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Mammalian nuclear function depends on the complex interaction of genetic and epi-genetic elements coordinated in space and time. Structure and function overlap to such a degree that they are usually considered as being inextricably linked. In this work I combine an experimental approach with a computational one in order to answer two main questions in the field of mammalian chromosome organization. In the first section of this thesis, I attempted to answer the question, to what extent does chromatin from different chromosome territories share the same space inside the nucleus? This is a relatively open question in the field of chromosome territories. It is well-known and accepted that interphase chromosomes are spatially constrained inside the nucleus and that they occupy their own territory, however, the degree of spatial interaction between neighbouring chromosomes is still under debate. Using labelling methods that directly incorporate halogenated DNA precursors into newly replicated DNA without the need for immuno-detection or in situ hybridization, we show that neighbouring chromosome territories colocalise at very low levels. We also found that the native structure of DNA foci is partially responsible for constraining the interaction of chromosome territories as disruption of the innate architecture of DNA foci by treatment with TSA resulted in increased colocalisation signal between adjacent chromosomes territories. The second major question I attempted to answer concerned the correlation between nuclear function and the banding pattern observed in human mitotic chromosomes. Human mitotic chromosomes display characteristic patterns of light and dark bands when visualized under the light microscope using specific chemical dyes such as Giemsa. Despite the long standing use of the Giemsa banding pattern in human genetics for identifying chromosome abnormalities and mapping genes, little is known about the molecular mechanisms that generate the Giemsa banding pattern or its biological relevance. The recent availability of many genetic and epigenetic features mapped to the human genome permit a high-resolution investigation of the molecular correlates of Giemsa banding. Here I investigate the relationship of more than 50 genomic and epigenomic features with light (R) and dark (G) bands. My results confirm many classical results, such as the low gene density of the most darkly staining G bands and their late replication time, using genome-wide data. Surprisingly, I found that for virtually all features investigated, R bands show intermediate properties between the lightest and darkest G bands, suggesting that many R bands contain G-like sequences within them. To identify R bands that show properties of G bands, I employed an unsupervised learning approach to classify R bands on their genomic and epigenomic properties and show that the smallest R bands show a tendency to have characteristics typical of G bands. I revisit the evidence supporting the boundaries of G and R bands in the current cytogenomic map and conclude that inaccurate placement of weakly supported band boundaries can explain the intermediate pattern of R bands. Finally, I propose an approach based on aggregating data from multiple genomic and epigenomic features to improve the positioning of band boundaries in the human cytogenomic map. My results suggest that contiguous domains showing a high degree of uniformity in the ratio of heterochromatin and euchromatin sub-domains define the Giemsa banding pattern in human chromosomes.

Phosphoramidite derivatives of a nucleoside analogue bearing photoswitchable ortho-, meta- and para azobenzene moieties were prepared and used to incorporate the azobenzene groups into DNA.8mers. The photochemical E-Z isomerisation of the azobenzene-appended 8mers was investigated by UV/vis spectroscopy and RP-HPLC. In order to investigate the stabilities of the irradiated-8mers towards thermal Z - E isomerisation, Arrhenius and Eyring parameters for the photoisomerisation were determined. The meta-isomer was found to be the most thermally stable. An initial investigation into the stability of duplexes containing a para-azobenzene-modified 8mer was carried out using melting studies. The duplex-forming activity of the oligonucleotide was modulated by the E- Z photoisomerisation of the para-azobenzene residue. A divergent methodology for the preparation of a novel structural class of photoswitchable oligonucleotide has been described. A novel anthracene methyl phosphoramidite derivative suitable for the preparation of end- labelled oligonucleotides under solid-phase directed-Arbusov conditions was prepared and its reactivity investigated. A comparison of the utility of this anthracene methyl phosphorarnidite with a related benzyl phosphorarnidite in a model reaction with the 5'-hydroxyl of support-bound decathymidylate was made. Directed Michaelis-Arbusov reactions of the putative phosphite triester intermediates with primary and secondary amines in the presence of 0.01 M iodine gave the corresponding phosphoramidate diesters in high yields. This reactivity was also demonstrated using commercially available phosphoramidites for the preparation of inter-nucleotide phosphoramidates bearing terminal primary amines. Derivatisation of these primary amine-functionalised oligomers was accomplished in solution-phase following treatment with the meta-azobenzene NHS ester.

