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Polypeptide synthesis during protoplasmic incompatibility in the fungus Podospora anserina.

In Podospora anserina, self-lysis resulting from the combination of the R and V incompatibility genes is accompanied by the appearance, in lysing cells, of specific enzyme activities, among which is a laccase exoenzyme, and by a quenching of ribonucleic acid synthesis. Present results show that the occurrence of the laccase is the result of de novo synthesis. By means of two-dimensional gel electrophoresis it was shown that the onset of self-lysis is accompanied by the immediate shut-off of more than 60% of the pre-existing normal polypeptide synthesis and the occurrence of at least 20 new polypeptides. The synthesis of these new polypeptides is active for several hours after the cessation of RNA synthesis, concurrently with the synthesis of about 30 normal polypeptides which is maintained. These modifications of protein synthesis are not accompanied by a concomitant variation in the level of polysomes. It is deduced that incompatibility genes are involved in the control of both transcription and translation.

Ascomycota↗

The phenoloxidases of the ascomycete Podospora anserina. XI. The state of copper of laccases I, II and III.

1. Laccases I, II and III were (EC 1.14.18.1) prepared from the mycelium of the ascomycete Podospora anserina. The tetrameric laccase I(mol. wt 340 000, 16 copper atoms) and the monomeric laccases II and II (mol. wt 80 000, 4 copper atoms) have been studied by optical absorption-, circular dichroism-(CD)and electron paramagnetic resonance spectroscopy (EPR). 2. The visible and near ultraviolet difference absorption spectrum, which is apparently identical for all three laccases, shows two maxima at 330 and 610 nm and a shoulder at about 725 nm. The molar extinction coefficients of these bands are 4 times larger for the tetrameric laccase I compared to the monomeric laccases II and III which show values similar to other blue copper-containing oxidases. 3. CD spectra between 300 and 730 nm of the tree laccases are similar and contain at least 5-bands in the oxidized enzyme. If the enzyme is reduced, only a band at 307 nm remains. The molar ellipticity values of these bands are 4 times larger for laccase I than the corresponding bands of laccases II and III. It is inferred that the reducible bands are associated with the Type 1 Cu-2+. 4. In all three laccases the EPR-detectable copper accounts for only about 50% of the total copper content. The 9-GHz and 35-GHz spectra, which are identical for all three laccases, consist of two components of equal intensity. One component shows a rather small copper hyperfine coupling and a small deviation from axial symmetry. It is suggested that this copper is associated with the blue chromophore in analogy to Type 1 Cu-2+ in other blue copper proteins. The other component has a broader hyperfine coupling similar to Type 2 Cu-2+ as found in other copper proteins. The assumption that the experimental spectra result from a superposition of the spectra of equal amounts of Type 1 and Type 2 Cu-2+ has been verified by computer simulation. 5. It is suggested that the copper ions which are not detected by EPR are connected to the absorption band at 330 nm and that these ions are also essential for the function of these laccases.

Ascomycota↗

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, alpha, beta and epsilon 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 alpha, the 5.5 kb epsilon and about 3.4 kb of the 9.8 kb beta 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 alpha senDNA has the characteristics of a group II intron, epsilon senDNA contains three group I introns, and beta senDNA did not show relevant sequences in the 3.4 kb examined. Comparison of the 5' and 3'-flanking sequences of alpha 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 alpha senDNA itself consists entirely of an intron. Upstream from the oxi 3 gene a transfer RNA cysteine sequence was detected. beta 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. epsilon senDNA contained sequences homologous to ATPase 8 and URFl; URFl was interrupted by three group I introns. The excision site sequences, as located by S1 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.

Amino Acid Sequence↗

Sequence analysis of mitochondrial DNA from Podospora anserina. Pervasiveness of a class I intron in three separate genes.

