Nickoloff J A, Reynolds R J
Life Sciences Division, Los Alamos National Laboratory, New Mexico 87545.
Mol Cell Biol. 1990 Sep;10(9):4837-45. doi: 10.1128/mcb.10.9.4837-4845.1990.
Transcription stimulates homologous recombination in Saccharomyces cerevisiae and has been implicated in the control of recombinational events during the development of mammalian immune systems. Here, we describe a plasmid-based system in which an inducible promoter from the mouse mammary tumor virus is located upstream of heteroallelic neomycin genes carried on two plasmids. Pairs of plasmids are introduced into Chinese hamster ovary cells by electroporation, and recombination is monitored by scoring colonies resistant to the aminoglycoside G418. When transcription is induced with dexamethasone, a synthetic glucocorticoid hormone, and double-strand breaks are introduced at mutation sites, recombination is stimulated sixfold over noninduced levels. Inducing transcription in circular substrates or in substrates cleaved at sites distant from the mutations has no detectable effect on recombination between neomycin genes. Results are presented that are consistent with the observed stimulation of recombination occurring before plasmids integrate into the cellular DNA. Our results are discussed in relation to molecular models for extrachromosomal recombination in mammalian cells.
转录可刺激酿酒酵母中的同源重组，并且在哺乳动物免疫系统发育过程中的重组事件控制中发挥作用。在此，我们描述了一种基于质粒的系统，其中来自小鼠乳腺肿瘤病毒的可诱导启动子位于两个质粒上携带的异等位新霉素基因的上游。通过电穿孔将质粒对导入中国仓鼠卵巢细胞，并通过对氨基糖苷类G418抗性菌落进行评分来监测重组。当用合成糖皮质激素地塞米松诱导转录，并在突变位点引入双链断裂时，重组比未诱导水平增加了六倍。在环状底物中或在远离突变位点切割的底物中诱导转录对新霉素基因之间的重组没有可检测到的影响。呈现的结果与观察到的在质粒整合到细胞DNA之前发生的重组刺激相一致。我们的结果将结合哺乳动物细胞中染色体外重组的分子模型进行讨论。
