Tsukamoto Y, Kato J, Ikeda H
Department of Molecular Biology, Univesity of Tokyo, Japan.
Genetics. 1996 Feb;142(2):383-91. doi: 10.1093/genetics/142.2.383.
To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rad51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.
为了研究酿酒酵母中异常重组的机制，我们开发了一种用于定量分析缺失形成的质粒系统。在YCp质粒上携带两个负选择标记CAN1和CYH2基因的can1 cyh2细胞对刀豆氨酸和环己酰亚胺敏感，但当质粒在CAN1和CYH2基因上发生缺失时，该细胞对这两种药物都产生抗性。对从抗性细胞中获得的重组质粒进行结构分析表明，质粒在CAN1 - CYH2区域的各个位点发生了缺失，并且在重组连接处只有短的同源区域（1 - 5个碱基对）。结果表明，在该系统中检测到的缺失是由异常重组形成的。对几种rad突变效应的研究表明，在rad52、rad50、mre11和xrs2突变体中，重组率分别降低了30倍、10倍、10倍和10倍，而在rad51、54、55和57突变体中，重组率与野生型菌株相当。rad52突变不影响异常重组连接处同源序列的长度。
