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DNA topological stress impedes normal DNA replication. If topological stress is allowed to build up in front of the replication fork, the fork rotates to overcome the stress, leading to formation of DNA pre-catenanes. The formation of DNA pre-catenanes is therefore a marker of DNA topological stress. In this study, I have examined how transcription linked DNA topological stress impacts on fork rotation and on endogenous DNA damage. Transcription, similar to replication, affects the topology of the DNA; and collision between the two machineries is likely to lead to high levels of DNA topological stress. I found that the frequency of fork rotation during DNA replication, increases with the number of genes present on a plasmid. Interestingly, I also found that this increase in pre-catenation is dependent on the cohesin complex. Cohesin and transcription are known to be linked, as transcription leads to the translocation of cohesin along budding yeast DNA away from its loading sites. Cohesin plays a major role in establishing chromosomal structure, influencing gene expression and genetic inheritance. In this work, I have analysed the relationship between cohesin and the generation of topological stress and found that topological stress associated with cohesin can lead to DNA replication stress and DNA damage.

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Hundreds of thousands of putative small ORFs (smORFs) sequences are present in eukaryotic genomes, potentially coding for peptides less than 100 amino acids. smORFs have been deemed non-coding on the basis of their high numbers and their small size that makes it extremely challenging to assess their functionality both bioinformatically and biochemically. The recently developed Ribo-Seq technique, which is the deep sequencing of ribosome footprints, has generated significant controversy by showing extensive translation of smORFs outside of annotated protein coding regions, including putative non-coding RNAs.. Our lab adapted the Ribo-Seq technique by combining it with the polysome fractionation in order to assess smORF translation in Drosophila S2 cells. This thesis provides a high-throughput assessment of smORF translation in Drosophila melanogaster by firstly implementing complementary techniques such as transfection-tagging and Mass spectrometry methods in order to provide an independent corroboration of the S2 cell data (Chapter 3). Secondly, the in order to expand the catalogue of smORFs that are translated, I significantly improve upon the yield and sequencing efficiency of the Poly-Ribo-Seq protocol while adapting it to Drosophila embryos and then implementing it across embryogenesis divided in to Early, Mid and Late stages (Chapter 4). Currently, there is still a lot of debate in the field with regards to Ribo-Seq data analysis, and various computational metrics have been developed aimed at discerning 'real' translation events to background noise. Chapter 5 explores some of the metrics developed and establishes a translation cut-off suitable for designating small ORFs as translated. Altogether, the improvements introduced to the protocol and my data analysis shows the translation of 500 annotated smORFs, 500 smORFs in long non-coding RNAs and 5,000 uORFs, of which only one-third of each type of smORF has previous evidence of translation. These findings strengthen the establishment of smORFs as a distinct class of genes that significantly expand the protein coding complement of the genome.

One of the proteins that lie at the heart of the DNA Damage Response (DDR) is Topoisomerase II-binding protein I (TopBP1). TopBP1 was initially identified and has been extensively studied in the yeast model organisms. However, the lack of readily available tools, including genetically defined mutant cell lines, has rendered the characterisation of TopBP1 in higher eukaryotes more challenging. Sequence information obtained from the characterisation of the gallus gallus TopBP1 mRNA revealed a different splicing pattern at the 5'end to the one reported in the Genome Browser. Our assembled TopBP1 mRNA sequence containing a novel open reading frame (ORF) enabled the creation of a conditional knockout cell line of TopBP1 in DT40, which has been impossible with the use of the annotated cDNA sequence. Thus the avian TopBP1 ORF identified herein contained the necessary function(s) to sustain viability of DT40 cells in the absence of the endogenous protein. Additionally, the establishment of an isogenic set of stable cell lines from the chicken B cell line DT40 by targeted deletion of the TopBP1 alleles revealed a gene dosage-dependent reduction of the TopBP1 protein levels and functions. This work establishes a novel gene-dosage system that can be used for the knock in of point mutations within the endogenous TopBP1 locus. Using this system, a novel characterisation of knock-in point mutants of the ATR Activation Domain (AAD) of TopBP1 was carried out, providing in vivo evidence of its DDR function(s). Finally, a stably integrated overexpression system (SIOS) capable of producing increased amounts of a protein of interest has been established in DT40 cells. SIOS represents an easy to use versatile system for various experimental purposes in the field of DT40. The work presented in this thesis represents a novel characterisation of the avian TopBP1 mRNA and the TopBP1 protein and its functions. This is crucial to gain insight into the mechanistics of the DDR network and the genetic instability characterising cancer development.

