Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
9140039 | Molecular and Biochemical Parasitology | 2005 | 11 Pages |
Abstract
Expression of the Plasmodium falciparum calmodulin gene (pfcam) is developmentally regulated throughout the blood-stage cycle. The promoter lies within â¼1Â kb of intergenic sequence that separates the pfcam open reading frame (ORF) from an upstream inverted ORF encoding a product homologous to the co-chaperone STI1. Using the oligo-capping method, which selectively reverse-transcribes cDNA from only full-length, capped transcript, we have mapped multiple transcription-initiation sites for both genes. Transcription of the pfSTI1 gene initiates over a 150Â bp region centred â¼350Â bp upstream of the ORF. The pfcam transcription start sites cluster into four â¼30Â bp regions lying within 180Â bp upstream of the pfcam ORF, generating transcripts with 5â² untranslated regions (UTR) of 3-173 nucleotides in length. Remarkably, splicing was found to be related to UTR length, with apparent preferential splicing of longer transcripts. Activity of the pfcam promoter diminished in a linear fashion to undetectable levels upon step-wise removal of sequence between 625 and 230Â bp upstream of the start ATG. Electromobility-shift assays demonstrated nuclear factor binding to eight oligonucleotide probes spanning 657Â bp of the pfcam ORF proximal upstream sequence. The degree of binding correlated with the density of poly(dA)poly(dT) tracts within the probes, and in all cases could be inhibited by excess synthetic poly(dA)poly(dT), but not by poly(dAdT)poly(dAdT). The multiple transcription-initiation sites of both pfSTI1 and pfcam genes lie just downstream of 25Â bp-long poly(dA)poly(dT) tracts, and the intergenic region contains over 20 poly(dA)poly(dT) tracts of 4Â bp or more. Our results suggest that the basal pfcam promoter is situated between approximately â300 and â230Â bp upstream of the pfcam ORF and that the P. falciparum transcription-initiation complex has a low degree of sequence-specificity for the sites of initiation but preferentially acts downstream of long poly(dA)poly(dT) tracts.
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Authors
Hannah E.J. Polson, Michael J. Blackman,