Theileria annulata: Identification, by differential mRNA display, of modulated host and parasite gene expression in cell lines that are competent or attenuated for differentiation to the merozoite

To identify both host and parasite genes that show altered expression durin g differentiation of Theileria annulata from the macroschizont to the meroz oite stage of the life cycle, the RNA profiles of two T. annulata-infected clonal cell lines (D7 and D7B12) with the same genetic background have been compared by RNA display. In the. cloned cell line D7, T. annulata differen tiates from the macroschizont to the merozoite at 41 degreesC, whereas in t he cell Line D7B12, which was derived by recloning D7, the parasite does no t differentiate. Therefore, genes, that show altered expression levels in e ither clone could be modulated by the differentiation event and are possibl e candidates for regulators of this process. Differential display was carri ed out initially on RNA extracted from D7 and D7B12 macroschizont-infected cells cultured at 37 degreesC and secondly on RNA extracted from the two ce ll lines incubated at 41 degreesC to induce differentiation to the merozoit e. The first procedure identified 29 cDNA fragments that displayed altered levels between D7 and D7B12, 9 of which were confirmed to be differentially expressed by Northern blot analysis. Of these 9 gene fragments, 8 were fou nd to be of host origin, while I was parasite derived. The second RNA displ ay analysis identified 14 transcripts that showed altered levels during a d ifferentiation time course, of which 6 were confirmed to be differentially expressed between D7B12 cells and differentiating D7 cells by Northern blot analysis. Of these 6 gene fragments, I was of host and 5 were of parasite origin. The parasite genes either showed levels of RNA consistent with cons titutive gene expression or, in one case, a genuine upregulation of mRNA as sociated with the differentiation process. (C) 2001 Academic Press.