Gene regulation id controlled by transcription factor proteins that bind to specific DNA sequences, known as transcription factor binding sites (TFBSs). Combinations of transcription factors working, co-operatively in cis-regulatory modules (CRMs), play a role in regulating gene expression. Current computational methods for TFBS prediction cannot distinguish between functional and non-functional sites, and predict very large numbers of false positives. The thesis focuses on the development of a novel computational model, based on artificial neural networks (ANNs), for the identification of functional TFBSs, and the CRMs within which they operate in the human genome. Datasets of 12,239 experimentally verified true positive (TP) TFBSs and 130,199 false positive (FP) TFBSs were extracted using a combination of position weight matrices from the JASPAR database and experimentally verified sites from the Encyclopedia of DNA elements (ENCODE). A number of machine learning alsgorithms were tested using a range of genetic information including gene expression, necleosome positioning, DNA methylation states and DNA entropy. The best model, that gave a mean area under the curve under a receiver operator characteristic curve of 0.800, was based on a feedforward ANN using backpropagation. This model was then used to predict functional TFBSs in a number of gene sets from the human genome. The predictions, combined with experimentally proven TFBSs from ENCODE, were used to investigate combinatorial patterns of TFBSs operating in CRMs. CRM patterns have been analysed in disease-associated genes located in linkage disequilibrium blocks containing SNPs obtained from Genome Wide Association Studies (GWAS). The potential for the model to make functional TFBS predictions to aid in the annotation of orphan genes of unknown function is discussed. In addition this thesis presents computational work on a number of smaller published studies.

