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.

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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.

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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.