Serial-Analysis-Gene-Expression (SAGE) in Toxoplasma. The laboratory has been active for nearly ten years in functional genomics starting with the first Eimeria bovis subtractive libraries made in 1992 and the pursuit of developmental gene expression by differential display that followed.  Recently, we established serial-analysis-of-gene-expression (SAGE) in Toxoplasma and developed new informatic methods to extract and analyze SAGE data.  SAGE libraries made, or in construction, are directed at gene expression in the intermediate life cycle of Toxoplasma—sporozoite, tachyzoite, bradyzoite; and at changes in gene expression in synchronized populations in order to identify genes expressed uniquely in the tachyzoite cell cycle.  SAGE was selected because of its strengths in gene discovery, flexibility in data comparison (not limited to pairwise comparisons), and cost effectiveness (1/50^th of a conventional EST project).  In addition to evaluating developmental and cell cycle gene expression, SAGE data will also be used to demarcate the boundaries of expressed genes in Toxoplasma genomic sequence (in conjunction with annotation efforts of Univ. Penn).  Recently, we have entered into a collaboration with the Aijoka laboratory to validate and compare SAGE data to the results obtained from microarrays.  These efforts combined with the current EST program spearheaded by David Sibley, will greatly aid in the understanding the expressed Toxoplasma genome. 

We have also used SAGE to sort parasite from host mRNAs using the 10 mer SAGE tag as a unique identifier.  This has worked remarkably well with only 22 out of the initial 20,000 tags unassignable to host or parasite (Radke, Larsen, White, 2001).  Libraries from parasite, uninfected-fibroblasts and infected-fibroblasts are now constructed and the sequencing of 60,000 tags is in progress (20,000 tags from each library).  This line of research has the potential to provide insight into the question of how tissue tropism may yield the distinct pathologies caused by Toxoplasma infections.