is present in a lower eucaryote.

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.

DNA Double-strand Break in vivo at the 3′ Extremity of Exons Located Upstream of Group II Introns: Senescence and Circular DNA Introns in Podospora Mitochondria

In the filamentous fungus Podospora anserina, the unavoidable phenomenon of senescence is associated with the amplification of the first intron of the mitochondrial cox1 that accumulates as circular DNA molecules consisting of tandem repeats. This group II intron (cox1-i1 or α) is able to transpose and contains an open reading frame with significant amino acid similarity with reverse transcriptases. The generation of these intronic circular DNA molecules, their amplification and their involvement in the senescence process are unresolved questions. We demonstrate here that: (1) another group II intron, the fourth intron of gene cox1, cox1-i4, is also able to give precise DNA end to end junctions; (2) this intronic sequence can be found amplified during senescence, although to a lesser extent that cox1-i1; (3) the amplification of the DNA multimeric cox1-i1 molecules likely does not proceed by autonomous replication; (4) the generation of the DNA intronic circles does not require efficient intron splicing; (5) a DNA double-strand break occurs in vivo at the 3′ extremity of the cox1 -e1 and cox1-e4 exons preceding the group II introns that form circular DNAs.On the whole, these results show that the ability to form DNA circular molecules is a property of some group II introns and they demonstrate the occurrence of a specific DNA cleavage at or near the integration site of these group II introns. The results strongly suggest that this cleavage is involved in the formation of the group II intronic DNA circles and could also be involved in the phenomenon of group II intron homing.

The S12 ribosomal protein of Podospora anserina belongs to the S19 bacterial family and controls the mitochondrial genome integrity through cytoplasmic translation.

In the filamentous fungus Podospora anserina, two nuclear genes are involved in the premature death syndrome associated with a site-specific deletion of the mitochondrial DNA: a mutant allele of the AS1 gene, encoding the cytoplasmic ribosomal protein S12, and an uncharacterized gene closely linked to the mating-type locus. We describe here the cloning and the sequencing of the wild-type and two mutant alleles of the AS1 gene. The P. anserina S12 protein belongs to the bacterial S19 ribosomal protein family and shows 72% identity with the S15 human ribosomal protein. Transformation experiments have shown that the AS1-4 mutation itself is responsible for the premature death phenotype and that it corresponds to a Gly to Asp change in the highly conserved COOH-terminal part of the protein. Use of antibodies directed against S12 did not permit detection of the mutant ribosomal protein inside the mitochondria. However, cross-reactions were observed with at least one mitochondrial ribosomal protein displaying a higher molecular weight than S12. The mitochondrial protein does not seem to be a by-product of the AS1 gene but is more likely the mitochondrial homologue of S12. These results strongly suggest that the mutant S12 protein acts indirectly to promote the mitochondrial deletion, via the cytoplasmic translation.

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).

Isolation of telomeric DNA from the filamentous fungus Podospora anserina and construction of a self-replicating linear plasmid showing high transformation frequency.

It has been previously shown that linear plasmids bearing Tetrahymena telomeric sequences are able to replicate autonomously in the filamentous fungus Podospora anserina (1). However, autonomous replication occurs in only 50-70% of the transformants, suggesting a defect in the recognition of the Tetrahymena telomeric template by the putative P. anserina telomerase so that only a fraction of entering DNA is stabilized into linear extrachromosomal molecules. We have cloned DNA sequences added to the Tetrahymena (T2G4)n ends of the linear plasmid. Nucleotide sequencing showed that these sequences are exclusively composed of T2AG3 repeat units. Hybridization experiments of Bal31 treated DNA showed that T2AG3 repeats are confined within 200 bp in chromosomal P. anserina telomeres. A new plasmid has been constructed so that after linearization, the terminal sequences contain T2AG3 repeats. This linear molecule transforms P. anserina with a high frequency (up to 1.75 x 10(4) transformants/micrograms), autonomous replication occurs in 100% of the transformants and the plasmid copy number is about 2-3 per nucleus. These results underscore the importance of the telomeric repeat nucleotide sequence for efficient recognition as functional telomeric DNA in vivo and provide the first step toward the development of an artificial chromosome cloning system for filamentous fungi.

Cloning the mating types of the heterothallic fungus Podospora anserina: developmental features of haploid transformants carrying both mating types.

