title: Characterisation of two members of a macroschizont gene family, Tashat1 and Tashat2, from Theileria annulata
Creators: name: Stern, Rowena F.
Publisher: name: University of Glasgow
Year: 2003

description: Theileria annulata is a protozoan parasite of cattle, that causes the disease tropical theileriosis throughout sub-tropical regions of the Old World. Theileria parasites have the ability to immortalise the host leukocyte they infect causing clonal expansion and dissemination of infected leukocytes throughout the host. This property has allowed the development of an in vitro system for the culture of bovine cells infected by the macroschizont stage of the parasite. In addition, differentiation of the parasite towards the next life cycle stage, the merozoite, can be induced in culture. The signals that cause the macroschizont to differentiate into merozoites are not fully understood, although it is known that this event is associated with a major elevation in merozoite gene expression (Shiels et al., 1994). Recently a small family of parasite genes that are negatively regulated early during differentiation to the merozoite were identified. One member, known as TashAT2 contained predicted AT hook DNA binding motifs and was shown to be localised to the host cell nucleus. It has been postulated that the TashAT2 polypeptide may play a role in the regulation of macroschizont or modulation of host cell gene expression (Swan et al., 1999). The focus of this project was to characterise TashAT1, a second member of the TashAT gene family. To this end, the TashAT1 gene was sub-cloned and sequenced and mapped to a region of the genome containing TashAT2 and a third Task AT gene, TashAT3. The 1.4kb open reading frame of TashAT1 was virtually identical to the five prime end of TashAT3, indicating that TashAT1 or TashAT3 (TashAT1/3) were derived from a recent duplication event. The predicted amino acid sequence of TashAT1/3 contained four AT hook motifs, a nuclear localisation signal and a signal sequence. Northern blot analysis revealed that TashAT1, TashAT2 and TashAT3 mRNA were down regulated early, during differentiation to the merozoite in vitro. However, no down regulation was observed for any of the TashAT transcripts in a cell line that was severely attenuated with respect to parasite differentiation. Sequence analysis of the upstream regions of TashAT1/3 identified a motif element (TashUM) located 43bp upstream of the putative transcription start site of TashAT1/3 that was highly related to a sequence upstream of TashAT1 and another, unrelated macroschizont gene, Tash1. Preliminary electromobility band shift analysis of TashUM revealed that it bound to a factor found in host and parasite enriched nuclear extract, which appeared to decrease in abundance as the parasite differentiated towards merogony. Antisera generated against a region of TashAT1 failed to recognise a TashAT1 polypeptide by Western blot analysis. However, a 180kDa polypeptide that was down regulated with respect to merogony and co-localised to the host nucleus was specifically recognised. The detected polypeptide was identified as TashAT3 on the basis of size, sequence identity and predicted expression profile. Immunofluorescence analysis showed that the anti-TashAT1 antisera reacted against both the host nucleus and parasite. This reactivity was lost as the parasite differentiated to the merozoite. The host reactivity was probably due to recognition of TashAT3, while it could not be concluded that the parasite reactivity was directed against TashAT1. Taken together, the results indicated that TashAT3 and possibly TashAT1 are additional candidates for parasite encoded factors that are translocated to the host nucleus, bind to DNA and alter host cell gene expression. This modulation of gene expression could directly or indirectly alter the phenotype of the host cell and be involved in parasite dependent regulation of leukocyte cell division.