A 48 kb region of the 95 kb mitochondrial genome of Podospora anserina has been mapped and sequenced (1 kb = 10(3) base-pairs). The DNA sequence of the genes for ND2, 3, 4, ATPase 6 and URFC are presented here. As in Neurospora crassa, the ND2 and 3 genes consist of a unit separated by one TAA stop codon. ND3, 4 and ATPase 6 are interrupted by class I introns. All three introns are remarkably similar in the C-domain of their secondary structure, sufficient enough to designate them as new subgroup, class IC introns. The open reading frames of the ND3 and 4 introns bear a high sequence similarity to the open reading frame of the class IB introns of ATPase 6 from N. crassa and ND1 from Neurospora intermedia Varkud. We also show that the tRNA Met-2 gene is duplicated and is involved in a recombinational event. The 5' region of URFC is also duplicated but no involvement of this gene with recombination or formation of plasmids is known. The evolutionary significance of the similarities of intron secondary structures and open reading frames of the ND3, 4 and ATPase 6 genes is discussed, including the possible separate evolution of structural and coding sequences.

Adenosine Triphosphatases↗

DNA sequence analysis of the mitochondrial ND4L-ND5 gene complex from Podospora anserina. Duplication of the ND4L gene within its intron.

A 15 kb region of the 100 kb mitochondrial genome of Podospora anserina has been mapped and sequenced (1 kb = 10(3) base-pairs). The genes for ND4L and ND5 are identified as contiguous genes with overlapping termination and initiation codons. In race A (101 kb) the gene for ND4L (4.3 kb) has a gene duplication within an intron including a second subgroup IC intron. Race s (95 kb) lacks this second gene complex. Each intron has the identical 5' exon boundary. Secondary structure analysis showed that the closest relative of the second intron is the first intron itself. The open reading frames of the two introns are also closely related to each other as well as to their counterpart in the ND4L gene of Neurospora crassa. The 9.9 kb ND5 gene starts immediately at the termination codon of ND4L and is split by two group IB introns, one group IC intron and one group II intron. The group II intron is closely related to other group II introns although its open reading frame sequence similarity with retroviral reverse transcriptase appears to be more divergent. The similarities in secondary structure and open reading frames for these six introns are discussed.

Amino Acid Sequence↗

Mitochondrial DNA sequence analysis of the cytochrome oxidase subunit II gene from Podospora anserina. A group IA intron with a putative alternative splice site.

A 5 kb region of the 95 kb mitochondrial genome of Podospora anserina race s has been mapped and sequenced (1 kb = 10(3) base-pairs). This DNA region is continuous with the sequence for the ND4L and ND5 gene complex in the accompanying paper. We show that this sequence contains the gene for cytochrome oxidase subunit II (COII). This gene is 4 kb in length and is interrupted by a subgroup IB intron (1267 base-pairs (bp) in length) and a subgroup IA intron (1992 bp in length). This group IA intron has a long open reading frame (ORF; 472 amino acid residues) discontinuous with the upstream exon sequence. A putative alternative splice site is present, which brings the ORF into phase with the 5' exon sequence. The 5'- and 3'-flanking regions of the COII gene contain G + C-rich palindromic sequences that resemble similar sequences flanking many Neurospora crassa mitochondrial genes.

Amino Acid Sequence↗

Antioxidants prolong life span and inhibit the senescence-dependent accumulation of fluorescent pigment (lipofuscin) in clones, of Podospora anserina.

Culture of clones of Podospora anserina s+ with either nordihydroguaiaretic acid or reduced glutathione (GSH) at concentrations that were not inhibitory to growth significantly prolonged the average time to onset of senscence. GSH also prolonged the average time to onset of clonal death. The specific concentration of chloroform-methanol soluble fluorescent pigment was larger in senescent than in pre-senescent cells. The pigment exhibited fluorescence excitation and emission spectra and fluorescence polarization numbers characteristic of lipofuscin, an end-product of lipid peroxidation. Analyses of the lipofuscin concentration in either sub-clonal fractions of different times of origin from senescent clones, or in sub-clonal fractions of identical age in time of origin from parent clones of different age, revealed a similar concentrations distribution. Although pre-senscent cells contained rather large concentrations, a massive increase occurred during senescence prior to the time of onset of clonal death. Culture with GSH not only prolonged clonal life span but aslo inhibited the formation of lipofuscin by an average factor of 30. Furthermore, unlike untreated clones, the sub-clonal distribution of the pigment was not only low but was also independent of their age.

