Keall, Rebecca M. (2005) Molecular and genetic analysis of Drosophila Rad21: a gene and protein involved in sister chromatid cohesion. PhD thesis, James Cook University.

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Abstract

The accurate and efficient dissemination of replicated chromosomes into daughter cells is fundamental to all aspects of biology. Chromosomal missegregation can lead to aneuploid chromosome configurations which are a hallmark of cancer cells and also a leading cause of birth defects and infertility in humans. Given that chromosome missegregation can result in such disastrous consequences, cells have evolved mechanisms to ensure the faithful segregation of chromosomes, one of which is sister-chromatid cohesion which is mediated by the cohesin complex.

Cohesin is a multi-protein complex thought to be the primary effector of sister chromatid cohesion in all eukaryotes. In yeast, cohesin is loaded onto chromosome arms in S-phase where it maintains sister chromatid cohesion until the metaphaseanaphase transition. Sister chromatid separation is then triggered by the site-specific cleavage of the RAD21 cohesin subunit. In metazoan species, including Drosophila, the bulk of cohesin dissociates form chromosomes in prophase, leaving a minor pool of centromere-associated cohesin to maintain sister-chromatid cohesion until anaphase. Exactly how the various cohesin subunits and their regulators orchestrate these events has yet to be fully elucidated. Meiotic cohesin complexes are subjected to additional levels of regulation to accommodate the different types of cell division that occur to produce haploid gametes. In humans, premature loss of meiotic sister chromatid cohesion has been proposed as the most likely molecular cause for sporadic aneuploidy linked to advanced maternal age.

The results presented in this thesis begin with a description of the rationale and approach used to identify the DRAD21 separase cleavage sites, and subsequently mutate them using site-directed mutagenesis. Characterisation of the dominant alleles generated is described, as is the first evidence of DRAD21 proteolysis. The over expression of non-cleavable DRAD21 isoforms was investigated in a range of different tissues and developmental stages, and was shown to dominantly reduce the size of adult tissue. These data suggested that overexpression of non-cleavable isoforms of DRAD21 in dividing cells increased levels of cell death. Analysis of the cellular effects of non-cleavable DRAD21 overexpression in the developing eye imaginal disc confirmed that the level of apoptosis was increased in cells expressing non-cleavable DRAD21, and that this DRAD21 isoform induced mitotic delay or arrest, consistent with a defect in mitotic progression. This is the first description of a Drad21 mutant phenotype. The reduced and roughened eye phenotype generated as a result of non-cleavable DRAD21 expression in the eye imaginal discs provided a tool to use in genetic studies of DRAD21 function.

Genetic analysis showed that known and predicted cohesin regulators are capable of modulating the DRAD21 eye phenotype, therefore establishing the suitability of this phenotype for use in a genetic screen. The entire Drosophila genome was screened for genetic modifiers of the DRAD21 eye phenotype. In total 62 interacting genomic regions were identified, spanning chromosomes two, three and four. Analysis of these interactions revealed both enhancers and suppressors of the DRAD21 eye phenotype, and genetic dissection of some of the interacting regions allowed 13 modifier loci to be unequivocally identified at the molecular level. Specifically, ten distinct interacting regions were examined at the molecular level and ten suppressor loci and three enhancer loci were identified and a mechanism by which these interactions may be occurring was proposed for each.

These studies are likely to significantly influence our current understanding of metazoan chromosome dynamics and identify novel regulators of chromosome segregation. To date, some interacting loci identified at the molecular level have established roles in chromosome cohesion, while for others this study provides the first evidence for their role in this process. These studies will identify both novel regulators of chromosome segregation and hopefully provide a shortlist of genes that when functionally impaired may incrementally increase the risk of chromosome missegregation and aneuploidy in humans.

Item ID:	2088
Item Type:	Thesis (PhD)
Keywords:	genetics, DNA, Drosophila Rad21, chromatid cohesion, chromosome segregation, sister-chromatid cohesion, gene mutation, apoptosis, cohesin complex, alleles, identification methods, molecular genetics, aneuploidy, separase, genetic modifiers, genetic dissection, mitosis
Date Deposited:	26 Feb 2009 02:52
FoR Codes:	06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060103 Cell Development, Proliferation and Death @ 0% 06 BIOLOGICAL SCIENCES > 0604 Genetics > 060409 Molecular Evolution @ 0% 06 BIOLOGICAL SCIENCES > 0604 Genetics > 060402 Cell and Nuclear Division @ 0%
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