Biosynthesis of mitochondrial DNA. Is 8 S DNA an artifact?
Biosynthesis of mitochondrial DNA. Is 8 S DNA an artifact?
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线粒体 DNA 的生物合成。
DOI：
10.1016/s0021-9258(17)40460-1
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发表时间：
1977
期刊：
影响因子：
--
通讯作者：
M. Simpson
中科院分区：
文献类型：
--
作者：
G. V. Van Tuyle;F. D. Hamilton;F. F. Vissering;M. Simpson
分离的大鼠肝脏线粒体 DNA 的蔗糖密度梯度分级通常会产生两个峰，一个在 39 S，另一个在 27 S。然而，当这些线粒体首先与标记的 DNA 前体一起孵育时，也会在约 8 S 处观察到标记峰。这种低分子量 8 S DNA 仅仅是污染或分解的产物，还是线粒体基因组的功能部分？它不是核污染物的表现是： (a) 活性线粒体制剂中不存在细胞核或核碎片； (b) 8S DNA 合成对用 DNase 和 RNase 处理线粒体不敏感； (c) 内膜制剂合成该 DNA 的能力； (d)白术苷同等地抑制[3H]dATP掺入8S和39S或27SDNA的能力； (e)体内给予[3H]胸苷后，标记8S DNA（以及39S和27SDNA），但不标记核DNA。 8 S DNA 不是线粒体孵育或 DNA 分离过程中 DNA 分解产生的伪影的证据如下： (a) 8 S DNA 可以从未孵育的线粒体中分离出来； (b) 当标记的DNA前体被施用到体内时，8S DNA被标记； (c) 8 S DNA 生物合成在完全不存在标记的 39 S 或 27 S DNA（其合成受到溴化乙锭抑制）的情况下继续进行，使得 8 S DNA 不可能由 39 S 或 27 S DNA 的分解形成； (d) 替代较温和的 DNA 提取方法不会降低 8 S DNA 标记；此外，当应用于纯化的 39 S 和 27 S DNA 时，通常的提取条件不会生成 8 S DNA，额外的线粒体洗涤循环也不会生成； (e) 8 S DNA 的比放射性高于 39 S 或 27 S DNA，使得后者不太可能是 8 S DNA 的前体。由于 8S DNA 是双链，因此它与 D 环 DNA 的 7S 片段不同。测试了含有相反 D 环的 DNA 分子的人为切口导致双链片段释放的假设。释放的 DNA 主要（并且可能完全）是单链的。我们的结论是 8 S DNA 可能不是人工制品，对其功能的研究正在进行中。
Sucrose density gradient fractionation of isolated rat liver mitochondrial DNA ordinarily yields two peaks, one at 39 S, the other at 27 S. However, when these mitochondria are first incubated with a labeled DNA precursor, a labeled peak at about 8 S is also observed. Is this low molecular weight 8 S DNA merely an artifact of contamination or breakdown, or is it a functioning part of the mitochondrial genome? That it is not a nuclear contaminant is shown by: (a) the absence of nuclei or nuclear fragments in active mitochondrial preparations; (b) the insensitivity of 8 S DNA synthesis to treatment of mitochondria with DNase and RNase; (c) the ability of inner membrane preparations to synthesize this DNA; (d) the ability of atractyloside to inhibit incorporation of [3H]dATP into 8 S and 39 S or 27 S DNA equally; (e) the labeling of 8 S DNA (as well as 39 S and 27 S DNA) but not of nuclear DNA after the administration in vivo of [3H]thymidine. The evidence that 8 S DNA is not an artifact resulting from DNA breakdown during mitochondrial incubation or DNA isolation is as follows: (a) 8 S DNA can be isolated from unincubated mitochondrial; (b) 8 S DNA becomes labeled when labeled DNA precursors are administered in vivo; (c) 8 S DNA biosynthesis continues in the complete absence of labeled 39 S or 27 S DNA (whose synthesis is repressed by ethidium bromide), making it unlikely that 8 S DNA is formed from the breakdown of 39 S or 27 S DNA; (d) substitution of milder methods of DNA extraction does not decrease 8 S DNA labeling; moreover, the usual conditions of extraction, when applied to purified 39 S and 27 S DNA, do not generate 8 S DNA, nor does an additional mitochondrial washing cycle; (e) the specific radioactivity of 8 S DNA is higher than that of 39 S or 27 S DNA, making it improbable that the latter forms are precursors of 8 S DNA. Since 8 S DNA is double-stranded, it is not identical to the 7 S fragment of D loop DNA. The hypothesis that the artifactual nicking of those DNA molecules which contain opposing D loops leads to the release of double-stranded fragments was tested. The DNA which was released was predominantly (and probably completely) single-stranded. We conclude that 8 S DNA is probably not an artifact and studies are in progress on its function.