HOMOLOGOUS RECOMBINATION AND THE REPAIR OF DOUBLE-STRAND BREAKS DURING COTRANSFORMATION OF DICTYOSTELIUM-DISCOIDEUM
HOMOLOGOUS RECOMBINATION AND THE REPAIR OF DOUBLE-STRAND BREAKS DURING COTRANSFORMATION OF DICTYOSTELIUM-DISCOIDEUM
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DOI：
10.1128/mcb.8.7.2779
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发表时间：
1988-07-01
影响因子：
5.3
通讯作者：
RATNER, DI
中科院分区：
文献类型：
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作者：
KATZ, KS;RATNER, DI
我们检测了非连锁非复制质粒分子在变形虫Dictyostelium amoebae共转化过程中进行同源重组的能力。转化载体B10S对抗生素G418具有抗性，并一直以封闭的圆圈呈现给变形虫。含有黏菌cDNA和同源序列的共转化DNA，在质粒的三个不同区域之一内切的限制性内切酶消化后，被呈现为封闭的圆形或线性分子。值得注意的是，在所检查的每个克隆中都发生了同源重组。此外，重组产物在所有情况下都是相同的，无论线性化末端的存在或位置如何。线性模板的末端不发生重组。引入的双链断裂的修复在重组过程中频繁发生。修复可能发生在分子间，或者更可能发生在分子内，即通过再循环。许多重组事件都是非相互的。尽管同源重组开始频繁出现，但使用与所选载体不具有同源性的共转化DNA确定了共转化不需要这种重组。
We examined the ability of unlinked nonreplicating plasmid molecules to undergo homologous recombination during cotransformation of Dictyostelium amoebae. The transformation vector B10S confers resistance to the antibiotic G418 and was always presented to amoebae as a closed circle. Cotransforming DNA, containing a slime mold cDNA and sequences homologous in the primary vector, was presented either as a closed circle or as a linear molecule after digestion with restriction endonucleases which cut within one of three distinct regions of the plasmid. Remarkably, homologous recombination occurred in every clone examined. Moreover, the products of recombination were identical in all instances, irrespective or the presence or position of linearized ends. The ends of the linear templates were not recombinogenic. Repair of the introduced double-strand break occurred frequently during recombination. The repair could occur intermolecularly or, more likely, intramolecularly, i.e., by recircularization. Many of the recombination events were of a nonreciprocal nature. Despite the startingly frequent level of homologous recombination, the use of contransforming DNA which contains no homology to the selected vector established that such recombination was not required for cotransformation.