Month/Year of Graduation
Bachelor of Science (B.S.)
Paul H. Davis, Ph.D.
Like other eukaryotic organisms, Toxoplasma gondii promoters feature both constitutive and life-stage regulated cis-elements. Using a transcriptomic microarray approach, a cluster of transcripts upregulated early during bradyzoite differentiation was identified. Computational analysis of the promoter regions of these “up-early” transcripts identified a shared upstream consensus motif, a putative transcription factor binding site. Using a dual luciferase assay adapted for recombinational cloning and reporter gene quantification by qPCR, we demonstrate developmental stage-specific expression of the luciferase reporter gene inserted downstream of the transcription factor binding site. The shared consensus motif was found to be an autonomous cis-element by conversion of a constitutive promoter into a bradyzoite growth condition-inducible promoter. A gel-shift experiment showed the cis-element’s binding to bradyzoite nuclear proteins. Mass spectrometry of the shifted gel band identified a protein possessing an apicomplexan AP2 transcription factor domain. Much detail concerning the mechanism of differentiation is yet to be discovered and this work highlights the mediation of life cycle progression by bradyzoite differential gene expression. Taken together, these data demonstrate the control an early bradyzoite promoter element exercises on stage differentiation.
Won, Harim I. and Davis, Paul H. Ph.D., "Investigation of bradyzoite differentiation initiation in Toxoplasma gondii" (2018). Theses/Capstones/Creative Projects. 7.