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首页> 外文期刊>Molecular and Cellular Biology >Effect of insertions, deletions, and double-strand breaks on homologous recombination in mouse L cells.
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Effect of insertions, deletions, and double-strand breaks on homologous recombination in mouse L cells.

机译：插入，缺失和双链断裂对小鼠L细胞中同源重组的影响。

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摘要

We have used DNA-mediated gene transfer to study homologous recombination in cultured mammalian cells. A family of plasmids with insertion and deletion mutations in the coding region of the herpes simplex type 1 thymidine kinase (tk) gene served as substrates for DNA-mediated gene transfer into mouse Ltk- cells by the calcium phosphate technique. Intermolecular recombination events were scored by the number of colonies in hypoxanthine-aminopterin-thymidine selective medium. We used supercoiled plasmids containing tk gene fragments to demonstrate that an overlap of 62 base pairs (bp) of homologous DNA was sufficient for intermolecular recombination. Addition of 598 bp of flanking homology separated from the region of recombination by a double-strand gap, deletion, or insertion of heterologous DNA increased the frequency of recombination by 300-, 20-, or 40-fold, respectively. Linearizing one of the mutant plasmids in a pair before cotransfer by cutting in the area of homology flanking a deletion of 104 bp or an insertion of less than 24 bp increased the frequency of recombination relative to that with uncut plasmids. However, cutting an insertion mutant of greater than or equal to 24 bp in the same manner did not increase the frequency. We show how our data are consistent with models that postulate at least two phases in the recombination process: homologous pairing and heteroduplex formation.
机译：我们已经使用DNA介导的基因转移来研究培养的哺乳动物细胞中的同源重组。在单纯疱疹1型胸苷激酶（tk）基因的编码区域中具有插入和缺失突变的质粒家族，是通过磷酸钙技术将DNA介导的基因转移到小鼠Ltk细胞中的底物。通过次黄嘌呤-氨基蝶呤-胸苷选择性培养基中的菌落数对分子间重组事件进行评分。我们使用包含tk基因片段的超螺旋质粒来证明同源DNA的62个碱基对（bp）的重叠足以进行分子间重组。通过双链缺口，缺失或插入异源DNA而从重组区域分离的598bp侧翼同源性分别使重组频率增加了300-，20-或40-倍。相对于未切割的质粒，通过切割侧翼缺失104 bp或插入少于24 bp的同源性区域来共转移之前，线性化一对突变质粒中的一个突变质粒可以增加重组的频率。但是，以相同方式切割大于或等于24 bp的插入突变体并不会增加频率。我们展示了我们的数据如何与在重组过程中假设至少两个阶段的模型一致：同源配对和异源双链体形成。

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