Cell degeneration in the model system Podospora anserina.

Podospora anserina is a filamentous fungus used in many studies of fundamental cell biology, including cell ageing. In this organism, ageing is defined as a diminution of cell ability to proliferate and/or differentiate. This may or may not culminate with cell death. Two different ageing processes are intensively studied. The 'Senescence' phenomenon is present in all wild-type strains, results in cell death and is caused by a yet undefined cytoplasmic and infectious element. It is associated with extensive mitochondrial DNA modifications. Longevity of the strains is controlled by a highly complex network of genes. Among these, those involved in cytosolic translation and respiratory metabolism are of special importance. The 'Crippled Growth' phenomenon is present only in strains with elevated translational accuracy. It does not result in cell death but in a severe impairment of cell growth, an acceleration of Senescence and a diminution of differentiation potencies. No mitochondrial DNA modification is associated with Crippled Growth. Another cytoplasmic and infectious element with peculiar properties, C, is causally involved in the set up of this cell degeneration. The study of P. anserina degenerative processes provides a conceptual framework to understand ageing in more complex organisms. Especially, it emphasises the complex control exerted by genes on longevity, the multiplicity of degenerative processes that may occur to cells with identical genotype and the potential role of non-conventional infectious elements in cell ageing.

What triggers senescence in Podospora anserina?

Senescence of Podospora anserina is triggered by a cytoplasmic and infectious factor (the determinant of senescence) and is always correlated with mitochondrial DNA modifications, especially with the accumulation of small circular subgenomic DNA molecules, the senDNAs. Several observations have suggested that the senDNAs could be the cytoplasmic and infectious determinant. However, we show here (1) that senDNA molecules can be transferred to a young culture without the cotransmission of the determinant of senescence and (2) that the determinant of senescence does not segregate as a mitochondrial DNA mutation. Overall, our data strongly argue that amplification of senDNA molecules in the mitochondria is not an intrinsic property of these small DNA molecules. They question the nature of the actual determinant of senescence.

Mutations in genes encoding the mitochondrial outer membrane proteins Tom70 and Mdm10 of Podospora anserina modify the spectrum of mitochondrial DNA rearrangements associated with cellular death.

Tom70 and Mdm10 are mitochondrial outer membrane proteins. Tom70 is implicated in the import of proteins from the cytosol into the mitochondria in Saccharomyces cerevisiae and Neurospora crassa. Mdm10 is involved in the morphology and distribution of mitochondria in S. cerevisiae. Here we report on the characterization of the genes encoding these proteins in the filamentous fungus Podospora anserina. The two genes were previously genetically identified through a systematic search for nuclear suppressors of a degenerative process displayed by the AS1-4 mutant. The PaTom70 protein shows 80% identity with its N. crassa homolog. The PaMdm10 protein displays 35.9% identity with its S. cerevisiae homolog, and cytological analyses show that the PaMDM10-1 mutant exhibits giant mitochondria, as does the S. cerevisiae mdm10-1 mutant. Mutations in PaTOM70 and PaMDM10 result in the accumulation of specific deleted mitochondrial genomes during the senescence process of the fungus. The phenotypic properties of the single- and double-mutant strains suggest a functional relationship between the Tom70 and Mdm10 proteins. These data emphasize the role of the mitochondrial outer membrane in the stability of the mitochondrial genome in an obligate aerobe, probably through the import process.

Intramolecular cross-overs generate deleted mitochondrial DNA molecules in Podospora anserina.

The unavoidable senescence process that limits the vegetative growth of Podospora anserina is always associated with an accumulation of various classes of circular, tandemly arranged, defective mitochondrial DNA molecules (senDNAs). The monomers of the senDNAs belonging to the so-called beta class share a common core, but differ in both their length and termini. To understand the mechanism leading to their formation, we have determined the junction sequence of 36 senDNA beta monomers present in various senescent cultures. In most cases, we observe that: (1) short direct repeats precisely bound the senDNA beta termini and (2) one copy of the repeats is retained in the senDNA sequence. Moreover, PCR analysis of the mitochondrial DNA of some of the senescent cultures, has allowed us to detect another genome which is exactly lacking the sequence of the senDNA beta found in the culture. These results demonstrate that an intramolecular unequal cross-over occurring between short direct repeats can generate deleted mtDNA molecules in P. anserina. In addition, the polymorphism displayed by one pair of repeats allows us to establish that this cross-over may be associated with a short conversion tract spanning a few (about 15) nucleotides.

