Odile Begel

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

SummaryThe 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/cm3) or in a band of higher density (1.699 g/cm3). 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.

Overexpression of the alternative oxidase restores senescence and fertility in a long-lived respiration-deficient mutant of Podospora anserina

Several lines of evidence have implicated reactive oxygen species (ROS) in the pathogenesis of various degenerative diseases and in organismal ageing. Furthermore, it has been shown recently that the alternative pathway respiration present in plants lowers ROS mitochondrial production. An alternative oxidase (AOXp) also occurs in the filamentous fungus Podospora anserina. We show here that overexpression of this oxidase does not decrease ROS production and has no effect on longevity, mitochondrial stability or ageing in this fungus. In the same way, inactivation of the gene has no effect on these parameters. In contrast, overexpression of the alternative oxidase in the long‐lived cox5::BLE mutant, deficient in cytochrome c oxidase, considerably increases ROS production of the mutant. It rescues slow growth rate and female sterility, indicating an improved energy level. This overexpression also restores senescence and mitochondrial DNA instability, demonstrating that these parameters are controlled by the energy level and not by the expression level of the alternative oxidase. We also suggest that expression of this oxidase in organisms naturally devoid of it could rescue respiratory defects resulting from cytochrome pathway dysfunctions.

Mitochondrial Group II Introns, Cytochrome c Oxidase, and Senescence in Podospora anserina

ABSTRACT Podospora anserina is a filamentous fungus with a limited life span. It expresses a degenerative syndrome called senescence, which is always associated with the accumulation of circular molecules (senDNAs) containing specific regions of the mitochondrial chromosome. A mobile group II intron (α) has been thought to play a prominent role in this syndrome. Intron α is the first intron of the cytochrome c oxidase subunit I gene (COX1). Mitochondrial mutants that escape the senescence process are missing this intron, as well as the first exon of theCOX1 gene. We describe here the first mutant of P. anserina that has the α sequence precisely deleted and whose cytochrome c oxidase activity is identical to that of wild-type cells. The integration site of the intron is slightly modified, and this change prevents efficient homing of intron α. We show here that this mutant displays a senescence syndrome similar to that of the wild type and that its life span is increased about twofold. The introduction of a related group II intron into the mitochondrial genome of the mutant does not restore the wild-type life span. These data clearly demonstrate that intron α is not the specific senescence factor but rather an accelerator or amplifier of the senescence process. They emphasize the role that intron α plays in the instability of the mitochondrial chromosome and the link between this instability and longevity. Our results strongly support the idea that in Podospora, “immortality” can be acquired not by the absence of intron α but rather by the lack of active cytochromec oxidase.

Lethal mitochondrial genotypes in Podospora anserina: A model for senescence

SummaryCrosses between spg1 and spg2, two mitochondrial mutants of Podospora anserina, yield a new type of strain, called pseudo wild-type (PSW), in addition to wild-type recombinants. PSW strains are characterized by a variable phenotype for germination of ascospores and a variable longevity. By autofecondation, PSW strains yield early lethal strains (which die soon after the germination of the spores and so cannot be used for further studies), short-lived strains (which stop their vegetative growth after several centimeters) and long-lived strains (which grow longer than 16 cm). Genetic analysis of the last two categories shows that the PSW phenotype corresponds to a new mitochondrial genotype resulting from the interaction of the two parental mitochondrial genomes.Variability in the longevity of PSW strains is interpretated as the result of a high rate of mutation of their mitochondrial genome into a lethal and suppressive genome, similar to that of the mitochondrial rho- suppressive mutant of yeast. Furthermore, on the basis of the striking similarities observed between short-lived PSW strains and senescent cultures of Podospora anserina, we propose that commitment and development of senescence in wild-type strains of Podospora anserina would result, in a similar way, of spontaneous suppressive rho--like mitochondrial mutations.

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

SummaryThe 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.

