Papadopoulou B, Dumas C
Centre de Recherche en Infectiologie, Centre Hospitalier de l'Université Laval and Département de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada.
Nucleic Acids Res. 1997 Nov 1;25(21):4278-86. doi: 10.1093/nar/25.21.4278.
In this study we investigated the role of several parameters governing the efficiency of gene targeting mediated by homologous recombination in the protozoan parasite Leishmania. We evaluated the relative targeting frequencies of different replacement vectors designed to target several sequences within the parasite genome. We found that a decrease in the length of homologous sequences <1 kb on one arm of the vector linearly influences the targeting frequency. No homologous recombination was detected, however, when the flanking homologous regions were <180 bp. A requirement for a very high degree of homology between donor and target sequences was found necessary for efficient gene targeting in Leishmania , as targeted recombination was strongly affected by base pair mismatches. Targeting frequency increased proportionally with copy number of the target only when the target was part of a linear amplicon, but remained unchanged when it was present on circles. Different chromosomal locations were found to be targeted with significantly variable levels of efficiency. Finally, different strains of the same species showed differences in gene targeting frequency. Overall, gene targeting mediated by homologous recombination in Leishmania shares similarities to both the yeast and the mammalian recombination systems.
在本研究中，我们调查了几个参数在原生动物寄生虫利什曼原虫中对同源重组介导的基因靶向效率的调控作用。我们评估了设计用于靶向寄生虫基因组内多个序列的不同置换载体的相对靶向频率。我们发现载体一条臂上同源序列长度<1 kb时，其长度的减少会线性影响靶向频率。然而，当侧翼同源区域<180 bp时，未检测到同源重组。在利什曼原虫中，发现供体和靶序列之间需要非常高的同源性才能实现高效基因靶向，因为靶向重组会受到碱基对错配的强烈影响。仅当靶标是线性扩增子的一部分时，靶向频率才会与靶标的拷贝数成比例增加，但当它存在于环状物上时，靶向频率保持不变。发现不同的染色体位置被靶向的效率水平存在显著差异。最后，同一物种的不同菌株在基因靶向频率上表现出差异。总体而言，利什曼原虫中同源重组介导的基因靶向与酵母和哺乳动物重组系统都有相似之处。
