NONRECIPROCAL EXCHANGES OF INFORMATION BETWEEN DNA DUPLEXES COINJECTED INTO MAMMALIAN-CELL NUCLEI
NONRECIPROCAL EXCHANGES OF INFORMATION BETWEEN DNA DUPLEXES COINJECTED INTO MAMMALIAN-CELL NUCLEI
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DOI：
10.1128/mcb.5.1.59
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发表时间：
1985-01-01
影响因子：
5.3
通讯作者：
CAPECCHI, MR
中科院分区：
文献类型：
--
作者：
FOLGER, KR;THOMAS, K;CAPECCHI, MR
共注射到培养的哺乳动物细胞中的质粒分子之间的同源重组的机制进行了检查。通过从Neor基因中携带不同琥珀突变的2个质粒中选择重建功能性Neor基因，获得含有重组DNA分子的细胞系。这些质粒在Neor基因内和附近含有限制性长度多态性。这些多态性不赋予可选择的表型，但用于鉴定和分类选择和非选择的重组DNA分子。短距离共注射质粒分子之间信息交换的主要机制（即，< 1内切酶）通过非相互同源重组进行。在培养的哺乳动物细胞中，共注射的质粒分子之间的同源重组频率非常高，接近统一。这种高频率既不需要每个细胞的质粒分子的高输入，也不需要核内的质粒DNA的局部高浓度。显然，质粒分子一旦被引入细胞核，即使在存在大量过量宿主DNA序列的情况下，也能毫不费力地相互寻找并参与同源重组。大多数同源重组事件发生在质粒DNA导入细胞核后1小时内。
The mechanism of homologous recombination between plasmid molecules coinjected into cultured mammalian cells was examined. Cell lines containing recombinant DNA molecules were obtained by selecting for the reconstruction of a functional Neor gene from 2 plasmids that bear different amber mutations in the Neor gene. These plasmids contain restriction-length polymorphisms within and near the Neor gene. These polymorphisms did not confer a selectable phenotype but was used to identify and categorize selected and nonselected recombinant DNA molecules. The predominant mechanism for the exchange of information between coinjected plasmid molecules over short distances (i.e., < 1 kilobase) proceeds via nonreciprocal homologous recombination. The frequency of homologous recombination between coinjected plasmid molecules in cultured mammalian cells is extremely high, approaching unity. This high frequency requires neither a high input of plasmid molecules per cell nor a localized high concentration of plasmid DNA within the nucleus. Apparently plasmid molecules, once introduced into the nucleus, have no difficulty seeking each other out and participating in homologous recombination even in the presence of a vast excess of host DNA sequences. Most of the homologous recombination events occur within a 1-h interval after the introduction of plasmid DNA into the cell nucleus.