Integration of a pAL2-1 homologous mitochondrial plasmid associated with life span extension in Podospora anserina.

We isolated and characterized a novel spontaneous longevity mutant of Podospora anserina strain Wa32 carrying one of the pAL2-1 homologous mitochondrial plasmids. This mutant is at least ten fold longer-lived than the wild type, and is hence a formal suppressor of both the regular and the 'plasmid-based' senescence process. We show that the longevity trait is maternally inherited and coincides with the presence of a copy of the plasmid integrated in the 5' UTR of the mitochondrial Complex I genes nd2 and nd3. This mutation is associated with complex alterations in the respiratory chain, including a dispensable induction of the alternative oxidase. It is also associated with a stabilization of the mitochondrial chromosome and a reduction of the overall cellular level of reactive oxygen species.

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.

A causal link between respiration and senescence in Podospora anserina.

Senescence, a progressive degenerative process leading to age-related increase in mortality, is found in most eukaryotes. However, the molecular events underlying aging remain largely unknown. Understanding how longevity is regulated is a fundamental problem. Here we demonstrate that the respiratory function is a key factor that contributes to shortening lifespan of the filamentous fungus Podospora anserina. In this organism, senescence is systematically associated with mitochondrial DNA instabilities. We show that inactivation of the nuclear COX5 gene encoding subunit V of the cytochrome c oxidase complex leads to the exclusive use of the alternative respiratory pathway and to a decrease in production of reactive oxygen species. This inactivation results in a striking increase of longevity associated with stabilization of the mitochondrial chromosome. Moreover, accumulation of several senescence-specific mitochondrial DNA molecules is prevented in this nuclear mutant. These findings provide direct evidence of a causal link between mitochondrial metabolism and longevity in Podospora anserina.

Recombinant mitochondrial DNA molecules suggest a template switching ability for group-II-intron reverse transcriptase.

Degenerative processes in the filamentous fungus Podospora anserina are strongly correlated with the instability of the mitochondrial genome. Among the sources of instability is the mobile group-II intron COX1-i1, also called intron alpha, which encodes a protein with a reverse transcriptase activity. In this paper we characterize, through PCR experiments, mitochondrial recombinant DNA molecules joining the 5' end of intron alpha to the 3' end of tRNA sequences including the CCA motif. The structure of these junctions led us to propose that they were most probably initiated by a RNA template switching of the reverse transcriptase encoded in COX1-i1. This activity might be involved in a number of mitochondrial rearrangements occurring in degenerative syndromes and in some long-lived mutants.

Mitochondrial group II introns, cytochrome c oxidase, and senescence in Podospora anserina.

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 (alpha) has been thought to play a prominent role in this syndrome. Intron alpha 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 the COX1 gene. We describe here the first mutant of P. anserina that has the alpha 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 alpha. 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 alpha is not the specific senescence factor but rather an accelerator or amplifier of the senescence process. They emphasize the role that intron alpha 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 alpha but rather by the lack of active cytochrome c oxidase.

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.

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 alpha) 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 alpha 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 alpha 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 alpha.

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 alpha) 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 than 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.

DNA deletion of mitochondrial introns is correlated with the process of senescence in Podospora anserina.

In the filamentous fungus Podospora anserina, the unavoidable phenomenon of senescence is associated with specific mitochondrial rearrangements and particularly with the amplification of some regions of the mitochondrial chromosome. Mechanisms responsible for these rearrangements are still unknown. The implication in this phenomenon, of the first intron of the mitochondrial gene cox1 (intron alpha), a class II intron that presents significant amino acid similarity with retroviral reverse transcriptases, was postulated several years ago. We demonstrate here by polymerase chain reaction experiments: (1) that senescent and young cultures contain DNA molecules precisely deleted for intronic sequences; (2) that these deletions are found to a much greater extent in senescent than in young cultures; (3) that DNA intron deletion likely results from a reverse transcriptase-mediated mechanism as indicated by the detection of copies of the gene 1 cox1 completely devoid of its 15 introns; (4) that the intron alpha-encoded protein could intervene in this process. On the whole, these results strongly suggest that in Podospora, an increase in a mitochondrial reverse transcriptase activity probably mediated by the intron alpha-encoded protein is involved in the process of senescence.

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).

Insertion of an LrDNA gene fragment and of filler DNA at a mitochondrial exon-intron junction in Podospora.

A rearrangement of the mitochondrial genome of a long lived mutant of Podospora anserina is presented. It consists in the insertion of 191 bp of the LrDNA gene (coding for the large ribosomal RNA) at the junction between exon1 and intron alpha of gene co1 (coding for subunit 1 of cytochrome oxidase). This insertion is accompanied by a 53 bp deletion of the junction and the presence of extra A and T nucleotides at both sides of the inserted sequence. We discuss possible mechanisms of production of this rearrangement. The presence of extra nucleotides at the recombination junctions suggests that it may pass through a stage of free DNA ends originating from a DNA break at the junction between exon1 and intron alpha of gene co1. The possibility that such a DNA break plays a major role in the instability of the mitochondrial genome is envisaged.

Absence of detectable mitochondrial recombination in Paramecium.

