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

我们发现从多头绒螯蟹中提取的mtDNA在弱离子溶液中会发生片段化。在STE（150 mM NaCl，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67.7 kbp（α带）和5.0 kbp（β带）处出现两条离散带，而在DW中最终观察到至少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片段来源于编码rRNA和ATP合酶（atpA）基因的区域。对幼年和老年疟原虫的mtDNA进行了等量的扩增，并测量了每个样品中β片段的频率。β带占总信号（包括背景）的比例在幼年疟原虫中估计为3.3 ~ 4.0%，而在老年疟原虫中增加到8.3 ~ 28.2%，α和β片段是体外DNA片段化的中间产物，在严酷的条件下（如DW）产生其余17个片段中的片段4、6、7和9。我们成功地测序了α和β片段的单链区域。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 bp之间，另外两个位于编码atpA的相对DNA链上的86 bp之间。这两个区域的DNA裂解在体内破坏这两个基因，而片段4在体外破坏。直接检测这种单链断裂的引物延伸分析表明，他们的频率增加疟原虫衰老。随着年龄的增长，多头绒螯蟹线粒体DNA的单链断裂可能会逐渐增加。

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.