Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays
Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays
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DOI：
10.1093/nar/gkg806
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发表时间：
2003-10-15
影响因子：
14.9
通讯作者：
Ouellette, M
中科院分区：
文献类型：
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作者：
Guimond, C;Trudel, N;Ouellette, M
在原生动物寄生虫利什曼原虫中，耐药性可能是一个复杂的现象。一些代谢途径和膜转运蛋白与耐药表型有关。为了监测这些基因的表达，我们生成了定制的 DNA 微阵列，其中包含与耐药性相关的 44 个基因相对应的 PCR 片段。使用这些阵列对亚砷酸盐和锑抗性突变体进行转录谱分析，精确定位了突变体中过度表达的许多基因，包括 ABC 转运蛋白 PGPA、谷胱甘肽生物合成基因 γ-谷氨酰半胱氨酸合成酶 (GSH1) 和谷胱甘肽合成酶 (GSH2)。与来自敏感细胞和甲氨蝶呤耐药细胞的总RNA的竞争性杂交揭示了编码二氢叶酸还原酶（DHFR-TS）、蝶啶还原酶（PTR1）和S-腺苷甲硫氨酸合酶（MAT2）的基因的过度表达以及叶酸转运蛋白（FT）家族的一个基因的下调。通过标记敏感和抗性寄生虫的 DNA，我们还可以使用 DNA 微阵列检测多种基因扩增事件，包括 S-腺苷高半胱氨酸水解酶基因 (SAHH) 的扩增。通过Northern印迹分析验证了微阵列检测到的基因表达的改变，而Southern印迹表明大多数过度表达的基因也被扩增，尽管也存在其他机制。微阵列可用于研究耐药性寄生虫，以查明与耐药性相关的几个基因。
In the protozoan parasite Leishmania, drug resistance can be a complex phenomenon. Several metabolic pathways and membrane transporters are implicated in the resistance phenotype. To monitor the expression of these genes, we generated custom DNA microarrays with PCR fragments corresponding to 44 genes involved with drug resistance. Transcript profiling of arsenite and antimony resistant mutants with these arrays pinpointed a number of genes overexpressed in mutants, including the ABC transporter PGPA, the glutathione biosynthesis genes gamma-glutamylcysteine synthetase (GSH1) and the glutathione synthetase (GSH2). Competitive hybridisations with total RNA derived from sensitive and methotrexate resistant cells revealed the overexpression of genes coding for dihydrofolate reductase (DHFR-TS), pteridine reductase (PTR1) and S-adenosylmethionine synthase (MAT2) and a down regulation of one gene of the folate transporter (FT) family. By labelling the DNA of sensitive and resistant parasites we could also detect several gene amplification events using DNA microarrays including the amplification of the S-adenosyl homocysteine hydrolase gene (SAHH). Alteration in gene expression detected by microarrays was validated by northern blot analysis, while Southern blots indicated that most genes overexpressed were also amplified, although other mechanisms were also present. The microarrays were useful in the study of resistant parasites to pinpoint several genes linked to drug resistance.