Leishmania mexicana amazonensis: Differential Display Analysis and Cloning of mRNAs from Attenuated and Infective Forms

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Abstract
The virulence of Leishmania mexicana is determined by the concerted action of several parasite molecules. These cells lose their infectivity to host macrophages after prolonged cultivation in axenic growth media. Both virulent and attenuated variants of the parasite cells were cloned. The differential display reverse transcription-polymerase chain reaction technique was employed to understand whether this natural attenuation of the parasite cells is accompanied by differential expression of selected genes in those cells. Twelve different dinucleotide-anchored oligo(dT) antisense primers were used to make cDNAs from poly(A)+ mRNAs isolated from a clonal population of virulent and avirulent cells following a protocol optimized for Leishmania mRNAs. Those cDNAs were subjected to amplifications using each of the three different arbitrary decanucleotide primers and the corresponding anchored oligo(dT) primer. This procedure revealed four virulent-specific cDNA probes and one avirulent-specific cDNA probe. Differential expressions of these genes were confirmed by northern hybridization using the cloned cDNA probes. These results indicate that differential expression of genes may be the key in determining the molecular basis of leishmanial virulence.