Identification of novel modifiers of chromosome inheritance: using a genetically sensitised drosophila model

Hunter, Sally M. (2009) Identification of novel modifiers of chromosome inheritance: using a genetically sensitised drosophila model. PhD thesis, James Cook University.

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Loss of chromosome cohesion has been implicated as a significant contributor to chromosome missegregation and aneuploidy. A central and essential figure mediating chromosome cohesion is the cohesin complex. This multi-subunit protein is conserved in all eukaryotes studied and related proteins are also present in bacteria. Reduction of cohesin subunits and cohesin regulators has been conclusively demonstrated to perturb normal chromosome cohesion and segregation. Cohesin not only has a central role in maintaining cohesion between sister-chromatids between S phase and the onset of anaphase in both mitosis and meiosis. The cohesin complex is also essential in the formation and maintenance of chiasmata between homologous chromosomes during meiosis.

The study presented in this thesis aimed to identify regulators of chromosome cohesion and segregation by employing a genetic screening approach using a modified cohesin subunit (Rad21NC) that perturbs chromosome segregation by impeding chromosome separation at anaphase. At the commencement of this study it was clear that much of the molecular network underlying chromosome segregation remained to be determined and that metazoan species, such as Drosophila, could provide unique insights into these processes. The aim of this study was to identify novel regulators of chromosome cohesion and segregation. To achieve this, a chromosome missegregation model was used that employed a non-cleavable form of the cohesin component Rad21 to impede chromosome segregation. When ectopically expressed in the replicating cells of the developing Drosophila eye this cleavage-resistant variant produced a reduced and disorganised eye phenotype. The underlying cellular phenotype demonstrated increased levels of tetraploidy, aneuploidy, lagging chromosomes and fragments of broken chromosomes. This eye phenotype proved modifiable by second site heterozygous mutations in genes encoding known regulators of cohesin and chromosome segregation, such as NippedB, Separase and Cyclin B. Following this characterisation the chromosome missegregation phenotype was utilised as a screening tool to identify regulators of chromosome segregation capable of modifying chromosome cohesion. The genetic screen performed as part of this study identified 133 candidate loci that were able to modify the GMR>Rad21NC chromosome missegregation model and thereby have been linked to chromosome segregation. The screen was carried out using mutant alleles of loci that fell within the breakpoints of deletions previously identified as GMR>Rad21NC modifiers. The number of modifying loci is greater than originally anticipated, however, this may reflect the molecular complexity of chromosome segregation, the inter-connectedness of cellular pathways and the multi-functional nature of proteins.

The presence of RAD21NC cohesin complexes on chromosomes has a detrimental effect on the cell following the onset of anaphase. The direct effects of the expression of Rad21NC on chromosome segregation were visualised using neuroblast chromosomes, with a significant number of tetraploid and aneuploid cells observed, as well as lagging and broken chromosomes. Impeding chromosome segregation during Drosophila eye development was shown to cause significant levels of cell death and altered cell cycle progression, producing a disorganised (rough) and reduced adult eye. This GMR>Rad21NC rough-eye phenotype was employed to perform a genome-wide screen for second site modifier loci capable of either suppressing or enhancing the RAD21NC eye phenotype. The genetic screen identified 133 individual modifier loci as candidate chromosome cohesion and segregation regulators. In-depth analyses of published studies revealed that many of the loci identified had established links to cohesin, chromosome segregation, mitosis and meiosis, indicating that the screen had successfully identified regulators of chromosome cohesion and segregation. However, several of these loci had not previously been implicated in chromosome cohesion and segregation and are very interesting for further investigation as novel regulators of these processes.

Item ID: 10926
Item Type: Thesis (PhD)
Keywords: Drosophila, chromosome cohesion, segregation, novel regulators, missegregation, aneuploidy, cohesin, mitosis, meiosis, RAD21
Date Deposited: 14 Oct 2010 05:47
FoR Codes: 06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060103 Cell Development, Proliferation and Death @ 34%
06 BIOLOGICAL SCIENCES > 0604 Genetics > 060403 Developmental Genetics (incl Sex Determination) @ 33%
06 BIOLOGICAL SCIENCES > 0604 Genetics > 060402 Cell and Nuclear Division @ 33%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 34%
92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920114 Reproductive System and Disorders @ 33%
97 EXPANDING KNOWLEDGE > 970111 Expanding Knowledge in the Medical and Health Sciences @ 33%
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