Life-span and Senescence in Podospora anserina: Effect of Mitochondrial Genes and Functions

Summary: The life-span of a mitochondrial mutant of Podospora anserina resistant to chloramphenicol was at least five times that of isonuclear chloramphenicol-sensitive strains. This property was maternally inherited. A study of the segregation of heteroplasmic mycelia showed that, in addition to the mitochondrial alleles cap r I/cap s I, another cytoplasmic factor, whose nature is discussed, controlled the life-span. Cycloheximide decreased the life-spans of all the strains studied, whereas they were greatly increased by chloramphenicol and ethidium bromide. Chloramphenicol seemed to act mainly by lowering the probability of commitment to senescence, while ethidium bromide seemed to affect both the commitment probability and the incubation period. Furthermore, chloramphenicol and ethidium bromide were able to rejuvenate senescent mycelia. These results are discussed in connection with previous results on the mitochondrial origin of senescence in Podospora anserina.

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

Abstract 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.

Two co-existing mechanisms account for the large-scale deletions of mitochondrial DNA in Podospora anserina that involve the 5′ border of a group-II intron

Abstract A degenerative syndrome associated with the accumulation of site-specific deletions within mitochondrial chromosomes occurs in strains of Podospora anserina carrying the AS1-4 nuclear mutation. The site-specific deletion event has been assumed to result from the transposition of a group-II intron (intron α) behind an IBS motif, followed by recombination between the two intron repeats. We show here that a number of distinct deletions can accumulate in AS1-4 strains. Most of them are present in low amounts in wild-type cells where they are only detectable in PCR experiments. The deletions can be divided into two classes. In class I, intron α is joined to an IBS motif. In class II, the intron is not joined to an IBS site, it can be truncated or contain a few upstream exonic nucleotides; some junctions carry non-templated nucleotides. These results indicate that at least two mechanisms are involved in the generation of large-scale mitochondrial deletions in Podospora. One of them seems to be based on the transposition properties of the group-II α intron, the other one on illegitimate recombination. We propose that these two mechanisms use DNA double-strand breaks occurring within the 5′ region of intron α.

Insertion of short poly d(A) d(T) sequences at recombination junctions in mitochondrial DNA of Podospora.

SummaryWe have characterized the DNA sequences at recombination points in the mitochondrial DNA of two independent mitochondrial mutants of Podospora anserina. These sequences reveal the presence of foreign DNA at each recombination border, consisting of short stretches of A and T residues. We discuss the possible origin of this DNA and suggest the involvement of a reverse transcriptase activity.

Does Senescence in Podospora anserina Result from Instability of the Mitochondrial Genome

There has been remarkable progress over the last few years in the knowledge of the organization and functions of mitochondrial genomes in organisms ranging from yeast to human cells. In this paper we propose an additional function for mtDNA: a role in the control of life span, at least for obligate aerobes. In the filamentous fungus Podospora anserina, genetic, physiological, and biochemical data gathered over the years have shown that the senescent state is correlated with modifications of the mitochondria. It has thus been suggested that senescence results from suppressive mitochondrial mutations (Belcour and Begel 1978). We present here new data on several mitochondrial mutations whose properties and effects on senescence and life span support this hypothesis. MITOCHONDRIAL CHANGES IN SENESCENT CULTURES In P. anserina, senescence and subsequent arrest of growth unavoidably occur when mycelia are maintained in continuous vegetative growth (Rizet 1953). Senescence is under cytoplasmic control and is infectious through anastomoses (Rizet 1957; Marcou 1961). Senescence is determined by the random appearance of a cytoplasmic factor but is expressed long after this event; during this incubation period the concentration of the cytoplasmic factor increases exponentially (Marcou 1961; Smith and Rubenstein 1973). Physiological evidence indicates the involvement of mitochondria in senescence. The cytochrome- aa 3 content gradually decreases as the mycelium approaches arrest of growth (Belcour and Begel 1978; Cummings et al. 1979). Inhibitors of mitochondrial functions, such as chloramphenicol and ethidium bromide, prolong the life span of mycelia and furthermore can rejuvenate senescent mycelia (Tudzynski and Esser 1979; Belcour and...