Understanding the molecular mechanisms that allow splice site selection is of fundamental importance to the study of alternative splicing. The study presented here has focused on the interactions of two spliceosomal trans-acting factors with pre-mRNA. The U1 snRNP is very important in 5’ splice site selection. However, how binding of U1 snRNP to pre-mRNAs containing multiple 5’ splice sites leads to 5’ splice site selection is unknown. To investigate the role of U1 snRNP in 5’splice site selection, single molecule microscopy was used to determine the number of U1 snRNPs bound to a single molecule of pre-mRNA. In cases where multiple strong 5’ splice sites are competing the furthest downstream 5’ss is used. In these cases, as many U1 snRNPs were observed bound to the pre-mRNA at an early stage of the reaction as there were 5’ splice sites, demonstrating that multiple sites were occupied. However, later pre-spliceosomal complexes contained only one U1 snRNP, indicating that the surplus U1 snRNP had been removed. A novel model of 5’ splice site selection is presented, in which U1snRNP stimulates the binding of SR proteins to the pre-mRNA and thus increases exon rigidity, enforcing use of the downstream 5’ splice site. The U2 Auxiliary Factor (U2AF), binds to the polypyrimidine tract and 3’ splice site of an intron and is a hetrodimer of the U2AF65 and U2AF35 subunits. The length of the polypyrimidine tract has been shown to influence the efficiency with which a 3’splice site is spliced. To investigate the role of polypyrimidine tract length on U2AF binding, experiments have been conducted in which the binding of U2AF65 to pre-mRNAs has been analysed using single molecule microscopy. Through the analysis of constructs derived from the Globin C pre-mRNA, the mechanisms of U2AF65 association with pre-mRNA have been shown to comprise; (i) a non-specific association that occurs with all RNA tested in the absence of ATP, (ii) a U1 snRNP-dependent association with pre-mRNAs in a polypyrimidine tract-independent manner, and (iii) an association with pre-mRNA in a polypyrimidine tract-dependent manner.

It is widely accepted that alternative splicing is often a catalyst for the development and progression of disease. Recent advances in oligonucleotide therapeutics have seen these alternative splicing patterns forcibly changed, providing a specific and versatile attack against such diseases. This study has focused on the use of bifunctional oligonucleotides for the treatment of spinal muscular atrophy (SMA) and cancer. In SMA the bifunctional oligonucleotide is targeted to inhibit exon 7 skipping during splicing of SMN2. The current study has confirmed that this oligonucleotide is able to stimulate SMN2 exon 7 splicing, as has been seen previously (Skordis et al., 2003; Owen et al., 2011). Analysis of the action of this oligonucleotide indicated that it specifically recruits spliceosomal factors to the 3’ splice site of SMN2 intron 6. The tail domain was seen to enhance binding of U2 snRNP to this region and stabilize protein complex formation, while the annealing domain was seen to enhance U2AF65-RNA binding. A greater understanding of the mechanism of action of the SMN2 bifunctional oligonucleotide also highlighted flaws in the design of the previously tested RON exon 11 bifunctional oligonucleotide (Ghigna et al., 2010). This oligonucleotide was designed to combat the production of RON Δ165 mRNA, in which RON exon 11 is skipped. Expression of this RON isoform has been linked with the onset of metastasis in some epithelial cell cancers. A systematic analysis of the splicing characteristics of this region in RON pre-mRNA, suggested that weak 3’ splice site sequences limit RON exon 11 splicing. hnRNP H was also shown to bind RON introns 10 and 11 and may play a role in the regulation of RON exon 11 splicing. A new bifunctional oligonucleotide targeted combat RON Δ165 mRNA production, through the stimulation of RON exon 11 splicing, was designed with the weakness in RON exon 11 splicing and the mechanism of action of the SMN2 bifunctional oligonucleotide in mind.

