Léon Belcour

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.

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.

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 (α) 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 α 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 α which allows us to identify, in senescent cultures, mitochondrial chromosomes lacking sequence α. These results taken together suggest that excision of intron α 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.

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 capnrnI/capnsnI, 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.

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.

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

SummaryIn the filamentous fungus Podospora anserina, the amplification as circular DNA molecules of the first intron (intron α) 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 α, was detected in five senescent subclones which all showed strong amplification of the intronic α sequence (Sen DNA α).

Mobile group II introns, DNA circles, reverse transcriptase and senescence (group II introns, transposition, aging, mitochondria, fungi).

Group II introns are widely distributed as attested by their recent discovery in several bacterial species (Ferat & Michel, 1993; Ferat, Le Gouar & Michel, 1994; Knoop & Brennicke, 1994). They are supposed to be relics of the RNA world and ancestors of the nuclear spliceosomal machinery (Cavalier-Smith, 1991; Sharp, 1991). Recent progress on their molecular biology has shed light on some of their interesting properties as mobile genetic sequences. They display ribozymic properties. Self-splicing, reverse splicing and erroneous reverse splicing have been demonstrated at the RNA level by in vitro experiments (Peebles et al., 1986; Augustin, MUller & Schweyen, 1990; M6rl & Schmelzer, 1990). They often contain an open reading frame related to reverse transcriptase (Michel & Lang, 1985) and recent results have shown that the Saccharomyces cerevisiae mitochondrial coxl-i2 intronic ORF indeed encodes a protein endowed with reverse transcriptase activity (Kennell et al., 1993). Like group I introns, they are able of homing, i.e. of integration into a homologous gene devoid of them. This has been demonstrated for the Saccharomyces cerevisiae coxl-il and coxl-i2 (Meunier et al., 1990), Kluyveromyces lactis coxl=il (Skelly, Hardy & ClarkWalker, 1991) and Podospora anserina coxl-i4 (our unpublished results) mitochondrial introns. Homing of group II introns is dependent upon the expression of their internal ORF (Meunier et al., 1990). More recently, their ability to transpose, i.e. to move to a novel location, has been demonstrated in yeast and in Podospora anserina (Mueller et al., 1993; Sellem, Lecellier & Belcour, 1993). These recurrent events of transposition would account for site-specific deletions of the mitochondrial chromosome leading to respiration deficient mutants in yeast and to the premature death syndrome in Podospora anserina (Mueller et al., 1993; Sellem, Lecellier & Belcour, 1993). Transposition of group II introns could explain their spreading in the course of evolution.

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.