Browsing by Author "Kumar, Dhiraj"

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    Studies of Gene Expression in Drug Resistant Leishmania donovani using DNA Microarrays
    (BITS Pilani, 2010) Kumar, Dhiraj
    In the present study, microarray experiments were carried out to compare gene expression in drug sensitive and resistant Leishmania donovani. Two genes that showed significantly higher expression at both RNA and protein level in a number of resistant field isolates of L.donovani were selected for further characterization. newline1. Histone 2A (H2A). newline2. Parasite Surface Antigen-2. newlineParasites cultures were set up from bone marrow aspirates of KA patients (n=19), including 4 SAG responsive and 15 non responsive cases. In search of antimony resistance related genes, the gene expression profiling was undertaken using highly sensitive microarray technology. We developed a microarray chip for L. donovani comprising of 8448 PCR amplified inserts (1.0 to 1.5 kb) from genomic library clones, 24 positive and 12 negative controls, each printed in duplicate. Two clinical isolates of L.donovani, K80 and K135 were used for the microarray study on the basis of their antimony susceptibility. The isolate K80 was adapted as SbIII resistant parasite (K80SbIII) by in vitro passages with a stepwise increase in the concentration of SbIII from 10 to 125 and#956;g/ml (K80SbIII). Microarray experiment was performed between antimony sensitive (K135 with ED50 for SAG 4.22 ± 0.38 and#956;g/ml) and resistant (K80SbIII; ED50 above 100 and#956;g/ml). We identified several genes that were uniquely expressed or showed altered expression in the drug resistant strain, indicating their potential role in drug resistance. Genes coding for Histone 1 (H1), Histone 2A (H2A), Histone 4 (H4), MAP-kinase newlineConclusions and future scope of work newline133 newline(MAPK), and two hypothetical proteins were transcribed more abundantly in the antimony resistant parasite in comparison to the sensitive parasite, while genes encoding amino acid transporter showed consistent over-expression in the sensitive parasite. Differential expression of all of these genes was validated by semi-quantitative reverse transcriptase (RT)-PCR assay and at protein level by western blotting for H1, H2A and MAPK.