Isolation and characterization of kinetoplast DNA from bloodstream form of Trypanosoma brucei.
Isolation and characterization of kinetoplast DNA from bloodstream form of Trypanosoma brucei.
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DOI：
10.1083/jcb.76.2.293
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发表时间：
1978-02
期刊：
影响因子：
--
通讯作者：
Borst P
中科院分区：
文献类型：
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作者：
Fairlamb AH;Weislogel PO;Hoeijmakers JH;Borst P
我们使用限制性内切酶 PstI、EcoRI、HapII、HhaI 和 S1 核酸酶来证明，除了布氏锥虫动质体 DNA (kDNA) 网络中的主要小环成分（东非锥虫病研究组织 [EATRO] 427）之外，还存在大型复杂成分（大环）。核酸内切酶 PstI 和 S1 核酸酶在单个位点切割最大环，使其以线性形式分离，摩尔重量为 12.2 x 10(6)（通过电子显微镜测定）。其他酶产生多个大环片段，通过凝胶电泳测定其添加摩尔重量为12-13 x 10(6)。另一种 T. brucei 分离物 (EATRO 1125) 中的大环产生类似的片段，但似乎包含约 0.7 x 10(6) 道尔顿的缺失。 kDNA 的电子显微镜显示 DNA 的存在比小圆轮廓长度（0.3 微米）长得多，无论是在网络中还是作为从边缘延伸的环。这种长 DNA 永远不会超过最大环长度（6.3 微米），并通过核酸内切酶 PstI 消化而完全去除。 5-10% 的网络是双联体，在迷你环网络的两半之间聚集了多达 40 个 DNA 环，可能代表了 kDNA 的分裂阶段。使用 PstI 进行消化可以选择性地去除这些环，而不会显着改变小环网络。我们得出的结论是，单网络和双网络中的长 DNA 代表大环，而小环的长串联重复寡聚体（实际上）不存在。来自马匹锥虫（一种无法合成功能齐全的线粒体的锥虫物种）的 kDNA 包含尺寸与布氏锥虫相似的单网络和双网络，但没有任何超过小圆轮廓长度的 DNA。我们得出结论，锥虫的最大环是其他生物体线粒体 DNA (mtDNA) 的遗传等价物。
We have used restriction endonucleases PstI, EcoRI, HapII, HhaI, and S1 nuclease to demonstrate the presence of a large complex component, the maxi-circle, in addition to the major mini-circle component in kinetoplast DNA (kDNA) networks of Trypanosoma brucei (East African Trypanosomiasis Research Organization [EATRO] 427). Endonuclease PstI and S1 nuclease cut the maxi-circle at a single site, allowing its isolation in a linear form with a mol wt of 12.2 x 10(6), determined by electron microscopy. The other enzymes give multiple maxi-circle fragments, whose added mol wt is 12-13 x 10(6), determined by gel electrophoresis. The maxi-circle in another T. brucei isolate (EATRO 1125) yields similar fragments but appears to contain a deletion of about 0.7 x 10(6) daltons. Electron microscopy of kDNA shows the presence of DNA considerably longer than the mini-circle contour length (0.3 micron) either in the network or as loops extending from the edge. This long DNA never exceeds the maxi-circle length (6.3 microns) and is completely removed by digestion with endonuclease PstI. 5-10% of the networks are doublets with up to 40 loops of DNA clustered between the two halves of the mini-circle network and probably represent a division stage of the kDNA. Digestion with PstI selectively removes these loops without markedly altering the mini-circle network. We conclude that the long DNA in both single and double networks represents maxi-circles and that long tandemly repeated oligomers of mini-circles are (virtually) absent. kDNA from Trypanosoma equiperdum, a trypanosome species incapable of synthesizing a fully functional mitochondrion, contains single and double networks of dimensions similar to those from T. brucei but without any DNA longer than mini-circle contour length. We conclude that the maxi-circle of trypanosomes is the genetic equivalent of the mitochondrial DNA (mtDNA) of other organisms.