Characterisation of cdc2-Related Kinases From Trypanosoma brucei

Glasssmith, Gareth (1997) Characterisation of cdc2-Related Kinases From Trypanosoma brucei. PhD thesis, University of Glasgow.

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Trypanosoma brucei is a kinetoplastid protozoan parasite of man and other mammals in Africa, that is transmitted by the bite of the tsetse fly. T. brucei has a complicated life cycle involving both dividing and non-dividing stages. The nondividing stages are to some extent pre-adapted to the environment of the next host, and as such are essential for efficient transmission from both tsetse fly to mammal and mammal to tsetse fly. It was the aim of this project to initiate the analysis of cell cycle controlling genes from T. brucei, with a view to assessing their involvement in the regulation of the division status of the various life cycle stages. The Cyclin Dependant Kinases (CDKs) are a highly conserved family of serine/threonine protein kinases, many members of which are vital for the regulation of the cell cycle in eukaryotes. The archetypal CDK is cdc2, a protein that regulates both S phase and mitosis in yeast; the homologous protein in higher eukaryotes regulates mitosis, while a closely related protein, CDK2, is involved in S phase control. The kinase activity of the CDK proteins is tightly regulated by phosphorylation (both negative and positive) and by the association of a regulatory subunit (a cyclin). Two cdc2-Related Kinase genes {crks) from T. brucei had already been cloned and sequenced at the start of the Ph.D. {tbcrkl and tbcrkl) and another gene fragment (tbcrkS) had been isolated by PCR. The genomic locus of the tbcrk3 gene was subcloned and sequenced. Analysis of the predicted protein sequences for the TbCRK proteins revealed no obvious candidate for the role of cdc2 homologue. The three TbCRK proteins were expressed in E. coli, and used to raise antisera, as well as to assess the cross reactivity of the antisera available in the laboratory. These antibodies were then used to analyse the expression of the TbCRK proteins in three of the parasite's life cycle stages. No important differences in expression were observed between dividing and non-dividing stages. TbCRK3 was found to be in the insoluble fraction of the protein extracts made, implying an association with either DNA or the XX cytoskeleton. sucl Another component of the cdc2/cycHn complex in fission yeast, pl3 , has previously been used to purify cdc2 related kinases from a variety of organisms. This approach was followed using both and a homologue from the kinetoplastid parasite, Leishmaniamexicana. Kinase activity could be bound to the p12LmmCKSi TbCRKl and TbCRKZ were shown by Western blotting to be part of the protein fraction purified on the beads. An attempt was made to analyse TbCRKB function by reverse genetics. Null mutants were created by homologous recombination and their phenotype assayed. The mutant cultures did not survive longer than three months. Microscopy using DNA binding dyes show a phenotype of aberrant cytokinesis, morphological abnormalities and a breakdown in cell polarity. The relevance of this phenotype to the insoluble nature of TbCRKS is discussed.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Molecular biology, Parasitology
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-75357
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 20:26
Last Modified: 19 Nov 2019 20:26

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