Figure 4

Groups of co-regulated mRNAs are associated with the major developmental transitions. Stage-specific gene expression as defined by normalized fold-change within the six developmental libraries was determined without regard to the level of expression in the laboratory-adapted strain libraries. SAGE tag clusters for the combined sporozoite and Day-4 post-sporozoite infection, Day-6, Day-7 and Day-15/pH-shifted and their respective plots were generated using GeneSpring 7.2 software. Similar distributions were obtained using standard k-means clustering (not shown). Note that expression of each SAGE tag within the four co-regulated sets was plotted for all nine libraries including the laboratory strains with expression color-coded by the level in the relevant developmental libraries. [A] Parasites emerging from sporozoite-infections at Day-4 post inoculation possessed high levels of sporozoite-specific gene expression (0.728 correlation between these libraries) that were nearly all down-regulated by Day-6 post-sporozoite infection and beyond. Note that with the exception of a few SAGE tags in the Type III (VEGmsj) library there is little expression of these genes in the laboratory strains. [B] Genes specifically expressed >3-fold in Day-6 post-sporozoite SAGE libraries (120 total) were also uniquely elevated in the Type I-RH library suggesting these parasites have a common gene expression that may reflect shared biology. Note that the expression of these genes is largely exclusive to RH and Day-6 and that the levels of expression are remarkably similar in each library. [C] Parasites post-growth shift at Day-7 (15 h doubling) represent a developmentally unique stage positioned at the initiation of bradyzoite development. Fully 5% of the total SAGE tags expressed in this stage showed regulation (15 % difference between Day-7 and Day-6) with relatively few known bradyzoite-specific genes expressed at this stage. Note that there was considerable shared expression of Day-7 SAGE tags with the laboratory strains, although expression levels showed higher or lower expression in lab strains as reflected in the color scale of individual curves that were fixed by the Day-7 levels in this gene plot. [D] Based on SAGE tag frequencies, 698 genes were found to be expressed >3-fold higher in the Day-15/pH-shift libraries. Genes identified in this set included the known bradyzoite genes BAG1, LDH2, SAG4.2, enolase 1, cyst-wall protein and a novel bradyzoite-specific NTPase. A number of the SAGE tags contained in this cluster were also elevated in the laboratory strains VEGmsj and Me49B7, while the RH strain showed little or no expression of the tags from this gene set as was found for the other developmental libraries including Day-6.