Synthetic biology aims to engineer biological systems and components, ultimately for applications as diverse as medical therapeutics and energy production. A major focus in synthetic biology has been the design and construction of relatively simple synthetic gene networks which display interesting dynamics, or functions. Many synthetic gene networks have been designed, and in some cases constructed in vivo, and can display a variety of dynamics, such as oscillations and switching. The most studied network has been the oscillator, and a number of different designs now exist. This thesis first reviews the oscillator field, the review forming the motivation for designing synthetic gene networks that are capable of either using the output from oscillators, or responding to other sources of oscillations. Two novel, theoretical synthetic gene networks are then presented which are designed to use oscillations as an input. The first network is a frequency multiplier, designed to use oscillations of the type produced by the current synthetic oscillators. Further analysis of this network shows that it possesses the property of multi-functionality, and can additionally function as a switch or an oscillator depending on the nature of the input it receives. The second network is a pulse generator, designed to use temperature as an input. In addition to a theoretical analysis, the pulse generator was constructed in vivo in E. coli. Experimental testing of the pulse generator suggested that the degradation conferred by ssrA tags, used to ensure rapid degradation of the network's proteins, is highly temperature dependent. Subsequent experiments confirmed this, highlighting an important weakness in using ssrA tags, the standard means of achieving rapid protein degradation in synthetic gene networks.

TDP-43 is an RNA-binding protein with particular predilection for UG repeats that was shown to regulate transcription, to mediate mRNA stability and to inhibit splicing in the CFTR and Apo All genes.Recently, TDP-43 has been found to be the major disease protein in Amyotrophic Lateral Sclerosis (ALS) and Fronto-temporal Lobar dementia [ftld]. Objective of this thesis was to characterize the function of TDP-43 with in-vitro and in vivostudies.

The external environment and internal cellular processes generate DNA double strand breaks (DSBs), a particularly toxic form of DNA damage that can result in chromosome fragmentation, replication failure, mutagenesis and cell death. Cells have evolved effective mechanisms to preserve the mtegrity of the genome including DNA damage signalling, cell cycle checkpoint activation and DNA repair.

Exonic mutations can result in altered protein function by affecting exon recognition during splicing. To understand this mechanism I have extensively evaluated the exonic regulatory elements affected by the disease G to T mutation at position +6 of BRCA1 exon 18. This substitution induces the exclusion of the exon and it has been suggested that it disrupts an ASF/SF2-dependent enhancer. Using a pulldown assay with an internal standard, I show that WT and T6 sequences bind ASF/SF2 with similar efficiency, which is significantly lower compared to the binding to a typical enhancer derived from the fibronectin EDA exon. Consistent with the absence of an ASF/SF2 enhancing effect, siRNA depletion of ASF/SF2 did not induce exon WT exclusion indicating that ASF/SF2 is not essential for BRCA1 exon 18 splicing. However, depletion or overexpression of ASF/SF2 resulted in a decrease or increase of T6 exon 18 inclusion, respectively. By a series of exonic deletions in the WT and T6 BRCA1 minigenes, an enhancer sequence was identified in position 23-32 which mediates the observed T6-specific, ASF/SF2-dependent splicing activation. Furthermore, extensive mutations analysis indicated that the T6 mutant creates a sequence with a predominantly inhibitory function. Double site-directed mutations showed that the point mutations affecting the "TAG" sequence, between +6 and +8 positions, completely restore normal splicing. Indeed, RNA protein interaction and siRNA experiments showed that the skipping of T6 BRCA1 exon 18 is due to the creation of a silencer element. This sequence specifically binds to the proteins hnRNP A1/A2 and to DAZAP1. By siRNA experiments, DAZAP1 and hnRNP A1/A2 are shown to be involved redundantly in the regulation of the defective BRCA1 exon 18. The present results support a gain-of-function model for the BRCA1 T6 exon 18 and indicate that the binding of the hnRNP A1/A2 and DAZAP1 is the primary determinant of exon skipping.

