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Title: Genetic and RNAi analysis of INCENP (inner centromere protein) and DNA Topoisomerase II in Drosophila melanogaster
Author: Chang, Chih-Jui
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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This thesis is divided into two parts. In part I, I carried out a genetic screen to obtain new INCENP alleles and interacts. After screening 5000 chromosomes, I isolated 18 mutants, one of them was a new allele of INCENP that we called incenp3747. Two mutants (3322, 4330) were dominant female sterile. All of the mutants were homozygous lethal. Some of the mutants showed defects in eyes, wings, bristle when combined with incenpP(EP)2340. The sequence of the coding region of INCENP in incenp3747 homozygous mutants showed a point mutation. A single base change in exon 4 resulted in a stop codon. The predicted molecular weight of the putative truncated gene product would be 61kD. The truncated protein was not detected by Western blot, which could suggest that it is unstable although I cannot rule out the unlikely possibility that our antibody failed to detect the truncated product. The phenotypic analysis of incenp3747 showed that the homozygous embryos exhibit chromosome segregation defects. In mixed populations of early embryos (incenp3747/CyO-Ftz-LacZ) I found chromosome segregation defects as early as cycle 3. Some of the nuclei also failed to follow the globally synchronous mitotic oscillator in the early divisions. My analysis revealed that these early defects resulted from a maternal contribution to the phenotype, due to genetic interaction with the balancer chromosome. Later in development, homozygous mutant embryos showed aberrant neuronal morphology and the homozygous mutant first instar larvae escapers exhibit abnormal behaviour. In part II, I used dsRNAi in Drosophila S2 cells to carry out a detailed functional analysis of the role of Topoisomerase II (Topo II) during mitosis. I found that Topo II was not required for the assembly of a functional kinetochore or the targeting of chromosomal passenger proteins, nonetheless, it was essential for anaphase sister chromatid separation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available