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首页> 外文期刊>Molecular and Cellular Biology >Selection against the dihydrofolate reductase-thymidylate synthase (DHFR-TS) locus as a probe of genetic alterations in Leishmania major.
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Selection against the dihydrofolate reductase-thymidylate synthase (DHFR-TS) locus as a probe of genetic alterations in Leishmania major.

机译：针对二氢叶酸还原酶-胸苷酸合酶（DHFR-TS）位点的选择，作为大利什曼原虫遗传变异的探针。

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The genome of the trypanosomatid protozoan genus Leishmania has been shown to undergo a number of changes relevant to drug resistance and virulence, such as gene amplification, chromosomal rearrangement, and variation in ploidy. Experimental approaches to the study of genomic changes have in some cases been limited by the fact that Leishmania cells are asexual diploids, as are some other trypanosomatids, pathogenic fungi, and cultured mammalian cells. Here we report upon a system which permits the measurement of several types of genomic change occurring at the dihydrofolate reductase-thymidylate synthase (DHFR-TS) locus. First, we show that DHFR-TS can function as a positiveegative marker. We used selection against DHFR-TS on a heterozygous line (+/HYG) to generate colonies exhibiting both loss of heterozygosity and structural mutations in DHFR-TS, permitting the first measurement of mutation frequencies in this parasite. Loss of heterozygosity occurred at a frequency ranging from 10(-4) to 10(-6) and was elevated 24-fold by treatment with gamma-irradiation, while the frequency of other events was less than 10(-6) and was increased more than 1,000-fold by nitrosoguanidine treatment. The frequency of loss of heterozygosity relative to other processes such as mutation and gene replacement has important implications for genetic variability in natural Leishmania populations and the generation of both targeted and random mutations. We also developed a protocol for null targeting of diploid cells, in which transfection of a DHFR-TS deletion construct into Leishmania cells followed by negative selection yielded parasites lacking DHFR-TS or foreign sequences. The null-targeting method can be applied to any diploid cell, at any locus for which a negative selection exists. Such marker-free auxotrophic Leishmania cells show potential as an attenuated vaccine, and the methods developed here provide a new approach for manipulating and characterizing the plasticity of the Leishmania genome.
机译：锥虫原虫属利什曼原虫属的基因组已显示出与耐药性和毒力有关的许多变化，例如基因扩增，染色体重排和倍性变化。研究基因组变化的实验方法在某些情况下受到以下事实的限制：利什曼原虫细胞是无性二倍体，其他一些锥虫，致病真菌和培养的哺乳动物细胞也是如此。在这里，我们报告一个系统，该系统允许测量发生在二氢叶酸还原酶-胸苷酸合酶（DHFR-TS）位点的几种类型的基因组变化。首先，我们表明DHFR-TS可以充当阳性/阴性标记。我们在杂合子系（+ / HYG）上使用针对DHFR-TS的选择来生成在DHFR-TS中显示杂合性丧失和结构突变的菌落，从而允许首次测量该寄生虫的突变频率。杂合性的丧失发生在10（-4）到10（-6）的频率上，通过伽马射线辐照处理增加了24倍，而其他事件的频率小于10（-6）并增加了通过亚硝基胍处理超过1000倍。相对于突变和基因置换等其他过程而言，杂合性丧失的频率对天然利什曼原虫种群的遗传变异以及靶向突变和随机突变的产生都具有重要意义。我们还开发了用于二倍体细胞无效靶向的方案，其中将DHFR-TS缺失构建体转染到利什曼原虫细胞中，然后进行阴性选择，产生缺少DHFR-TS或外源序列的寄生虫。空靶方法可以应用于任何二倍体细胞，在存在负选择的任何基因座上。这种无标记的营养缺陷型利什曼原虫细胞显示出作为减毒疫苗的潜力，这里开发的方法为操纵和表征利什曼原虫基因组的可塑性提供了一种新方法。

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