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Title: Regulation of TamS1 gene expression during differentiation to the merozoite in Theileria annulata
Author: Fox, Mark
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1997
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Much of the pathogenicity of the bovine disease tropical theileriosis, caused by the protozoan parasite Theileria annulata, results from the parasite differentiating from one life cycle stage to another. Given the observation, by Shiels et al., (1994) that the timing of differentiation from macroschizont to merozoite can be altered and that this correlates with changes in the expression of the 30kDa merozoite surface polypeptide (TamS1); the aim of this study was to identify the molecular mechanisms controlling the expression of the TamS1 gene. Furthermore, the identification of the factors controlling TamS1expression could lead to the identification of a factor involved in the timing of differentiation events. Therefore, it was intended to clone and sequence the regulatory regions controlling the expression of the TamS1 gene and to develop a gel retardation mobility assay in order to analyse transcription factors interacting with TamS1 promoter elements particularly with respect to factors binding during induction of differentiation. Isolation and sequencing of the 5' and 3' regions flanking the TamSl protein coding sequence, identified three additional open reading frames. Attempts to identify each gene or functional motifs within each gene by sequence comparisons with polypeptides in known databases was unsuccessful. Nuclear run analysis demonstrated that the TamS1 gene is monocistronically transcribed with definite start and termination signals. Additionally, the orientation of the other open reading frames would suggest that all of the genes within the isolated contig are monocistronically transcribed. In an attempt to identify the promoter region involved in the regulation of the TamS1 gene the RNA start site was mapped. Two strategies were used in an attempt to identify the promoter elements; firstly the 5' intergenic regions from related genes in other species of Theileria were cloned and sequenced to allow for comparisons to be made and the 5' region of the TamS1 gene was compared with that of known promoter elements. However, neither of these approaches was successful in highlighting possible functional promoter elements. Theileria annulata macroschizont infected cell lines can be induced to produce merozoites by culture at an elevated temperature (41°C). This in vitro system, enabled the isolation of host-free parasite nuclear extracts for use in the development of a gel retardation mobility shift assay. This technique enable the identification of a promoter element which bound three specific mobility bands. Analysis of parasite nuclear extracts made from two types of clonal cell lines (enhanced and diminished differentiates) showed that two electrophoretic bands were only present during a specific stage of parasite differentiation corresponding either to commitment or a significant increase in expression of the TamSl gene. Determination of the size and number of factors interacting with the promoter element were attempted using two strategies, by UV crosslinking and South-Western blotting. However, neither of these approaches were successful and the possible reasons for this are explained.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available