Genetics - Theses
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ItemNo Preview AvailableRegulation of the G2(University of Melbourne, 2000)In order to survive DNA damage, cells respond with a range of mechanisms, including DNA repair, checkpoint responses, and survival mechanisms. The DNA damage checkpoint cannot be viewed as a simple signal transduction pathway. It is divided into several steps that are genetically separable. These observations have lead to the questions that are addressed in this project. Firstly, how does the checkpoint, interact with the core G2/M cell cycle machinery. I address this with a biochemical study of the affects of a DNA damage checkpoint on Y15 regulation of p34cdc2/cyclinB. This study allows the two pathways to be linked. Secondly, do we know all the proteins that are involved in the checkpoint. We do not know how the checkpoint detects DNA damage, nor how the checkpoint integrates with signals from DNA repair pathways, and so other, as yet unidentified, checkpoint genes may fill in these gaps. In this study I used a genetic screen to identify other members of the pathway. The mutants isolated in this screen allowed the posing of new questions about the DNA damage checkpoint pathway. It is becoming increasingly clear, through this and other studies, that essential genes will play roles in the checkpoint pathway. This allows us to link the checkpoint pathway with essential pathways such as DNA replication or chromatin organisation. In a screen for new genes involved in the DNA damage checkpoint, I isolated two essential genes that play roles in G2 checkpoint responses. cut5 provides a link between DNA replication and the G2 checkpoints responding to DNA damage and blocks to DNA replication. The isolation of a novel allele of rad18 in this project is the first evidence in S. pombe of a gene linking the DNA damage checkpoint and DNA repair. Several pieces of evidence point to rad18 playing an essential role in chromatin organisation and this then links chromatin regulation to the regulation of both repair and checkpoint responses to DNA. An allele-specific suppressor of this rad18 allele, brc1, suggested a link between checkpoint and repair pathways and independent regulators of genomic stability. The novel rad18 allele also allows us to address the issue of checkpoint maintenance and checkpoint initiation. The final gene isolated in the screen was a DNA repair gene that is not absolutely required for the ability to repair. As it is not clear what repair pathway this gene is involved in, it suggests a broad-range defect, which may also link the processes of chromatin organisation and DNA repair. The isolation of these genes links together multiple pathways of DNA damage responses. It suggests that genes with essential roles in processes such as DNA replication, chromatin organisation and maintenance of genomic stability may also play roles in checkpoint and other responses. Further screening, taking advantage of the types of phenotypes discovered in this screen, will be needed to identify these genes.
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ItemCharacterization of the tamA gene of Aspergillus nidulans(University of Melbourne, 2000)In Aspergillus nidulans, the GATA zinc finger protein AreA activates the expression of enzymes that metabolize less favoured nitrogen sources in the absence of preferred nitrogen sources, such as ammonium or glutamine. The amount and activity of AreA are modulated by a number of mechanisms, including an interaction with the NmrA protein through the GATA zinc finger and C-terminal regions of AreA to inhibit DNA binding under nitrogen-sufficient conditions. The TamA protein has also been implicated in nitrogen regulation, with mutants described as having reduced levels of a number of nitrogen metabolic enzymes. This thesis describes the characterization of the tamA gene and investigates its role in nitrogen regulation. tamA encodes a 739 amino acid protein that contains features common to DNA-binding transcription factors, including a potential Zn(II)2Cys6 DNA-binding domain. Uga35p of S. cerevisiae shows some similarity in both structure and function to TamA, and remarkably the Zn(II)2Cys6-like domains of both proteins are not required for function. To define important regions of TamA, sequence changes in a number of tamA mutants were determined and constructs containing deletions of various regions were tested for function. While the most N-terminal and C-terminal regions of TamA were dispensable for function, changes affecting even small parts of other regions of the protein abolished function. This suggests that the overall protein conformation is critical. Constructs encoding the TamA protein fused to DNA-binding domains were shown to activate gene expression in A. nidulans by recruitment of AreA. The Aspergillus oryzae AreA and Neurospora crassa NIT2 proteins were able to substitute for A. nidulans AreA in this interaction. Although the GATA zinc finger did not seem to be involved, the 12 amino acids at the AreA C-terminus were essential for interaction with TamA. This region is also involved in the interaction with NmrA, suggesting that competition for binding to the AreA C-terminus may be a part of the function of TamA. Uga35p was not able to interact with AreA and also could not complement a tamA? mutation, demonstrating differences in the coevolution of nitrogen regulatory mechanisms between different species.
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ItemNo Preview AvailableRegulation of the G2(University of Melbourne, 2000)In order to survive DNA damage, cells respond with a range of mechanisms, including DNA repair, checkpoint responses, and survival mechanisms. The DNA damage checkpoint cannot be viewed as a simple signal transduction pathway. It is divided into several steps that are genetically separable. These observations have lead to the questions that are addressed in this project. Firstly, how does the checkpoint, interact with the core G2/M cell cycle machinery. I address this with a biochemical study of the affects of a DNA damage checkpoint on Y15 regulation of p34cdc2/cyclinB. This study allows the two pathways to be linked. Secondly, do we know all the proteins that are involved in the checkpoint. We do not know how the checkpoint detects DNA damage, nor how the checkpoint integrates with signals from DNA repair pathways, and so other, as yet unidentified, checkpoint genes may fill in these gaps. In this study I used a genetic screen to identify other members of the pathway. The mutants isolated in this screen allowed the posing of new questions about the DNA damage checkpoint pathway. It is becoming increasingly clear, through this and other studies, that essential genes will play roles in the checkpoint pathway. This allows us to link the checkpoint pathway with essential pathways such as DNA replication or chromatin organisation. In a screen for new genes involved in the DNA damage checkpoint, I isolated two essential genes that play roles in G2 checkpoint responses. cut5 provides a link between DNA replication and the G2 checkpoints responding to DNA damage and blocks to DNA replication. The isolation of a novel allele of rad18 in this project is the first evidence in S. pombe of a gene linking the DNA damage checkpoint and DNA repair. Several pieces of evidence point to rad18 playing an essential role in chromatin organisation and this then links chromatin regulation to the regulation of both repair and checkpoint responses to DNA. An allele-specific suppressor of this rad18 allele, brc1, suggested a link between checkpoint and repair pathways and independent regulators of genomic stability. The novel rad18 allele also allows us to address the issue of checkpoint maintenance and checkpoint initiation. The final gene isolated in the screen was a DNA repair gene that is not absolutely required for the ability to repair. As it is not clear what repair pathway this gene is involved in, it suggests a broad-range defect, which may also link the processes of chromatin organisation and DNA repair. The isolation of these genes links together multiple pathways of DNA damage responses. It suggests that genes with essential roles in processes such as DNA replication, chromatin organisation and maintenance of genomic stability may also play roles in checkpoint and other responses. Further screening, taking advantage of the types of phenotypes discovered in this screen, will be needed to identify these genes.