Cyclin Dependent Kinases and Cell Cycle Control in Trypanosoma brucei

Ford, Jack Ragnar (1999) Cyclin Dependent Kinases and Cell Cycle Control in Trypanosoma brucei. PhD thesis, University of Glasgow.

Abstract

In all eukaryotic organisms, from yeast to mammals, progression through the cell cycle is ultimately regulated by the actions of a group of highly conserved serine/threonine protein kinases named the cyclin dependent kinases (CDKs). CDK activity is controlled by the association of CDK subunits with regulatory protein subunits named cyclins to form active CDK-cyclin complexes. The trypanosomatids represent one of the earliest known divergent branches of the eukaryotic phylogenetic tree. At the start of this project several putative CDK and cyclin homologues had been described in Trypanosoma hrucei, tbCRKl-3 and tbCYC1. During the project tbCRK2 and tbCYC1 were analysed and a new CRK (tbCRK4) isolated. The function of the tbCRK2 protein and the tbCRK2 gene was investigated using biochemical and molecular genetic approaches. Use of anti-CRK2 antisera showed that tbCRK2 is expressed in both proliferative and nonproliferative life cycle stages of T. brucei. Attempts to create null mutants at the tbCRK2 locus repeatedly failed, with the wild-type allele preserved in all transgenic lines generated. Ectopic expression of a cloned copy of tbCRKl did not allow deletion of wild-type alleles, although the cloned copy of tbCRK1 was demonstrated to generate abnormally high cellular levels of tbCRK2. These results may suggest that tbCRK2 is an essential gene in the procyclic form of the parasite; however, control experiments are needed to verify this. The tbCYC1 protein was previously reported to be a mitotic-type cyclin in T. brucei. Biochemical and molecular genetic approaches were taken to investigate tbCYC1 function. A specific anti-CYC 1 antiserum was generated, which demonstrated that in T. brucei strain STIB 247 tbCYC1 is expressed in the short stumpy bloodstream and procyclic forms, but not in the long slender bloodstream form, suggesting a function in differentiation rather than cell cycle control per se. No significant tbCYC1-associated kinase activity was found in procyclic extracts. In addition, no association of tbCYC1 with any of the known T. brucei CRKs or with the yeast protein p13SUCI or the leishmanial protein pl2CKSI was found in procyclic extracts. These data suggests that tbCYC1 does not fulfil the function of a mitotic-type cyclin. Attempts to generate null mutants for the tbCYC1 locus failed, which may provide preliminary evidence that tbCYC1 serves an essential function in the procyclic form. However, further experiments are required to clarify this. A full-length genomic clone of the tbCRK4 gene was isolated and demonstrated to be homologous to the Crithidia fasciculata cfCRK gene. Computer analysis revealed the presence of two large, highly hydrophilic insert dpmains between the conserved kinase catalysis domains VIb and VII, and X and XI. Specific anti-CRK4 antisera were generated and used to demonstrate that in strain STIB 247, tbCRK4 is expressed in the short stumpy bloodstream and procyclic forms, but not in the long bloodstream form. No tbCRK4-associated kinase activity was detected from procyclic extracts, and no interactions with the yeast protein p13SUCI or the leishmanial protein pl2CKSI was found from procyclic extracts. This study has provided evidence that mechanisms of cell cycle control in trypanosomatids may be based upon the functional CDK-cyclin paradigm observed in higher eukaryotes yet have diverged to serve the parasite's unique biology. If sufficient divergence in terms of biochemical function between trypanosome CRKs and cyclins and the CDKs and cyclin of mammals could be demonstrated, they would provide a tempting drug target.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Jeremy Mottram
• Keywords: Parasitology
• Date of Award: 1999
• Depositing User: Enlighten Team
• Unique ID: glathesis:1999-76127
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Dec 2019 09:15
• Last Modified: 19 Dec 2019 09:15
• URI: https://theses.gla.ac.uk/id/eprint/76127