Studies of gene expression control in the apicomplexan parasite Toxoplasma gondii
Behnke, Michael Sean.
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Our understanding of global gene expression patterns and control of both developmental and strain specific aspects of Toxoplasma gondii has broadened in the past few years. A global approach was initially undertaken to construct the "transcriptome" for the Toxoplasma intermediate life cycle using serial-analysis-of-gene expression (SAGE). From this analysis, we confirmed the increased expression of known as well as novel mRNAs associated with the tachyzoite-to-bradyzoite transition. Accumulation of bradyzoite specific mRNAs in the bradyzoite SAGE libraries raises the possibility that transcriptional mechanisms play a key role in the developmental switch. To investigate this question, we adapted the dual luciferase model to recombinational cloning in order to construct a high throughput model for testing and mapping multiple bradyzoite promoters. Expression of luciferase from constructs matched previously published results indicating that developmental gene expression in Toxoplasma is controlled by elements contained in the 5'-flanking regions upstream of the protein coding regions. Promoter cis-elements from two genes, BAG1 and B-NTPase, have been fine mapped to 6-8 bp. EMSA assays confirm that these elements form sequence specific protein/DNA complexes. These data suggest that gene proximal cis-elements are required to initiate developmental gene expression, most likely by the binding of gene-specific trans-acting factors. Although strategies to identify these putative trans factors such as yeast one-hybrid are ongoing, we describe here a more global approach to identifying controllers of gene expression. We conducted an expression Quantitative Trait Locus (eQTL) mapping project that combines the power of Affymetrix microarray technology and QTL mapping to explore the genetic basis of differences in stage-specific gene expression in Type I versus III parasite strains and in F1 progeny from the I X III cross. Gene expression QTLs discovered in this analysis were either local or non-local to the associated transcriptional change and reflect proximal cis-mutations, transcription factors or central mechanisms that co-regulate gene expression during tachyzoite differentiation. Additionally, we identified segmental duplication events in various parents and progeny of the Type I X III cross that have a gene dosage effect on the level of gene expression in those parasites.