DNAs that encode the mating-type functions (mat+ and mat-) of the filamentous fungus Podospora anserina were cloned with the use of the mating-type A probe from Neurospora crassa. Cloning the full mat information was ascertained through gene replacement experiments. Molecular and functional analyses of haploid transformants carrying both mating types lead to several striking conclusions. Mat+ mat- strains are dual maters. However, the resident mat information is dominant to the mat information added by transformation with respect to fruiting body development and ascus production. Moreover, when dual mating mat+ mat- strains are crossed to mat+ or mat- testers, there is strong selection, after fertilization, that leads to the loss from the mat+ mat- nucleus of the mat information that matches that of the tester. Finally, the mat locus contains at least two domains, one sufficient for fertilization, the other necessary for sporulation.

A site-specific deletion in mitochondrial DNA of Podospora is under the control of nuclear genes.

In the filamentous fungus Podospora anserina, the association of two nuclear genes inevitably leads to a "premature death" phenotype consisting of an early end of vegetative growth a few days after ascospore germination. Mycelia showing this phenotype contain a mitochondrial chromosome that always bears the same deletion. One of the break points is exactly at the 5' splice site of a particular mitochondrial intron, suggesting that the deletion event could result from molecular mechanisms also involved in intron mobility. One of the nuclear genes involved in triggering this site-specific event belongs to the mating-type minus haplotype; the other is a mutant allele of a gene encoding a cytosolic ribosomal protein.

Detection of a protein encoded by a class II mitochondrial intron of Podospora anserina

In the filamentous fungus Podospora anserina, the amplification as circular DNA molecules of the first intron (intron alpha) of the CO1 mitochondrial gene, encoding the cytochrome oxidase subunit 1, is known to be strongly associated with aging of strains. In this study we have attempted to detect the protein potentially encoded by the open reading frame (ORF) contained in this intron. This was done by the Western blot technique using specific antisera raised against three polypeptides encoded by three non-overlapping fragments of this ORF adapted to the universal code and overexpressed in Escherichia coli. We examined about thirty independent subclones of Podospora derived from two different geographic races (A, s), using wild-type and mutant strains, young and senescent cultures. A 100 kDa polypeptide, encoded by the class II intron alpha, was detected in five senescent subclones which all showed strong amplification of the intronic alpha sequence (Sen DNA alpha).

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).

Heat shock at an elevated temperature improves transformation efficiency of protoplasts from Podospora anserina.

We have developed an improved transformation procedure for the filamentous fungus Podospora anserina. This procedure is based on the observation that a heat shock at an elevated temperature (48 degrees C) improves the competence of P. anserina protoplasts for transformation 5- to 10-fold. This is observable only if the heat shock is applied before the addition of transforming DNA. An increase in competence is observed immediately after the heat shock, and heat-shocked cells are still competent after 20-30 min. The mechanism by which heat shock improves competence remains unclear. The modified transformation procedure gives as many as 200-500 stable transformants per microgram of plasmid DNA containing the P. anserina ura5 gene. This should allow direct cloning of P. anserina genes from a cosmid library.

Chromosome walking towards a centromere in the filamentous fungus Podospora anserina: cloning of a sequence lethal at a two-copy state

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.

Nonintegrative transformation in the filamentous fungus Podospora anserina: stabilization of a linear vector by the chromosomal ends of Tetrahymena thermophila.

The effect of the chromosomal ends of Tetrahymena thermophila on the stability of linear transforming molecules in the filamentous fungus Podospora anserina was tested. A derivative of an integrative vector for this fungus has been constructed, so that after linearization, the ends of the plasmid are the telomeric sequences of T. thermophila. After transformation, this linear molecule was maintained as an extrachromosomal plasmid with no integrated copies in about 50% of the transformants. Under selective conditions, there was approximately one linear molecule per 5 to 10 nuclei, and these extrachromosomal molecules were rapidly lost under nonselective conditions. The circular plasmid carrying an inverted repeat of T. thermophila telomeres could be linearized and processed in vivo.

Nonintegrative transformation in the filamentous fungus Podospora anserina: stabilization of a linear vector by the chromosomal ends of Tetrahymena thermophila.

The effect of the chromosomal ends of Tetrahymena thermophila on the stability of linear transforming molecules in the filamentous fungus Podospora anserina was tested. A derivative of an integrative vector for this fungus has been constructed, so that after linearization, the ends of the plasmid are the telomeric sequences of T. thermophila. After transformation, this linear molecule was maintained as an extrachromosomal plasmid with no integrated copies in about 50% of the transformants. Under selective conditions, there was approximately one linear molecule per 5 to 10 nuclei, and these extrachromosomal molecules were rapidly lost under nonselective conditions. The circular plasmid carrying an inverted repeat of T. thermophila telomeres could be linearized and processed in vivo.

