Senescence In Podospora Anserina

14.3.1 Deletion and Rearrangements in Mitochondrial DNA

Genetic tests in Podospora anserina localized the senescence determinant in mitochondria. A restriction enzyme analysis by agarose-gel electrophoresis revealed that mitochondrial DNA (Figure 14.3) from the senescing cultures contained unique circular DNA molecules comprising head-to-tail of monomer 2.05 ^M, dimer, trimer, tetramer or pentamer sizes which were termed the senDNA (Jamet-Vierny et al., 1980; Wright and Cummings, 1983).

4000 bp 2300 bp

Young Senescent culture culture

Figure 14.3 Diagram of electrophoresis patterns of mitochondrial DNA from young and senescent cultures of Podospora anserina. Arrows point to unique 4000 and 2000 bp fragments in senescent strain after HaeIII digestion. Based on Jamet-Vierny et al. (1980).

senDNA hybridized to restriction fragments of the mitochondrial DNA but not of nuclear DNA, revealing that it is homologous to mitochondrial DNA (Figure 14.4). Several senDNAs, named a, ß, y, and so on, may be produced by the deletion and amplification of separate regions of mitochondrial DNA, of which senDNAa is produced regularly. senDNAa results from the site-specific deletion and amplification of the first intron of the mitochondrial COX1 gene which encodes subunit I of the respiratory enzyme, cytochrome c oxidase. Wright and Cummings (1983) reported that senDNAa probe hybridized to nuclear DNA from the senescing mycelium and hypothesized that a mitochondrial genetic element is transposed to the nucleus and is integrated into nuclear DNA. However, this observation has not been confirmed—rather, it is likely that the result obtained was due to contamination of nuclear DNA by mitochondrial DNA.

Comparison of nucleotide sequences of mitochondrial DNA fragments revealed intron at new locations in the mitochondrial genome (Sellem et al., 1993). Somehow the defective mitochondria, lacking a portion of mitochondrial genome, increase in number and dominate the cytoplasm during hyphal growth (Figure 14.5)—a condition known as suppressivity that results in the loss of ATP production, slowing down of the growth rate and finally in the total cessation of growth. The ß and the y senDNAs are highly variable in size and contain, respectively, an intergenic region downstream of coxl gene near rRNA. Their numbers also increase during the progression of senescence but since senDNAa

I Subculture

I Subculture

Figure 14.4 A diagram of suppressivity. Defective (closed circle) and functional (open circle) mitochondria.

appears regularly during senescence, it is believed to be the primary determinant of senescence. However, this is controversial because senDNAa was found in nonsenescent mutant incoloris vivax (Tudzynski et al., 1982). The uncertainty regarding the role of senDNA is also due to an observation that when senescent mycelium was placed over juvenile mycelium, the senDNAa molecules were transmitted to the recipient mycelium but senescence did not occur (Jamet-Vierny et al., 1999). Moreover, senescence occurred in certain nuclear-gene mutants in which senDNAa amplification was impaired (Dujon


Mauriceville 3581 bp

Reverse I transcriptase o

VSDNA 881 bp


Reverse transcriptase

Terminal inverted DNA polymerase Kalilo RNA polymerase Terminal inverted repeat 8643 bp repeat □-----□

Terminal inverted RNA polymerase Maranhar DNA polymerase Terminal inverted repeat 7052 bp repeat

Figure 14.5 pMAU, pVAR, pKAL and pMAR. Heavy lines are open reading frames, with direction of transcription. From Griffiths (1995).

and Belcour, 1989). Because of the inconsequential role of senDNA in senescence, another senDNA — senDNAß—is suspected to be involved in senescence. Nevertheless, a point of interest is how specific regions of mitochondrial DNA are deleted. PCR analysis of junction sequences of senDNAß monomers, recovered from several senescent cultures, indicate that the break points are bound by repeats of up to 27 base pairs. It has been hypothesized that deleted mitochondrial DNA molecules arise from unequal intramolecular cross-overs between short repeats that occur in the mitochondrial DNA (Jamet-Vierny et al., 1997a or b).

14.3.2 Nuclear Gene Control of Mitochondrial DNA Deletions

Although senescence in P. anserina showed extranuclear (maternal) inheritance, the time of senescence is altered in certain nuclear gene mutants, suggesting that nuclear genes control mitochondrial DNA rearrangements. For example, the double mutant incoloris vivax (i viv) remained alive for at least four years in contrast to the wild type that died in less than 21 days (Tudzynski and Esser, 1979). The mutant grisea also had an extended life span (Borghouts et al., 1997), whereas the mutant AS1-4 died prematurely in five to six days (Belcour et al., 1991). Since the amplification of senDNAa in the mutant grisea was minimal, this suggests that the wild-type GRISEA gene controls the amplification of senDNAa. These observations predict that several nuclear genes control the synthesis of factors which are imported into mitochondria and function in the stabilization of the mitochondrial genome, a point that will be addressed later.

14.4 PLASMID-BASED SENESCENCE IN NEUROSPORA 14.4.1 Mitochondrial Plasmids

Plasmids are small molecular weight, autonomously replicating extrachromosomal DNA molecules first discovered in bacteria. In fungi, plasmids are found in the mitochondria where they were discovered in a screen of natural populations of Neurospora for structural variants of the mitochondrial chromosome. The presence of plasmids is manifested by a brightly staining band on gels after electrophoresis of restriction enzyme digests of mito-chondrial DNA preparations stained with ethidium bromide (Collins et al., 1981). Plasmids (Figure 14.6) are implicated in senescence because they are co-inherited maternally with the senescence character and are integrated into mitochondrial DNA in senescing mycelia (Griffiths, 1995). However, the mere presence of plasmid does not identify a strain as senescence-prone because strains can harbor harmless plasmids.

A survey of Neurospora species by the Southern hybridization method using plasmid probes demonstrated that both senescent and nonsenescent strains have two types of plasmids: linear and circular (Griffiths, 1995). The first plasmid discovered was named Mauriceville plasmid after the place from where the host strain was collected. It is a closed-circular DNA molecule (a concatamer of up to six repeats of a monomer of about 3.6 kb). In genetic terminology for plasmid, the letter "p" is added before the first three letters of the name of plasmid in capital letters. In Southern hybridization, the Mauriceville plasmid (pMAU) hybridized neither to mitochondrial DNA nor to nuclear DNA, refuting its origin from either of these DNAs.



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