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

我们发现，从多头拟青霉中分离到的线粒体DNA在弱离子溶液中发生碎裂。在STE(150 mM氯化钠，TE)中观察到完整的mtDNA条带，但在TE(10 mM Tris-HCl，1 mM EDTA)和DW中出现了几条新的条带。在TE中，在67.7kBP(α带)和5.0kBP(β带)处出现两条离散带，而在DW中至少观察到17条离散带。对这些片段的定位表明，它们的切割位点是线粒体DNA的S1核酸酶敏感区。α和β片段来自编码RNA和三磷酸腺苷合成酶基因的区域。对幼年疟原虫和老年疟原虫等量的线粒体DNA进行电泳法，测量每个样本中β片段的频率。幼体原虫β条带占总信号的比例为3.3%~4.0%，老化原虫为8.3%~28.2%。α和β片段是体外扩增片段的中间产物，在DW等恶劣条件下，最终17个片段中有4、6、7和9个片段。我们成功地对α和β片段的单链区进行了测序。在α片段的特定序列上至少有4个单链断裂。其中两个位于编码LrRNA的相对DNA链上的110个核苷酸之间，另两个位于编码ATPA的相反DNA链上的86个核苷酸之间。这两个区域的DNA裂解在体内破坏了这两个基因，而片段4在体外破坏了这两个基因。通过引物延伸分析直接检测到这样的单链断裂，表明它们的频率随着原生质体的衰老而增加。多头拟青霉线粒体DNA的单链断裂可能随年龄的增长而积累。

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.