The ura5 gene of the filamentous fungus Podospora anserina: nucleotide sequence and expression in transformed strains

We have sequenced the ura5 gene of the filamentous fungus Podospora anserina. The deduced sequence for the orotidylic acid pyrophosphorylase (OMPppase) has been compared with the Escherichia coli enzyme which is the only known sequence for this enzyme. This comparison shows extensive blocks of homology. The expression of the ura5 gene has been studied in a ura5 mutant which has been transformed by a recombinant plasmid carrying the ura5 gene. We observed that strains carrying integrated multicopies of the transforming vector exhibit higher specific activity for OMPppase than wild type (wt). By recombination we have constructed a strain in which the level of this enzyme is 32 times higher than in the wt strain.

At least seven ribosomal proteins are involved in the control of translational accuracy in a eukaryotic organism

In the filamentous fungus Podospora anserina, ribosomal proteins of 60 mutants impaired in the control of translational fidelity have been submitted to electrophoretic analysis. The “four corners” system combining four different two-dimensional polyacrylamide gel electrophoretic systems has been used. An altered electrophoretic pattern has been observed for 12 mutants. In mutants su3, su12 and su11 (decreased translational fidelity), proteins S1, S7 and S8, respectively, are altered. For AS mutants (increased translational fidelity), proteins S9, S12 and S19, respectively, are altered in AS9, AS1 and AS6 mutants, and protein S29 is lacking in AS3 mutants. The data suggest that five of these genes (at least) are the structural genes for the relevant proteins ( su3:S1, su12:S7, AS1:S12, AS6:S19, AS9:S9), while the AS3 gene may code for a modifying enzyme.

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

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.

A DNA polymerase activity with characteristics of a reverse transcriptase in Podospora anserina

In the filamentous ascomycete fungus Podospora anserina, senescence is associated with dramatic changes of the juvenile mitochondrial genome. During senescence specific regions of the non-senescent genome are excised, ligated and amplified as a plasmid. In this paper, we report our studies on nuclear-mitochondrial extracts for the presence of RNA-dependent DNA polymerase activity (reverse transcriptase). In so-called middle aged mycelia of race A, we detected a DNA polymerase possessing properties of a reverse transcriptase. It prefers the ribopolymer templates poly(rA)-oligo(dT)12-18 and poly(rCm)-oligo(dG)12-18 over the deoxypolymer template poly(dA)-oligo(dT)12-18. It also uses natural rabbit globin mRNA as a template. The enzymatic activity can clearly be distinguished from a second DNA polymerase activity found in the same extracts of both young and middle aged mycelia.

Excision-amplification of mitochondrial DNA during senescence in Podospora anserina: DNA sequence analysis of three unique “plasmids”

During senescence in the filamentous fungus Podospora anserina, specific regions of the mitochondrial genome, termed senDNA are excised, ligated and amplified. We have cloned in their entirety three such autonomously replicating plasmids, α, β and ε senDNA. None of these plasmids displayed cross-hybridization nor did we detect any significant DNA homology by computer analysis. The complete DNA sequence of the 2.5 kb α, the 5.5 kb ε and about 3.4 kb of the 9.8 kb β senDNA is presented (kb = 10 3 base-pairs). These sequences were analyzed for the presence of consensus sequences common to introns, and it was found that a senDNA has the characteristics of a group II intron, ε senDNA contains three group I introns, and β senDNA did not show relevant sequences in the 3.4 kb examined. Comparison of the 5′ and 3′-flanking sequences of α senDNA with oxi 3 (Co I) amino acid sequences from Neurospora crassa and Saccharomyces cerevisiae revealed significant homology and provided strong support that the excised α senDNA itself consists entirely of an intron. Upstream from the oxi 3 gene a transfer RNA cysteine sequence was detected. β senDNA contained four tRNA sequences, aspartic acid, serine, valine and tryptophan, and sequences homologous to URFC (untranslated reading frame C) as well as two new URFs. ε senDNA contained sequences homologous to ATPase 8 and URF1; URF1 was interrupted by three group I introns. The excision site sequences, as located by S 1 nuclease mapping were unique for each senDNA. Analysis for repeated units showed that each plasmid contained elements which could be involved in secondary structure required for the alignment of distal ends preparatory to excision. These results are interpreted in terms of the structural requirements of mobile elements including the possible involvement of reverse transcriptase in the excision-ligation-amplification process.