Polson, Hannah Emily Jane; (2002) Transcriptional control in the malaria parasite plasmodium falciparum: Characterisation of the calmodulin promoter. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Expression of the Plasmodium falciparum calmodulin (cam) gene is developmentally regulated within the erythrocytic stage of the parasite life cycle. The promoter lies within 1 kb of intergenic sequence that separates the cam open reading frame (ORF) and a predicted upstream inverted ORF encoding a gene with a high degree of similarity to the stress-inducible gene STI1 (pfSTI1). Multiple transcription initiation sites were mapped by RLM-RACE for both genes and the intervening sequence is approximately 400 bp. The cam transcription start sites identified lay within 200 bp of the first ATG of the cam ORF, generating transcripts with untranslated regions (UTRs) of 3-173 nucleotides. There are four areas in this 200 bp region, of approximately 30 bp, where transcription initiation activity is highest. Interestingly, in late schizonts, the two cam ORF-proximal areas produce transcripts that are predominantly detected in an unspliced state, and the two cam ORF-distal regions generate transcripts that are predominantly spliced. To identify sequence motifs within the cam upstream sequence involved in driving gene expression, deletion mutagenesis and electromobility shift assays (EMSA) were performed. Maximal expression of the CAT reporter gene was achieved with the full-length (625 bp) promoter and found to drop away in a linear fashion to undetectable levels over 350 bp. EMSA analysis of 658 bp of the intergenic sequence using eight 100 bp overlapping double-stranded DNA probes showed nuclear factor binding activity along the entire length of the region. The binding of nuclear factors could be inhibited by excess synthetic poly[dA]poly[dT], while poly[dAdT]poly[dAdT] had no effect. The multiple transcription initiation sites of pfSTI1 and cam both lie just downstream of a 25 bp poly[dA]poly[dT] tract, and the intergenic region contains over 20 poly[dA]poly[dT] tracts of 4 bp or more. Taken together, the data suggest that maximal transcription of cam is achieved by an accumulation of nuclear factor binding events occurring between 625 and 300 bp upstream of the start ATG. It would also appear that the transcription initiation complex has a high degree of promiscuity and may preferentially act downstream of poly[dA]poly[dT] tracts of 25 bp or more. The EMSA results strongly suggest the presence of nuclear factors able to interact with poly[dA]poly[dT], though their involvement in transcriptional regulation is unclear.

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