|Title:||Identification and molecular characterization of differentially expressed gene(s) among LeMDR1 (Leishmania enriettii Multidrug Resistance 1) mutants using suppression subtractive hybridization technology|
|Subject:||Hong Kong Polytechnic University -- Dissertations|
|Department:||Department of Applied Biology and Chemical Technology|
|Pages:||xvi, 299 leaves : ill. ; 30 cm|
|Abstract:||Drug resistance is emerging as a major problem in many parasitic diseases. It poses a significant problem for treatment and control of these diseases. Recent evidence has suggested that a major mechanism of drug resistance in protozoan parasites may be through overexpression of a P-glycoprotein molecule encoded by a multidrug resistance gene (mdr). In Leishmania enriettii, the role of LeMDR1 (Leishmania enriettii Multidrug Resistance 1) gene in mediating drug resistance has been demonstrated by stepwise selection, gene knockout and transfection approaches. Full level of resistance, however, may require other factors besides LeMDR1. The natural function of LeMDR1 remains unclear. The goal of this work is to understand the natural function of LeMDR1 and to identify the regulatory genes of LeMDR1. Here, a novel technique, suppression subtractive hybridization (SSH), was employed to study the differential gene expression among LeMDR1 +/+ parent, LeMDR1 -/-mutant and LeMDR1-amplified mutant (LeVl60) of L. enriettii. The differentially expressed gene(s) identified by SSH have been characterized by Northern analysis, RT-PCR, 5' and 3' RACE and functional analysis. In LeMDR1 -/- mutant, nine genes were found to be up-regulated (>1.8-fold) relative to the Le wild type including methionine synthase, ribosomal protein S16, ribosomal protein S23, cytochrome c oxidase subunit I (COX I), cytochrome c oxidase subunit VI (COX VI), stomatin-like protein, DNA J homolog and two novel genes, whereas three genes were found to be down-regulated (<0.6-fold) including p68-like RNA helicase and two novel genes. In cytochrome c oxidase activity and apoptosis assay, LeMDR1 -/- mutant consistently showed higher activity (2.5-fold) and became more resistant to apoptosis than the Le wild type. The findings of up-regulation of both COX I and COX VI and higher cytochrome c oxidase activity in LeMDR1 -/- mutant are consistent with our previous findings that LeMDR1 is located at the mitochondria by immunoelectron microscopy and its overexpression causes hypersensitivity to mitochondrial drugs like rhodamine 123. It further supports the notion that LeMDR1's normal function is in the mitochondria. In addition, methionine synthase, DNA J homolog and two novel genes possibly belong to LeMDR1 regulatory genes. In Northern analysis, the expression levels of methionine synthase, DNA J homolog and a novel gene were found to be negatively correlated with the LeMDR1 copy number, whereas another novel gene exhibited a positive relationship. In methionine synthase activity, LeMDR1 -/- mutant significantly exhibited a higher (2.0-fold), whereas LeV160 mutant showed a lower activity (0.76-fold) relative to the Le wild type. It suggests that alteration of methionine metabolism can compensate the loss-of-function of LeMDR1 and cause drug resistant phenotype in parasites. These differentially expressed genes identified by SSH represent valuable candidate genes for further functional analysis in drug resistance and should be informative in studying the natural function of LeMDR1: they seem to participate in a network between oxidative phosphorylation, ion-channel regulation, methionine metabolism, protein synthesis, apoptosis inhibition.|
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