Contribution of various classes of defective mitochondrial DNA molecules to senescence in Podospora anserina.

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 alpha) of the cox1 gene; nevertheless, other regions (called beta and gamma) 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 beta. In addition, we provide evidence that senDNA beta and senDNA alpha accumulate by different mechanisms, as previously proposed. This suggests that beta senDNAs have a lethal effect on the mycelium on their own and most likely have replicative properties independent of the presence of sequence alpha. These data do not fit well with the current opinion that gives an essential role to intron alpha in the senescence of P. anserina.

[Genetic analysis of two cellular degenerations in filamentous fungus Podospora anserina]. / Génétique de deux dégénérescences cellulaires chez le champignon filamenteux Podospora anserina.

The filamentous fungus Podopsora anserina presents an unavoidable arrest of vegetative growth (Senescence) determined by a cytoplasmic and infectious factor. Senescence is correlated with a disorganization of the mitochondrial DNA. This disorganization is caused by an event which is not the appearance of the first defective DNA molecules. These ones are generated constitutively and their accumulation during Senescence requires the presence of an additional factor. Life span of the strains is under nuclear and cytoplasmic genetic control. At least 600 nuclear genes influence longevity. Our analysis focuses on the role of the genes involved in cytosolic translation, since mutations in these genes seem to display the most drastic effects on longevity but also on the structure of the defective mitochondrial DNA molecules that accumulate during Senescence. We have detected in some Podospora anserina mutant strains (permissive strains) the presence of a novel cytoplasmic and infectious determinant that entails an easily discernible phenotype associated with a severe growth alteration (Crippled Growth). This growth alteration is not associated with mitochondrial DNA modifications. Only the strains that have an increased translational accuracy present Crippled Growth. However, the Crippled Growth Determinant is found in all the strains during the stationary phase; it is eliminated from the non permissive strains during the exit of the stationary phase. The mutants, that have an increased translational accuracy, probably lack a factor which is needed to eliminate the determinant when cells enter the growth phase.

Senescence-specific mitochondrial DNA molecules in P. anserina: evidence for transcription and normal processing of the RNA.

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

Comparative study of the symbiotic plasmid DNA in free living bacteria and bacteroids of Rhizobium leguminosarum.

The symbiotic plasmid (pSym) DNA present in bacteroids of strain RCR1001 of Rhizobium leguminosarum biovar viceae has been compared qualitatively and quantitatively with that present in free living bacteria by hybridization experiments with appropriate probes. A decrease in the relative amount of pSym DNA was observed in bacteroids as compared to bacteria. No rearrangements of the symbiotically expressed pSym borne genes were detected in bacteroids.

Variable DNA splicing sites of a mitochondrial intron: relationship to the senescence process in Podospora.

The unavoidable phenomenon of senescence in Podospora was previously shown to be correlated with the presence of a senescence-specific DNA originating from amplification of some regions of the mitochondrial chromosome. The most frequently amplified region (alpha) corresponds to the first intron of the gene coding for subunit one of cytochrome oxidase. Eleven long-lived mitochondrial mutants were isolated. Here we report sequencing experiments that show that three of them are deleted for most of intron alpha and for a few base pairs belonging to the upstream adjacent exon. We also report an analysis of the residual mitochondrial DNA associated with amplification of senescence-specific DNA alpha which allows us to identify, in senescent cultures, mitochondrial chromosomes lacking sequence alpha. These results taken together suggest that excision of intron alpha from the mitochondrial DNA occurs systematically during the aging process in Podospora. They furthermore provide the first example of inaccurate intron excision at the DNA level.

