Intramolecular recombination and deletions in mitochondrial DNA of senescent, a nuclear-gene mutant of Neurospora crassa exhibiting “death” phenotype

Abstract

In Neurospora crassa, a nuclear-gene mutant, senescent, derived from a phenotypically normal wild isolate of Neurospora intermedia exhibits a ‘death’ phenotype. Regardless of the composition of the culture medium, the mycelium ceases to grow in 2–6 subcultures at 26 °C and 1 or 2 subcultures at 34 °C. Senescence of vegetative mycelium is associated with deficiencies in cytochromes aa 3 and b and reduced oxygen uptake. The restriction fragment analysis of mitochondrial DNA from senescing mycelia showed deletions and gross sequence rearrangements. Analysis of mitochondrial DNA of ( sen + sen +) heterokaryons constructed with “excess” sen cytoplasm suggested correlation between mtDNA deletions and senescence. Three novel sen-specific EcoRI fragments of sizes 3.6, 3.9, and 4.4 kb were cloned, sequenced, and analyzed. Nucleotide sequences of the sen-specific EcoRI fragments suggested that deletions were a consequence of intramolecular recombination between EcoRI-5 and -10 and/or between EcoRI-8 and -10. The recombination junctions were close to stretches of GC-rich- PstI palindromic sequences that potentially form stable hairpin structures and might facilitate recombination between homologous repeats as short as 6–10 bp. These observations suggest that the wild-type ( sen +) allele encodes a factor that protects the mitochondrial genome from undergoing intramolecular recombination and deletions. In this respect sen + (linkage group V) has a function similar to nd + (linkage group I) and the two gene products probably have mutually exclusive roles in suppressing cruciform-associated and homologous recombination, respectively, thus safeguarding mitochondrial genome integrity. The sen + allele most likely codes for a factor involved in recombination, repair or replication of the mitochondrial genome, or a transcription factor that regulates the expression of genes affiliated with mitochondrial DNA metabolism.