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首页> 外文期刊>Molecular and Cellular Biology >Homologous plasmid recombination is elevated in immortally transformed cells.
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Homologous plasmid recombination is elevated in immortally transformed cells.

机译：在永生转化的细胞中同源质粒重组增加。

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摘要

The levels of intramolecular plasmid recombination, following transfection of a plasmid substrate for homologous recombination into normal and immortally transformed cells, have been examined by two independent assays. In the first assay, recovered plasmid was tested for DNA rearrangements which regenerate a functional neomycin resistance gene from two overlapping fragments. Following transformation of bacteria, frequencies of recombinationlike events were determined from the ratio of neomycin-resistant (recombinant) colonies to ampicillin-resistant colonies (indicating total plasmid recovery). Such events, yielding predominantly deletions between the directly repeated sequences, were substantially more frequent in five immortal cell lines than in any of three normal diploid cell strains tested. Effects of plasmid replication or interaction with T antigen and of bacterially mediated rejoining of linear molecules generated in mammalian cells were excluded by appropriate controls. The second assay used limited coamplification of a control segment of plasmid DNA, and of the predicted recombinant DNA region, primed by two sets of flanking oligonucleotides. Each amplified band was quantitated by reference to a near-linear standard curve generated concurrently, and recombination frequencies were determined from the ratio of recombinant/control DNA regions. The results confirmed that recombinant DNA structures were generated within human cells at direct repeats in the transfected plasmid and were markedly more abundant in an immortal cell line than in the diploid normal cells from which that line was derived.
机译：已经通过两个独立的测定法检查了将质粒底物转染用于同源重组的正常重组和永生转化的细胞后，分子内质粒重组的水平。在第一试验中，测试了回收的质粒的DNA重排，其从两个重叠的片段再生了功能性新霉素抗性基因。细菌转化后，从新霉素抗性（重组）菌落与氨苄青霉素抗性菌落的比率（表明总质粒回收率）确定了类似重组事件的频率。在五个永生细胞系中，此类事件主要在直接重复的序列之间产生缺失，其频率比所测试的三个正常二倍体细胞株中的任何一个更为频繁。适当的对照排除了质粒复制或与T抗原的相互作用以及在哺乳动物细胞中产生的细菌介导的线性分子的再结合的影响。第二种测定法使用了由两组侧翼寡核苷酸引发的质粒DNA控制片段和预测的重组DNA区域的有限共扩增。参照同时产生的近线性标准曲线对每个扩增的条带进行定量，并从重组/对照DNA区域的比率确定重组频率。结果证实，重组DNA结构是在人类细胞内直接转染的质粒中产生的，并且在永生细胞系中的DNA显着高于衍生自该系的二倍体正常细胞。

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