An extensive search for recombination between mitochondrial markers was carried out in Paramecium tetraurelia. Thirty-two combinations, altogether involving 24 different markers, were studied. The markers belonged to the three main categories of mitochondrial mutations presently available in this organism, (a) Spontaneous or UV-induced antibiotic resistance mutations, most probably affecting mitochondrial ribosomes, (b) nitrosoguanidine-induced antibiotic resistance markers displaying thermosensitivity or slow growth, enabling easy selection of possible wild-type recombinants, and (c) mitochondrial partial suppressors of a nuclear gene, probably corresponding to molecular alterations distinct from the preceding two categories. In addition, different genetic configurations were analyzed (i.e., mutant X mutant, double-mutant X wild-type, etc.).--None of the combinations yielded any evidence for the occurrence of recombined genomes despite the fact that: (1) all of them were studied on a large scale involving the screening of at least several thousand mitochondrial genomes (often several millions), (2) in many of them the detection level was sufficiently high to enable the isolation of spontaneous mutants in control cells, and (3) in several of them, reconstitution experiments carried out in parallel show that the conditions were fully adequate to detect recombinant genotypes. The results are in marked contrast with those obtained on the few other organisms in which mitochondrial recombination has been studied, particularly Saccharomyces cerevisiae, in which mitochondrial recombination is intense.--The most likely basis for the various manifestations of mitochondrial genetic autonomy in Paramecium, described in this as well as in previous publications, is that the chondriome of this organism is made up of thousands of structurally discrete, noninteracting units.

Restoration of nucleo-mitochondrial compatibility in paramecium.

In Paramecium, as previously described, the nuclear mutation cl(1) is incompatible with wild-type mitochondria (M(+)); however, all cl(1)/cl(1)M(+) cells eventually overcome this incompatibility (Sainsard, Claisse and Balmefrezol 1974). We have studied the kinetics and genetic basis of the spontaneous restoration of harmony between nucleus and mitochondria. We also studied the modification of these kinetics following microinjection of compatible mutant mitochondria into cl(1)/cl(1)M(+) cells. We demonstrate that nucleo-mitochondrial readjustment is always achieved by mitochondrial changes that fall into two classes. The first class corresponds to spontaneous mitochondrial mutations affecting the amount of cytochrome aa(3) and is similar to the previously described M(cl) and M(su) mutations (Sainsard-Chanet 1978; Sainsard 1975). The nature of the second class of modification is not yet understood; it may correspond either to a mitochondrial "adaptation" or to an unusual type of mutation arising and reverting at high frequency.

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1. I. Spectral properties and redox potentials.

1. Purified mitochondria have been prepared from wild type Paramecium tetraurelia and from the mutant Cl1 which lacks cytochrome aa3. Both mitochondrial preparations are characterized by cyanide insensitivity. Their spectral properties and their redox potentials have been studied. 2. Difference spectra (dithionite reduced minus oxidized) of mitochondria from wild type P. tetraurelia at 77 K revealed the alpha peaks of b-type cytochrome (s) at 553 and 557 nm, of c-type cytochrome at 549 nm and a-type cytochrome at 608 nm. Two alpha peaks at 549 and 545 nm could be distinguished in the isolated cytochrome c at 77 K. After cytochrome c extraction from wild type mitochondria, a new peak at 551 nm was unmasked, probably belonging to cytochdrome c1. The a-type cytochrome was characterized by a split Soret band with maxima at 441 and 450 nm. The mitochondria of the mutant Cl1 in exponential phase of growth differed from the wild type mitochondria in that cytochrome aa3 was absent while twice the quantity of cytochrome b was present. In stationary phase, mitochondria of the mutant were characterized by a new absorption peak at 590 nm. 3. Cytochrome aa3 was present at a concentration of 0.3 nmol/mg protein in wild type mitochondria and ubiquinone at a concentration of 8 nmol/mg protein both in mitochondria of the wild type and the mutant Cl1. Cytochrome aa3 was more susceptible to heat than cytochromes b and c,c1.

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1. II. Cyanide-insensitive respiration. Function and regulation.

1. The cyanide-insensitive respiration in Paramecium tetraurelia was found to be located in mitochondria. 2. Sensitivity of the mitochondrial respiration to cyanide depended on growth conditions. Under standard conditions of growth, 15--20% of respiration was insensitive to 1 mM cyanide. Full resistance to 1 mM cyanide was observed by growing cells in the presence of erythromycin (100--400 microgram/ml) 0.2 mM cyanide. The mitochondrial respiration of the mutant Cl1 harvested during the exponential phase of growth was largely insensitive to cyanide (more than 80%). 3. Pyruvate was oxidized at the same rate by wild type mitochondria and mitochondria of the mutant Cl1. In contrast, succinate oxidation was 2--3 times faster in mitochondria of the mutant Cl1 than in wild type mitochondria. 4. The cyanide-insensitive respiration was inhibited by 1 mM salicylhydroxamic acid to nearly 100%. Other efficient respiratory inhibitors included amytal and heptylhydroxyquinoline. Antimycin was not inhibitory even at concentrations as high as 5 microgram/mg protein, a finding consistent with the lack of antimycin binding sites.

Gene controlled selection of mitochondria in Paramecium.

The slow growing mutant cl1 of Paramecium, previously described (Sainsard, Claisse and Balmefrezol, 1974) differs from wild-type by a single recessive nuclear mutation and by a particular mitonchondrial phenotype (Mcl) that gene cl1 distinguishes from the wild-type mitochondrial phenotype (M+). A further analysis of these nucleo-mitochondrial interactions was carried out by confronting the genes cl1 and cl)+ with mixed populations of M+ and Mcl mitochondria obtained after cytoplasmic exchange at conjugation. The following results were obtained: 1. M+ and Mcl mitochondria introduced respectively into mutant and wild-type cells do not multiply easily; 2. when a mixed population (M+ + Mcl) is established, both mitochondrial types are maintained during the growth of the F1 heterozygous cl1/cl1+ clones; 3. when the nuclear segregation occurs in F2, the formation of homozygotes cl1/cl1 or cl1+/cl1+ is soon followed by the segregation of the two mitochondrial types, Mcl or M+, reconstituting the two parental nucleo-mitochondrial associations.