Characterisation of a cluster of genes encoding Theileria annulata AT hookDNA-binding proteins and evidence for localisation to the host cell nucleus

Infection of bovine leukocytes by the apicomplexan parasite Theileria annul ata results in alteration of host cell gene expression and stimulation of h ost cell proliferation. At present, the parasite-derived factors involved i n these processes are unknown. Recently, we described the characterisation of a parasite gene (TashAT2), whose polypeptide product bears AT hook DNA-b inding motifs and may be transported from the parasite to the host nucleus. We now describe the isolation of a further two genes (TashAT1 and TashAT3) that are very closely related to TashAT2. All three TashAT genes are locat ed together in a tight cluster, interspersed by two further small open read ing frames, all facing head to tail. TashAT2 was shown to be expressed in a ll T. annulata cell lines examined, whereas TashAT1 and TashAT3 were expres sed in the sporozoite stage of the parasite, and also in infected cell line s, where their expression was found to vary between different cell lines. E vidence for transport was provided by antisera raised against TashAT1 and T ashAT3 that reacted with the host nucleus of T. annulata-infected cells. Re activity was particularly strong against the host nuclei of the T annulata- infected cloned cell line D7B12, which is attenuated for differentiation. A polypeptide in the size range predicted for TashAT3 was preferentially det ected in host enriched D7B12 nuclear extracts. DNA-binding analysis demonst rated that fusion proteins containing the AT hook region of either TashAT1 or TashAT2 bound preferentially to AT rich DNA.