Antioxidants↗

Oxidative stress and ageing in the fungus Podospora anserina.

The ageing phenomenon exhibited by the ascomycetous fungus Podospora anserina can be either delayed or induced by either different carbon sources or effectors. As these effects seem to have analogy to catabolite-repression of respiratory genes, experiments concerning respiratory functions have been carried out. Ageing is parallelled by switching from cytochrome c-oxidase-mediated respiration to alternative, cyanide-resistant respiration for reasons still unknown. The latter is always accompanied by appearance of the phenol oxidizing enzyme laccase (EC 1.10.3.2), which seems to act as an alternative oxidase. The existence of a second, non-mitochondrially encoded respiratory pathway relieves the selective pressure on mitochondria leading to disintegrated, non-functional mtDNA and thereby whole mitochondria which accumulate in the hyphal cells. Mutants lacking cytochrome c-oxidase aa3 or laccase have stable mitochondrial populations and live eternally.

DNA, Mitochondrial↗

Genes that control longevity in Podospora anserina.

Two genetic methods were used to estimate the number of genes that potentially modulate longevity in the filamentous fungus Podospora anserina. First, life span of strains carrying mutations selected on criteria unrelated to senescence was measured. Second, strains bearing random mutations were generated by insertional mutagenesis. Life span of these strains was then measured. Surprisingly, both methods lead to the conclusion that a large number of genes (between 600 and 3000) can modulate life span. Among, the mutations that affect longevity, 50% increase life span and 50% diminish it.

Ascomycota↗

Senescence in Podospora anserina: amplification of a mitochondrial DNA sequence.

Senescence in Podospora anserina has long been shown to be under cytoplasmic control. Comparison of DNAs isolated from young and senescent cultures made it possible to detect the presence, in senescent cultures only, of a specific DNA (SEN-DNA). This DNA consists of repeated sequences arranged in a multimeric set of circular molecules. In this study we have examined one particular SEN-DNA whose monomer unit is 6300 bp long. Using the Southern hybridization technique, we have demonstrated that this SEN-DNA results from the amplication of a sequence of the mitochondrial chromosome. This amplification, which resembles the process leading to rho- ("petite") mutations in yeast, is discussed in relation to the determinism of senescence.

Ascomycota↗

Are mitochondrial structural genes selectively amplified during senescence in Podospora anserina?

Genetic and transcriptional maps have been constructed for the mitochondrial genome of the Ascomycete Podospora anserina. These data have been plotted on the restriction maps for Sal I, Xho I, Bam HI, Eco RI, BgI II and Hae III. We have characterized and cloned a new and unique senescent mitochondrial DNA (beta-event senDNA) and have organized all of the recognized senDNAs on the genomic maps. We make the observation that all of the known and characterized senDNAs are derived from specific genes or gene regions of the young mitochondrial genome. We have unambiguously assigned the alpha-event senDNA (the 2.6 kb monomer) to the oxi3 gene locus and the beta-event senDNA to the oxi2 gene locus.

Ascomycota↗

A 1100-bp sequence of mitochondrial DNA is involved in senescence process in Podospora: study of senescent and mutant cultures.

In Podospora, senescence is assumed to be caused by the amplification of short sequences of the mitochondrial genome (sen-DNAs). We have characterized a 1100-bp-long mitochondrial DNA sequence which could be directly involved in the phenomenon. Indeed, by hybridization experiments, we show that this sequence is both present in all the sen-DNA molecules which originate from the beta region of the mitochondrial chromosome and rearranged in the mitochondrial genome of two mitochondrial mutants selected as resistant to senescence.

Ascomycota↗

A mitochondrial DNA rearrangement and three new mitochondrial plasmids from long-lived strains of Podospora anserina.