A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation by the protein kinase ATM. H2AX is unevenly distributed throughout chromatin and is rapidly phosphorylated to form γH2AX up to 2 megabases either side of DSBs. Studies in yeast systems have shown that while γH2A can spread in cis surrounding the break site, it can also spread in trans onto unbroken chromosomes located in close spatial proximity. Although the majority of data in the current literature presents the well characterised in cis spread of γH2AX, there are strong indications that it can also occur in trans in mammalian systems; analogous to the findings shown in yeast. This thesis lays out the steps taken to develop a novel system to address the spatial distribution of γH2AX around a nascent DSB. Since the first published live imaging experiments of the dynamics of chromatin by in vivo single particle tracking there has been extensive investigation into the regulation and biological function of movement of damaged DNA. In yeast, a relative consensus exists that DSB induction increases the movement of a DSB. In contrast to yeast however, data published of DSB movement in higher eukaryotes has been controversial, caused by conflicting results. Here, I developed a cell-based system, and utilised timelapse live cell imaging to show that a chromosomal locus containing a single endonuclease-induced DSB shows confined movement in comparison to an undamaged locus. Furthermore, this confined movement of a damaged locus is compounded by treatment with an ATM kinase inhibitor but not a DNA-PKcs kinase inhibitor, suggesting that the kinase activity of ATM and not the kinase activity of DNA-PKcs plays a significant role in the dynamics of DSBs.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The olive industry has emerged as an important industry in Australia with increasing demand for both olive oil and table olives. To meet the domestic demand for olive products, it is necessary to increase production. Studies have shown that only 1-2% of olive flowers mature into fruits (Martin, 1990). Insufficient pollination due to self and cross incompatibility is a major factor affecting fruit set. The various methods used for studies on compatibility relationships have often shown conflicting results, with the same cultivar being found to be self-compatible in some studies, and self-incompatible in others (Sibbett et at., 1992; Caruso et al., 1993). Also, most of these studies have been conducted in the northern hemisphere where the environmental conditions and combination of cultivars growing nearby are expected to be different from Australia. It is therefore necessary to carry out studies on compatibility relationships under natural conditions in the Australian environment. The use of molecular markers has been found to be an effective and reliable method for paternity analysis studies. Using polymorphic and codominant markers, fingerprints of embryos may be compared to markers present in the mother plant, and therefore, the paternal contribution of alleles may be identified. By comparing these alleles with the genotype of all the potential pollen donors the pollinating genotype can be identified. The aim of this project was to identify the most compatible pollen donors for five olive cultivars (Barnea, Corregiola, Koroneiki, Kalamata, and Mission) and to observe the effect of morphological characters (bloom time, percentage pollen vitality, and percentage of complete flowers) and weather conditions (temperature, rainfall, and wind direction) on pollination. The study was conducted in a mixed olive orchard in Gumeracha, South Australia over the 2002-2003 and 2003-2004 growing seasons. Prior to the study, the genotypes of the trees were compared with the standards in the database (Guerin et al., 2002) and it was found that most of the trees matched with the standard cultivars. However, the trees considered to be Manaki by the grower did not match with the standard Manaki and were therefore referred to as atypical Manaki. Also, some Pendolino, Corregiola, and Kalamata trees did not match with the standard and were also referred to as atypical. The maximum and minimum temperatures, rainfall, and wind direction were recorded for the bloom period of both the years. The range of maximum temperature minimum temperatures during the bloom period was similar in both years. There was more rainfall in the bloom period during the first year than during the second year. Wind direction data during the bloom period showed that the wind direction was similar in both years. The winds were mainly easterly or westerly in the mornings and mainly westerly in the afternoon. However, there were winds of lower intensities blowing in the other directions as well, thus ensuring adequate wind movement for pollen dissemination. Dates of the start of bloom, full bloom and end of bloom for each cultivar were recorded for both years. It was observed that most of the cultivars overlapped in their bloom time, although some such as Kalamata flowered late in both years. Bloom time dates for replicate trees of a cultivar were similar, but there were differences in the dates between cultivars. The percentage of complete flowers was recorded for all cultivars in both years and it was observed that King Kalamata had the lowest value (42.5%) in the first year and Koroneiki had the lowest value (29%) in the second year. Leccino, atypical Manaki, and Corregiola had high percentages of complete flowers in both years. Percentage pollen vitality observations ranged from 23.5% in King Kalamata to 72.3% in Koroneiki in the first year. In the following year, UC13A6 had the lowest percentage pollen vitality (19.7%) and Leccino had the highest value (65.5%). The flowers sampled from Verdale and atypical Manaki did not contain pollen in both the years. Paternity analysis showed that: Barnea embryos were mainly fertilised by Pendolino and Mission; Corregiola embryos were mainly fertilised by Mission, Kalamata, and atypical Manaki; Koroneiki embryos were mainly fertilised by Mission; Kalamata embryos were mainly fertilised by Koroneiki; and Mission embryos were mainly fertilised by Koroneiki. There were also unidentified pollen donors pollinating a significant proportion of embryos. No apparent effect of direction of canopy and distance of pollen donors was observed and it was concluded that wind movement was not a limitation for movement of pollen in the orchard. Temperature and rainfall did not have any apparent effect on the overall bloom period. Pollen vitality, time of flowering, number of trees in the orchard, and tree age may have affected the effectiveness of some cultivars as pollen donors. The results highlighted the importance of cross-pollination for fruit set. Only two instances of self-pollination were observed suggesting that cross-pollination is more effective than selfing. The results also suggest that there is genetically controlled compatibility relationship operating among the cultivars and this determined which pollen type lead to successful fruit formation. However little is known about the mechanism of incompatibility operating in olives. There were differences in the effectiveness of some pollen donors over the two years which suggests that having more than one compatible pollen donor in the orchard is important. The results obtained in this study may be used as a basis for studying the mechanism of incompatibility in olives. The compatible pollen donors identified can be used to make recommendations to olive growers regarding the combinations of olive cultivars that will maximise yield and hence boost the production of olives in Australia. The method can also be extended to other cultivars to identify compatible pollen donors and also to compare the effect of different environmental conditions on pollination. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1148163 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2004

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