Molecular cytological studies on the relationship between mitochondrial genome damage and plasmodial senescence of the true slime mold, Physarum polycephalum

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We found that mtDNA isolated from P. polycephalum fragmented itself in weak ionic solutions. The intact mtDNA band was seen in STE (150 mM NaCl, TE), but several novel bands appeared in TE (10 mM Tris-HC1, 1 mM EDTA) and DW. In TE, two discrete bands appeared at 67.7 kbp (α band) and 5.0 kbp (β band), whereas at least 17 discrete bands were finally observed in DW. Mapping these fragments demonstrated that their cleavage sites are S1 nuclease sensitive regions of the mtDNA. The α and β fragments are derived from the region encoding rRNAs and the ATP synthase (atpA) gene. Equal amounts of mtDNA from juvenile and aged plasmodia were electrophoresed and the frequency of the β fragment in each sample was measured. The ratio of the β band to the total signal including background was estimated to be 3.3 to 4.0 % in juvenile plasmodia whereas it increased to 8.3 to 28.2 % in aged plasmodia.The α and β fragments are intermediates of in vitro DNA fragmentation and yield fragments 4, 6, 7 and 9 of the other final 17 fragments under severe condition such as DW. We succeeded to sequence the single stranded regions of the α and β fragments. There are at least 4 single stranded breakages on specific sequences of the α fragment. Two of them are located between 110 bp on opposite DNA strands coding LrRNA, and the other two are located between 86 bp on opposite DNA strands coding atpA. The DNA cleavages of these two regions disrupt the two genes in vivo while the fragment 4 in vitro. Direct detection of such single stranded breakages by a primer-extension analysis showed that the frequency of them increased as plasmodial senescence. The single-stranded breakage of mtDNA of P. polycephalum may accumulate with age.