The aim of the work described in this thesis was to develop novel methods for the extraction of gene regulatory networks (GRN) from gene expression data, and use these methods to capture previously unknown relationships between genes in specific biological applications. This has been accomplished through the application of Bayesian Networks (BN) through minimum description length (MDL) and taboo search for parameter and structure learning respectively to three large scale microarray datasets from Saccharomyeces cerevisae, Escherichia coli and human stem cells. The application of BNs for modelling the well characterised yeast cell cycle demonstrated the efficacy of the techniques employed. Using the cDNA microarray data from the yeast cell cycle project by Spellman et a l (1998), this study succeeded in extracting many biologically plausible genetic relationships, which were supported by evidence from publicly available genome and literature databases. Two novel knowledge extraction techniques were applied; Target Node (TN) analysis and learning through simulation. Further, it was demonstrated how the addition of prior knowledge to the extracted network can improve the network structure extracted purely from experimental data. The second part of this thesis demonstrated how the BN approach could be adapted to a data set of very high dimensionality, specifically data from a 54,634 probe array used to monitor human adipose tissue. Genetic networks extracted included insulin receptor (IR) and Fatty acid binding proteins (FABP) families that play key roles in fatty acid uptake, transport, and metabolism In the final part of this thesis, the genome-wide GRNs of a prokaryotic expression system were extracted from novel oligo cDNA microarray data from E-coli K12 to identify metabolic stress responsive genes during recombinant protein production. Also, detailed analysis of known metabolic stress related genes and the genes that are directly or indirectly associated in the GRN were used to establish possible markers for host system exhaustion. In conclusion, the BN methods developed proved to be a powerful and effective means of extracting GRNs in a variety of applications.

This thesis describes the generation, mapping, and characterisation of a mutant mouse strain derived from a genome-wide ENU mutagenesis programme in collaboration between Australian National University and Oxford University funded by the Wellcome Trust. Starting with the identification of the growth retardation and immunodeficiency phenotype in the G3 progeny of one mutagenised mouse, we mapped the mutation to the DNA Ligase IV (LigIV) gene. The enzyme is the final component of the nonhomologous end joining (NHEJ) pathway of DNA double strand break repair, which is involved in maintenance of genomic stability and in VDJ rearrangement of lymphocyte receptor genes. The mutation is a single base change that results in a Y288C amino acid substitution near the active site of the LigIV enzyme. The mutation causes an over 20- fold reduction in the LigIV enzymatic activity in vivo, partly though its effect on protein stability and expression levels. This mouse strain is the first viable animal model of LigIV deficiency, and therefore this work contributes to our understanding of the pathology of the human LigIV syndrome. Here we provide a detailed characterisation of the strain, addressing a number of questions about the physiological pathways in which LigIV activity is limiting. Lymphocyte development is impaired in the mutants and peripheral lymphocyte numbers are reduced nearly 100-fold, but a population of B1 cells develops in the peritoneal cavity. In addition to impaired VDJ rearrangement, lymphocyte survival in peripheral lymphoid organs is also affected by the mutation and this may contribute to the lymphopenia. We also demonstrate that LigIV is required for normal immunoglobulin class switching. Furthermore, the mutant bone marrow is deficient in reconstituting haematopoiesis in irradiated recipients and we observe an age-dependent decline in the stem and progenitor cell population in the bone marrow of the mutant mice, suggesting that LigIV activity may also be limiting in the maintenance and function of haematopoietic stem cells. In addition, the mutant mice are highly predisposed to haematopoietic malignancies. This demonstrates how ENU mutagenesis can be used to create hypomorphic alleles of genes essential for survival, contributing to our understanding of the function of mammalian genes and creating animal models of human genetic diseases.

