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Serial analysis of gene expression (SAGE) was applied to the malarial parasite Plasmodium falciparum to characterize the comprehensive transcriptional profile of erythrocytic stages. A SAGE library of ϳ8335 tags representing 4866 different genes was generated from 3D7 strain parasites. Basic local alignment search tool analysis of high abundance SAGE tags revealed that a majority (88%) corresponded to 3D7 sequence, and despite the low complexity of the genome, 70% of these highly abundant tags matched unique loci. Characterization of these suggested the major metabolic pathways that are used by the organism under normal culture conditions. Furthermore several tags expressed at high abundance (30% of tags matching to unique loci of the 3D7 genome) were derived from previously uncharacterized open reading frames, demonstrating the use of SAGE in genome annotation. The open platform "profiling" nature of SAGE also lead to the important discovery of a novel transcriptional phenomenon in the malarial pathogen: a significant number of highly abundant tags that were derived from annotated genes (17%) corresponded to antisense transcripts. These SAGE data were validated by two independent means, strand specific reverse transcription-polymerase chain reaction and Northern analysis, where antisense messages were detected in both asexual and sexual stages. This finding has implications for transcriptional regulation of Plasmodium gene expression.

SummaryThe Plasmodium falciparum multidrug resistance gene, pfmdr1 , has been shown to be involved in the mediation of the parasite ' s response to various antimalarial drugs. Previous studies of pfmdr1 expression have shown that transcript levels are increased in drug-resistant isolates. However, a detailed examination of the transcriptional regulation of this gene has not been completed. The aim of this study was to map the 5 ¢ ¢ ¢ ¢ UTR of pfmdr1 , and to examine the transcriptional profile of the gene in sensitive parasites treated with four different antimalarial drugs. RT-PCR and 5 ¢ ¢ ¢ ¢ -RACE mapping showed that the 5 ¢ ¢ ¢ ¢ UTR has a length of 1.94 kb. A putative promoter has been identified via transient transfection. Northern analysis revealed a 2.1-to 2.7-fold increase in pfmdr1 expression in 3D7 parasites treated with 50 nM chloroquine for 6 h, confirming results from Serial Analysis of Gene Expression. 3D7 parasites were subsequently treated with experimentally derived IC 50 concentrations of mefloquine, quinine and pyrimethamine. pfmdr1 transcript levels specifically increased 2.5-fold at 6 h in mefloquine-treated parasites and threefold in parasites treated with quinine for 30 min. There was no evidence of transcript induction in pyrimethamine-treated parasites. This is the first evidence of induction of pfmdr1 expression in sensitive cells; and suggests a novel method of transcriptional control for this gene.

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