Expression analysis of Theileria parva telomere-associated ORFs with an oligonucleotide microarray
Polymorphic, multicopy gene families encoding variant antigens are frequently located in subtelomeric regions of the genomes of parasitic protozoa, including Trypanosoma, Plasmodium and Giardia. These sub-telomeric regions are dynamic by nature and highly adaptive in that it allows rapid evolution of antigen genes in response to the immune response of the host.
Theileria parva, a protozoan that parasitises the lymphocytes of cattle, appears to have a similar genomic architecture. Several telomere-associated ORFs of T. parva were previously cloned and show extensive strain polymorphism (Bishop et al., Mol Biochem Parasitol, 110  359-371). In addition, it was recently found that the T. parva genome contains 138 telomere-associated ORFs, grouped into 7 gene families according to domain-based clustering (unpublished data). Predicted proteins encoded by 77 of the telomere-associated ORFs potentially have access to the parasite secretory pathway. Therefore, these proteins might be secreted into the cytoplasm of the bovine lymphocyte and interact with host proteins. As such, these proteins are of great interest as potential targets of host immune responses or as molecules that may facilitate parasite survival within the host.
Using a microarray approach we hope to answer the following questions:
- Which ORFs are expressed (at least at the mRNA level) during lymphocyte infection?
- Does the level of ORF expression change during the life of the lymphocyte stage of the parasite
We have begun to answer these questions by microarray analysis using mRNA from T. parva-infected lymphocytes grown in in vitro culture, and a microarray of 70mer oligonucleotides based on the telomere-associated ORFs. Our initial results indicate a subset of ORFs are expressed by the parasite, and that the expression profile is quite stable during long term culture of the parasite.
The figure below shows a microarray hybridisation result for one of our cultured parasite/lymphocyte cell lines at a single time point (A; green channel). Also shown is the hybridisation result for a reference pool control on the same microarray slide (B; red channel). The white arrow indicates the position of +ve control spots (oligo for bovine MHC). Each oligo is in triplicate.