Abstract
Gene expression and DNA copy number analyses using full genome oligonucleotide microarrays of Leishmania reveal molecular mechanisms of methotrexate resistance.
Highlights
Drug resistance can be complex, and several mutations responsible for it can coexist in a resistant cell
Relative gene expression data (RNA) expression profiling in methotrexate resistant Leishmania cells Completion of the L. major genome has allowed the generation of arrays containing 60-mer oligonucleotide probes designed by NimbleGen Systems [55,56] and in this work, we present the generation of a full genome DNA microarray composed of 70-mer oligonucleotide probes suitable for both L. major and L. infantum analysis
Microarrays are likely to be useful for studying resistance in Leishmania since it is often mediated by gene amplification [3,4] and we show here that DNA arrays hybridized to cDNAs were most valuable for detecting gene amplification events (Figures 2, 4, and 5)
Summary
Drug resistance can be complex, and several mutations responsible for it can coexist in a resistant cell. We generated full genome 70-mer oligonucleotide microarrays for all protein coding genes of the human protozoan parasites Leishmania major and Leishmania infantum. These arrays were used to monitor gene expression in methotrexate resistant parasites. Several studies dealing with drug resistance in Leishmania have highlighted the plasticity of the Leishmania genome [3,4]. Leishmania is a folic acid auxotroph and studies of MTX resistance mechanisms have highlighted several novel aspects of folate metabolism in this parasite that could be exploited for drug interventions [9,10]. The development of novel antifolate molecules for Leishmania and related parasites has been ongoing in several laboratories [1113]
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