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 (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.

FIG. 1

Restriction and hybridization analysis of the mtDNA of the mid26 strain. (A) mtDNAs of the mid26 (lanes 2, 4, and 6) and wild-type (lanes 1, 3, and 5) strains were digested with restriction enzymes HaeIII (lanes 1 and 2), BamHI (lanes 3 and 4), and HindIII (lanes 5 and 6). The fragments lacking in the mid26 mtDNA are indicated by circles. (B) A corresponding gel was hybridized with a ³²P-labeled probe, Pα (sequence α inserted into pBR322). The expected restriction fragments (HaeIII 1,900 and 840 bp, BamHI 3,280 and 40,000 bp, and HindIII 1,330, 1,250, and 5,400 bp) are revealed only in the wild-type lanes. (C) Restriction sites of the COX1 region. ●, HaeIII; ◊, BamHI; ★, HindIII. CYTb, cytochrome b gene.

FIG. 2

Nucleotide sequence of the COX1e1-COX1e2 junction of the mid26 strain. The upper line shows the wild-type (WT) sequence of the COX1e1 and COX1e2 exonic regions flanking the α intronic sequence, the middle line shows the sequence of the COX1e1-COX1e2 junction of the α deletion chromosome of the mid26 strain, and the lower line shows the sequence of the junction of the Δ1 deletion chromosome. The wild-type exonic sequence is shaded. The IBS1, IBS′1, and IBS2 motifs are indicated. The deduced amino acid sequence is shown below the nucleotide sequence. Variable bases E-4, E-8, E-11, E-12, and E-13 (indicating nucleotide positions in the spliced transcript, relative to the integration site of the intron) and variable amino acids are in boldface.

FIG. 3

Model of formation of the mid26 chromosome. The dark and hatched rectangles symbolize, respectively, the IBS1 sequence of intron α (located at the 3′ end of COX1e1) and the IBS′1 sequence (located in an intergenic region about 37,000 bp upstream of the COX1 gene [3]). The crosses symbolize recombination events. WT, wild type.

FIG. 4

DNA sequence of the amplified product obtained with primers surrounding the α sequence in the Δ1 chromosome. The amplification primers were located inside COX1i2 and inside the intergenic region upstream of the Δ1 endpoint, respectively. The junction between the 5′ end of COX1e2 and the IBS′-Δ1 deletion endpoint is indicated by an arrow. The positions of the nucleotides shown at the top and the bottom of the sequence are indicated by arrowheads.

FIG. 5

Life span curves of the mid26 and wild-type strains curves for s mat− (▵), s mat+ (▴), mid26 mat− (○), and mid26 mat+ (●) strains are shown. The data are reported as the frequency of living subcultures with respect to growth length. Data are taken from Table 2 (experiment I).

FIG. 6

Determination curves for a mid26 culture. Immediately after germination of a mid26 mat− spore (mid26 11−), one implant was grown in a culture tube. Fifteen implants were taken at various distances from the initial implant and analyzed for their longevity. Results for implants taken at 0.3 (■), 3 (□), 6 (▵), 9 (○), and 11.5 (×) cm are shown. For this mid26 11− culture, arrest of growth occurred at 18 cm. Longevity curves for implants taken at 0.3 and 3 cm are similar to each other and to the longevity curve for the mid26 mat− strain shown in Fig. 5. In contrast, longevity curves for implants taken after 3 cm from the initial implant show a linear decrease of longevity, as they were taken closer to the senescent edge.

FIG. 7

mtDNA content of senescent cultures from the mid26 strain. (A) HaeIII restriction patterns of the mtDNAs of nine senescent cultures of the mid26 strain (lanes 1 to 9) and of a young culture of the wild-type strain (lane 10). (B) Corresponding hybridization with a specific probe for region β. In the young wild-type culture (lane 10), this probe reveals the three encompassed HaeIII chromosomal fragments 1 (8,600 bp), 10 (3,400 bp), and 16 (2,100 bp) (∗). In the senescent mid26 cultures (lanes 1, 3, 6, 7, 8, and 9), it reveals the intact fragment 16 present in large amounts plus additional fragments corresponding to the junction fragments of β senDNAs; these are constituted by parts of fragments 1 and 10. In senescent cultures 4 and 5, the junction fragments involve the chromosomal fragment 16; in senescent culture 2, the gross amplification seen in ethidium bromide staining (panel A) does not correspond to a β senDNA. (C) Corresponding hybridization with a specific probe for region γ. In the young wild-type culture (lane 10), this probe reveals the four encompassed HaeIII fragments 2 (6,700 bp), 4 (4,900 bp), 17 (2,000 bp), and 30 (930 bp) (∗). In the mid26 senescent cultures 5, 6, 7, 8, and 9, the probe identifies an additional junction fragment whose size is greater than that of chromosomal fragment 2. In senescent culture 2, it reveals only a γ monomer whose size is about 5,000 bp; therefore, this culture contains a nonidentified gross amplification in addition to this senDNA (see panel A).