Deletion mutants of Neurospora crassa mitochondrial DNA and their relationship to the "stop-start" growth phenotype.

Abstract

ABSTRACT"Stoppers" are a class of Neurospora crassa extranuclear mutants characterized by gross deficiencies of cytochromes b and aa3 and an unusual growth phenotype which involves irregular periods of growth and nongrowth. In the present work, mtDNAs from all four stopper mutants were found to contain deletions or insertions detectable by restriction enzyme analysis. [stp] mtDNA consists predominantly of defective molecules which retain a 16-megadalton segment 4, [stp] mtDNA (i.e., EcoRI-1, 4, and -6). The findings suggest that production of defective mtDNAs in Neurospora is nonrandom with a preferred mechanism leading to retention of this segment. It may be significant that the retained segment contains both mitochondrial rRNA genes and most mitochondrial tRNA genes. These deletion mutants may provide a tool for genetic mapping of Neurospora mtDNA."Stoppers" are a class of Neurospora crassa extranuclear mutants distinguished by an unusual growth pattern on solid media: in contrast to wild-type strains which grow by continuous extension of hyphal tips, stopper mutants alternate between periods of growth and nongrowth, either of which may last for a few hours or several days (1-5). Stopper mutants are also characterized by gross alterations in mitochondrial phenotype including severe deficiencies of cytochromes b and aa3 (1-5). These findings suggest that the stopper phenotype results from a mutation in mtDNA, but the relationship between the mitochondrial defect and the growth characteristics has remained obscure.Recently, in the course of studying variant Neurospora mtDNAs which contain tandem reiterations, Mannella et al. (6)

Unstable mitochondrial DNA in natural-death nuclear mutants of Neurospora crassa.

“…These observations not only confirm the recessive nature of the nd mutation but also suggest that it confers loss of function, rather than altered specificity, to the product of the nd gene, most likely a protein that somehow is involved with maintenance, replication, repair, or recombination of the mitochondrial chromosome. Furthermore, the combination of our results with the observation by Sheng (32) that the natural-death syndrome is not acquired by nd+ strains through heterokaryosis with the nd mutant leads to the conclusion that the abnormal mtDNAs that are generated at a very rapid rate in nd homokaryons do not include molecules that accumulate in a suppressive mode as do the abnormal mitochondrial chromosomes of the stopper cytoplasmic mutants of N. crassa (5,6,14,17). If suppressive types of defective mtDNAs were generated, they would be introduced by the cytoplasm from the nd homokaryons into maintainer heterokaryons, which subsequently would develop senescence symptoms analogous to those associated with the natural-death syndrome.…”

“…With respect to aging in filamentous fungi, it appears that the nd mutant conforms to the idea that the senescence syndromes of these organisms involve mitochondrial dysfunction resulting from mutations affecting mtDNA (2,3,4,5,6,10,15,17). However, the nd mutant is different from all other systems that have been analyzed to date in that the instability in the mtDNA is caused by a recessive mutation in a nuclear gene rather than a mutation in the mitochondrial genome that mediates directly the suppressive accumulation of a specific class of mutant mtDNA molecules.…”

“…4. The main and most surprising observation that emerges from this figure is that the segment of the mtDNA that contains the genes for both rRNAs and most of the tRNAs, and is consistently conserved and maintained in high copy numbers in the death-prone stopper cytoplasmic mutants of N. crassa (5,14,17), was labile and conspicuously diminished in copy number in the nd nuclear mutant. The exact nature of these changes and their implications for the role of the mitochondrial genome in ndmediated senescence are under investigation.…”

“…In Podospora anserina, aging is associated with excision and amplification of small segments of mitochondrial DNA (mtDNA) (2,12,19,20). In certain strains of Neurospora spp., senescence is initiated by integration of linear (4,6) or circular (1) plasmids into the mitochondrial chromosome, whereas the stopper mutants of Neurospora crassa (5,14,17), the ragged mutants of Aspergillus amstelodami (24), and senescent cultures of Podospora curvicolla (10) all have large deletions in their mtDNAs. In all of these cases, the senescence phenotype is suppressive and inherited cytoplasmically.…”

Hyperactive recombination in the mitochondrial DNA of the natural death nuclear mutant of Neurospora crassa.

“…The (2,12,15,27,36), the magnitude of the recombinogenic activity in the mitochondria of the nd mutant is illustrated by the fact that two of the five novel EcoRI fragments that were characterized each contained three different rearrangements, one registered two instances of unequal crossing over, and only two contained single, albeit different, deletion junctions. All of these rearrangements had occurred within 3 weeks after extraction of the nd homokaryon from the maintainer heterokaryon, while it takes many months, often years, of growth for the appearance of a single rearrangement in the entire mitochondrial chromosome of wild-type strains (12,15 (2,5,12,26,36). For example, a reciprocal crossover between the two rep701 sequences which are located at opposite ends of EcoRI-1 (Fig.…”