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

真粘菌多头绒泡菌线粒体基因组损伤与疟原虫衰老关系的分子细胞学研究

基本信息

批准号：
11440242
负责人：
KAWANO Shigeyuki
金额：
$ 5.95万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11440242/
关键词：
Physarum polycephalum plasmodium aging senescence mitochondria single-stranded breakage dysfunction genome damage

项目摘要

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.

我们发现从 P.polycephalum 中分离的 mtDNA 在弱离子溶液中会自行断裂，在 STE（150 mM NaCl、TE）中观察到完整的 mtDNA 带，但在 TE（10 mM Tris-HC1、1 mM EDTA）中出现了一些新的带。在TE中，在67.7 kbp（α带）和5.0 kbp（β带）处出现了两个离散带，而最终至少有17个离散带。在 DW 中观察到这些片段表明它们的切割位点是 mtDNA 的 S1 核酸酶敏感区域，α 和 β 片段源自编码 rRNA 和 ATP 合酶 (atpA) 基因的区域。对疟原虫进行电泳并测量每个样品中β片段的频率。β带与包括背景的总信号的比率估计为3.3至4.0。幼年疟原虫中为 %，而老年疟原虫中增加到 8.3 至 28.2%。α 和 β 片段是体外 DNA 片段化的中间体，在 DW 等严酷条件下产生其他最终 17 个片段中的片段 4、6、7 和 9。我们成功对α和β片段的单链区域进行了测序，其中α片段的特定序列上至少有4处单链断裂。它们位于编码 LrRNA 的相反 DNA 链上的 110 bp 之间，另外两个位于编码 atpA 的相反 DNA 链上的 86 bp 之间。这两个区域的 DNA 切割在体内破坏这两个基因，而片段 4 在体外直接破坏。通过引物延伸分析检测此类单链断裂表明，它们的频率随着疟原虫线粒体衰老而增加。多头菌可能会随着年龄的增长而积累。