The cell lysis step is one of the most critical operations in large-scale plasmid DNA processing as fragments of chromosomal DNA and degraded plasmids remaining in solution have a detrimental effect on downstream operations. In the present thesis the effect of the engineering environment on the bacterial cell suspension used in the lysis step and on solutions containing gene vectors of up to 242 kb in size was investigated. An integral aspect of the thesis was to evaluate current analytic techniques relating to the characterisation of process streams containing plasmid vectors and to identify and develop characterisation techniques suitable for process development and manufacturing. Cell pastes derived from laboratory- and 450 litre-scale fermentations of plasmid-containing bacterial cells were characterised using a variety of analytical techniques. The impact of pilot scale continuous centrifugation equipment used for cell harvesting was then investigated. It was observed that equipment design and operating conditions had an impact on product yield and the molecular weight of contaminant DNA in the process stream. It was found that direct measurement of released nucleic acids at the cell resuspension stage can provide a simple analytical technology to characterise cells before the lysis stage. After cell lysis, the downstream options depend on the susceptibility of the product to chemical and mechanical damage in solution. In the case of circular DNA molecules, it is of particular importance to characterise the backbone integrity. A microwell-based high throughput method to evaluate this for pure double stranded DNA solutions was developed. The method was then validated for reproducibility, accuracy and sensitivity and was successfully adapted for use on a robotic liquid handling system. The method was utilised to investigate the response to hydrodynamic stresses of bacterial artificial chromosomes. It was observed that for a 116 kb vector, average shear rates 104 s-1 (comparable with standard process equipment), caused a 5 fold decrease in the integrity of the molecules in the sample. Finally, the thesis ends with a look towards future process analytical technologies and assay development within the context of DNA-based pharmaceuticals.

The EcoR124I NT restriction-modification (R-M) system contains the genes HsdS3, HsdM and HsdR. S3 encodes the N-terminal domain of the wild-type S subunit and has been shown to dimerise in solution (Smith et al., 1998). Following purification of the subunits of the EcoR124I NT R-M system, complexes of the methyltransferase S3/M and restriction endonuclease S3/M/R were formed and shown to have activity in vitro, methylating and hydrolysing a symmetrical DNA recognition sequence, respectively. The DNA mimic OCR (overcome classical restriction) protein inhibited the methyltransferase activity in vitro, with maximum inhibition at a 1: 2 molar ratio of (S3/M)2 to an ocr dimer. Dynamic light scattering (DLS), sedimentation equilibrium (SE) and sedimentation velocity (SV) experiments showed S3 to exist as a dimer and S11 (the central conserved domain of S) to exist as a tetramer in solution. M was found to be dimeric in solution, whilst the R protein was monomeric. A complex of S3/M was found to have a stoichiometry (S3/M)2 and a complex of S3/M/R had a stoichiometry of S3/M/R1, even when a 2: 1 molar ratio of R to S3/M, was added. Small angle neutron scattering (SANS) experiments provided values for the radius of gyration (Rg), which for S3 was comparable to that calculated for the recently published crystal structure of the S subunit from Methanococcus jannaschii (Kim et al., 2005). These experiments also showed a decrease in the Dmax in the presence of the 30 bp DNA recognition sequence from 200A to 140A, suggesting a similar conformational change in the positioning of the subunits as has been detected for the wild-type M. EcoR124I and a related type 1 1/2 system AhdI. This change following DNA binding was also observed by SV experiments. Furthermore ab initio modelling from the SANS data has provided a low-resolution structure for the EcoR124I NT MTase and its complex with DNA.

Regulatory authorities have stringent requirements for the manufacture of recombinant drugs intended for human recipients. Contaminating heterologous host-derived nucleic acids from product expression strains must be minimised to satisfactory levels in order to gain manufacturing and product approval. High molecular weight nucleic acid species also cause difficulties in large-scale production processes due to the high viscoelastic characteristics they display upon being released from the host cell into the process stream. One of the difficulties associated with the manufacture of plasmid DNA for gene therapy is the removal of the host cell-related impurity RNA following cell lysis, whilst host cell derived high molecular weight chromosomal DNA causes problems from both process and regulatory perspectives in the production of recombinant protein. This work describes the construction and characterisation of two modified Eschericia coli JM107 host stains, JMRNaseA and JMN, for application in the large-scale production of recombinant plasmid DNA and protein biotherapeutics, which contain bovine pancreatic ribonuclease and Staphylococcal nuclease expression cassettes integrated into the dif locus of the host chromosome respectively. The expressed nucleases are translocated to the periplasm of the cell, protecting the cytoplasmic nucleic acid from any toxic activity, and are released during cell lysis. Subsequently the auto-hydrolytic activity acts to hydrolyse host nucleic acid, providing a suitable regulatory methodology for the purification of RNA free plasmid DNA, and DNA free protein, and significantly decreasing lysate viscosity, which will ease process difficulties associated with filtration and chromatographic purification. The biological approach to downstream processing by host cell modification avoids the regulatory problems associated with the addition of exogenous animal sourced and prohibitively expensive recombinant nucleases in large-scale recombinant manufacturing processes.

