Studies on centromere organisation and function inTrypanosoma brucei
Abstract:
Trypanosomes are unicellular early divergent eukaryotic microorganisms that cause sleeping sickness and Chagas disease. Genome sequencing has revealed that these parasites lack most of the highly conserved proteins involved in chromosome segregation. However, kinetochore-associated chromosome segregation has been proposed to occur in Trypanosoma brucei and the existence of centromeres, the locus that interacts with the kinetochores, has been inferred in T. brucei and Trypansoma cruzi. This work aimed to characterise, at a structural and functional level, the centromeres of Trypanosoma sp.
Centromeres in T. brucei have been localised to chromosomal loci that contain an array of ~147 bp AT-rich tandem repeats. Using a long-range restriction endonuclease mapping approach, we found that the sizes of the arrays in T. brucei chromosomes 1-8 vary from 20 to 110 kb and present length heterogeneity between chromosome homologues.
Topoisomerase II-? (TOP2?) plays a major role in sister chromatid segregation. TOP2? specific centromeric activity has been demonstrated in trypanosomes. In this project, we systematically evaluated through different approaches, regulatory mechanisms for the TOP2? activity/recruitment to trypanosome centromeres. In other eukaryotes, the Small Ubiquitin-like MOdifier (SUMO) interaction with TOP2? is required for it to be recruited to centromeres during mitosis. In independent experiments, we found no evidence for this association in bloodstream form (BSF) T. brucei . Modifications to the carboxyl terminal region (CTR) of TOP2?, other than SUMOylation, have been described that affect cell cycle specific localisation on chromosomes. Here, we undertook experiments to further delineate regions of the CTR of trypanosome TOP2?. Using a sequential deletion approach, we found that amino acids 1223-1312 in the CTR of enzyme are not required for its centromeric-specific activity. Finally, we found that the condensin unit SMC2 is essential for growth of T. brucei BSFs and not for TOP2? specific centromeric activity.
Fuente:
Biblioteca Virtual Banco de la República
Formatos de contenido:
Tesis
