IN-VIVO AND IN-VITRO EVIDENCE FOR SLIPPED MISPAIRING IN MAMMALIAN MITOCHONDRIA
IN-VIVO AND IN-VITRO EVIDENCE FOR SLIPPED MISPAIRING IN MAMMALIAN MITOCHONDRIA
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DOI：
10.1073/pnas.90.16.7671
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发表时间：
1993-08-15
影响因子：
11.1
通讯作者：
HAUSWIRTH, WW
中科院分区：
文献类型：
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作者：
MADSEN, CS;GHIVIZZANI, SC;HAUSWIRTH, WW
DNA复制过程中重复序列间的错配是一种重要的致突变事件。它是被认为负责产生哺乳动物线粒体DNA中发现的多态性区域和大规模缺失的几种建议机制之一。在猪线粒体基因组中，携带10 bp串联重复序列的结构域显示出独特的重复拷贝数多态性的体内模式。在大肠杆菌中传代后，含有该结构域的重组质粒也显示出与动物中所见不同的独特多态性模式。为了检验这些多态性是滑动诱导的并且不同的多态性模式反映了复制模式的差异的假设，我们进行了一系列体外引物延伸反应。通过利用包含重复结构域的单链或双链模板，我们能够将体外产生的重复多态性模式分别与线粒体或细菌中观察到的重复多态性模式相关联，从而提供了实验证据，即滑移复制是哺乳动物突变的主要原因。
Slipped mispairing between repeated sequences during DNA replication is an important mutagenic event. It is one of several suggested mechanisms thought to be responsible for generating polymorphic regions and large-scale deletions found in mammalian mitochondrial DNA. In the porcine mitochondrial genome, a domain carrying a 10-bp tandemly repeated sequence displays a unique in vivo pattern of repeat copy number polymorphs. Upon passage in Escherichia coli, a recombinant plasmid containing this domain also displays a unique polymorphic pattern that is different from that seen in the animal. To test the hypothesis that these polymorphisms were slippage induced and that the different polymorphic patterns reflected differences in modes of replication, we performed a series of in vitro primer extension reactions. By utilizing either single- or double-stranded templates containing the repeat domain we were able to correlate in vitro generated repeat polymorphism patterns with those seen in the mitochondria or the bacteria, respectively, thus providing experimental evidence that slippage replication is responsible for a major class of mammalian mutations.