The excision-junction sites of a mtDNA rearrangement of a long-lived strain of Podospora anserina, Mn19, were cloned and sequenced. Analysis of sequence and hybridization data lead to the conclusion that the Mn19 mtDNA consists of two nonoverlapping circular molecules. Three plasmids, LMt-2, LMt-3, and LMt-4, cloned from long-lived progeny of crosses between the Mn19 strain and wild type were cloned and sequenced. These plasmids share features and excision-junction sites with previously described longevity and senescence plasmids. The Mn19 mtDNA rearrangement and plasmids LMt-2, LMt-3, and LMt-4 are described. The possible significance of similarities to previously described plasmids is discussed.

Ascomycota↗

Cloning gene ura5 for the orotidylic acid pyrophosphorylase of the filamentous fungus Podospora anserina: transformation of protoplasts.

From a genomic library of the filamentous fungus Podospora anserina, we have cloned a 4.9-kb fragment which complements an Escherichia coli mutant strain deficient for orotidylic acid pyrophosphorylase (pyrE gene). The recombinant plasmid pPAura5 also transforms to prototrophy a mutant strain of P. anserina carrying a mutation in the ura5 gene and lacking OMPppase activity.

Cloning, Molecular↗

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.

Amino Acid Sequence↗

Senescence in Podospora anserina: a possible role for nucleic acid interacting proteins suggested by the sequence analysis of a mitochondrial DNA region specifically amplified in senescent cultures.

In Podospora anserina, the phenomenon of senescence was previously shown to be correlated with the presence of a senescence-specific DNA (sen-DNA) resulting from the amplification of some regions (alpha, beta, gamma, epsilon) of the mitochondrial chromosome. The beta region gives rise to sen-DNAs with variable sizes and junctions which share a 1,100-bp common sequence. Here we report the complete nucleotide sequence of one 4-kb beta sen-DNA. Included in the sequence are a large part of the first intron open reading frame (ORF) of the gene ND4L and three short unidentified ORFs more precisely located in the common beta region. The primary structure of the polypeptide possibly encoded by one of them is very similar to the glycine-rich domains present in various single-stranded DNA-binding proteins. The comparison of the information content of this beta sen-DNA with that of other previously sequenced sen-DNAs suggests that the role in the senescence process attributed to the sen-DNAs could be related to the overproduction of a variety of proteins which interact with nucleic acids.

Amino Acid Sequence↗

Non-homologous integration of transforming vectors in the fungus Podospora anserina: sequences of junctions at the integration sites.

Transformation of the ura5-6 mutant strain of Podospora anserina with a recombinant vector carrying the ura5+ gene often results in the integration of the transforming plasmid by non-homologous recombination outside of the genomic ura5 locus. To investigate the mechanism of such integration, we rescued the integrated plasmid from three transformants. In two cases, the rescued plasmid was highly altered compared with the original transforming vector. We cloned the junctions between plasmidic DNA and genomic DNA of the transformants and determined their nucleotide sequences. It was found that there was little homology between plasmidic and genomic DNA sequences. Moreover, in all cases deletions of plasmid sequences at the integration site had occurred. These rearrangements can be explained by the formation of multimeric plasmids prior to integration.

Ascomycota↗

A gene responsible for vegetative incompatibility in the fungus Podospora anserina encodes a protein with a GTP-binding motif and G beta homologous domain.

The het-e-1 gene of the fungus Podospora anserina is responsible for vegetative incompatibility through specific interactions with different alleles of the unlinked gene, het-c. Coexpression of two incompatible genes triggers a cell death reaction that prevents heterokaryon formation. The het-e1 allele has been cloned to get information on the function of the locus. It encodes a putative 1356-amino-acid polypeptide that displays two sequence motifs that have not yet been reported to be present on a single polypeptide. They are a GTP-binding domain and a repeated region that shares similarity with that of the beta-transducin. Contrary to other members of the beta-transducin family, sequence conservation between the repeated units is very strong and the number of repeats is different in wild-type het-e alleles.

Alleles↗