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.

A 20 X 10(3)-base mosaic gene identified on the mitochondrial chromosome of Podospora anserina.

By DNA sequencing and hybridization experiments we have localized the genes cob and col on the mitochondrial chromosome of Podospora anserina. The positions we have determined for these two genes are different from those previously attributed to them. The presence in the gene col of at least two introns, belonging respectively to class I and II, has been demonstrated. This gene, with a size of about 20 X 10(3) bases, appears to be the longest known mitochondrial mosaic gene.

Ethidium bromide rejuvenation of senescent cultures of Podospora anserina : Loss of senescence-specific DNA and recovery of normal mitochondrial DNA.

The effect of ethidium bromide (EB) which is known to be able to "rejuvenate" senescent mycelia in Podospora anserina, has been investigated at the level of the mitochondrial DNA (mtDNA) by restriction analysis and molecular hybridization. While senescent mycelia display a very low growth ability and gross mtDNA modifications (tandem amplification of short sequences and disorganization of the mitochondrial chromosome: deletion of large sequences), the rejuvenated mycelia display a normal life span and contain a mtDNA in all respects identical to that of wild type mycelium (neither circular molecules nor amplified fragments could be detected). These results demonstrate a strict correlation between the senescent state and the presence of amplified mtDNA and suggest that EB rejuvenation could proceed by an efficient selection of intact mitochondrial chromosomes still present in senescent cultures.

Mitochondrial DNA amplification in senescent cultures of Podospora anserina: Variability between the retained, amplified sequences.

The non-nuclear DNA of a number of independent senescent cultures of Podospora anserina was extracted and studied. In all cases, a specific repetitive DNA (SEN-DNA) arranged in multimeric sets of circular molecules, was identified. Depending on the senescent culture, the SEN-DNA was found either in a band of about same density as the mitochondrial DNA from young mycelia (1.694 g/cm(3)) or in a band of higher density (1.699 g/cm(3)). Electron microscopy, restriction enzyme analysis and Southern hybridization experiments allowed us to establish that: (1) SEN-DNAs obtained from independent senescent cultures, both from the same strain and from different strains, can differ in the size of their monomer unit (from 2.5 to 6.3 kb). (2) All SEN-DNAs hybridize with mitochondrial DNA of a young culture and not with nuclear DNA. (3) These SEN-DNAs belong to two classes which hybridize with two non-overlapping regions of the mitochondrial chromosome.

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.

Regulation of surface antigens expression in Paramecium primaurelia: genetic and physiological factors involved in allelic exclusion.

In paramecium aurelia, allelic exclusion can be considered as a basic feature of the surface antigens system in the same way as intergenic exclusion. Our studies on allelic exclusion in G156/G168 heterozygotes show that (1) allelic exclusion does not depend on discrete regulatory genes dispersed throughout the genome; (2) it does not seem to be influenced by cytoplasmic factors; (3) it occurs regardless of the surface antigen expressed by the parental strains at the time of the cross. These results are discussed in relation to both intergenic and interallelic exclusion for which a common basis is proposed.

Induction and transmission of a merodiploid condition near the terminal area of the chromosome of Bacillus subtilis.

A small fraction (about 0.5%) of the transformants for a particular marker of B. subtilis (ilvA4; most probably a deletion) were found to be relatively unstable merodiploids. They possess a redundancy of the metB-ilvA chromosome segment. When their DNA is used as donor in transformation a merodiploid condition for the whole of this segment is created in all ilvA4+ transformants. For several of the duplicated loci both copies often are of recipient strain origin. Markers originally belonging to different copies of the diploidized region can be cotransferred in PBS1-mediated transduction. The data are well in agreement with the hypothesis that the merodiploids carry a tandem duplication. An alternative hypothesis which does not call for integration of the exogenote within the recipient chromosome was also considered. Models are proposed for interpreting the segregation of the merodiploids, the transmission of the diploid state and its generation during transformation of the ilvA4 marker by wild-type DNA.