Large scale mutagenesis programmes are crucial for both the identification of new genes and for understanding their function. Gene trapping provides identifiable mouse mutations and mutant phenotypes and the use of embryonic stem (ES) cells allows for large scale screens to be undertaken and several have been carried out (Skames et al., 1995; Chowdury et al., 1997; Hicks et al., 1997; Zambrowicz et al., 1998). Many of these studies employ promoterless plasmid vectors which are dependent upon the transcriptional regulation of the genes in which they integrate to drive their expression. As such, they are limited to trapping those genes whose level of expression at the ES cell stage of development is sufficient for their selection. The data presented here demonstrate that gene trapping dependent upon gene expression in ES cells does allow for the discovery of genes expressed in the adult brain. Using the secretory trap vector pGTl.Stm (Skames et al., 1995), six out of 37 genes trapped (16%) were found to be expressed in the adult brain. Thus a significant fraction of genes involved in adult brain function can be accessed by gene trapping. The complete adult brain mRNA expression patterns of six genes ɑ-mannosidase II, APLP2, SDR-1, laminin-y, glypican-4 and one protein of unknown function, have been analysed. Comparative studies of the ß-galactosidase staining patterns and in situ hybridisation data for the gene encoding APLP2, confirm that P-galactosidase expression in gene trap lines is a faithful representation of mRNA expression for splice variants incorporating the exon fused to the marker gene. The insertion into the gene encoding laminin-y1 has also proven lethal when breed to homozygosity, in concurrence with targeted disruptions of this gene (Smith et al, 1999; Murray and Edgar, 2000). Altogether these data provide evidence that gene trapping with promoterless vectors in embryonic stem cells is able to produce mutations of interest in highly differentiated tissue such as the brain. An insertion into the gene encoding glypican-4 was then selected for more detailed study. GPC4 was originally cloned from kidney cells (Watanabe et al., 1995) and is a member of a family of heparan sulfate cell surface proteoglycans. This insertion into GPC4 lies within the first intron of the gene and ß-galactosidase activity in the transgenic embryo matches that of the previously published in situ patterns. The expression pattern of GPC4 in the adult brain is highly limited, with high levels of ß-galactosidase activity observed in the dentate gyrus and in specific layers of the cerebral cortex. This mutation has proven viable and fertile and shows no behavioural abnormalities in the open field test or in the Morris water maze. RT- PCR analysis of heinizygous tissue, however, shows that this gene trap insertion has not provided a null allele; production of properly spliced endogenous transcript continues despite the insertion of the vector into intron 1 of the gene. Such incomplete disruptions of endogenous mRNA have been found in other gene trap insertions (eg-McClive et al., 1998; Voss et al., 1998). The Exl94 mutations most likely represents a hypomorphic allele of the gene GPC4. The full disruption of this gene has yet to be reported, and the line remains of value as an expression marker and may yet reveal subtle phenotypes.

Various aspects concerning the fate of novel DNA in rainbow trout were investigated. These included mosaicism, germ-line transmission, and the methylation status of a mouse metallothionein-1/rat growth hormone gene (mMTrGH) construct, in addition to the fate and temporal expression patterns of injected DNA sequences during development, using a carp β-actin/CAT gene (pFV1) construct. Widespread mosaicism was observed amongst the transgenic fish, resulting in an unequal distribution of transgene copies in the cells/tissues of the fish. Germ line transmission was, however, observed in 50% of the transgenic fish containing the mMTrGH gene construct, with transmission frequencies ranging from 14-81%. The lack of transmission from the rest of the founder transgenics could also be attributed to mosaicism. The methylation studies revealed tissue-specific differences in the methylation patterns of the mMTrGH transgene, but could not be correlated with expression due to the consistent lack of expression of this transgene in our founder transgenics. With respect to the fate of DNA during development, prolonged persistence of injected DNA sequences appeared likely, probably contributing to the mosaicism observed. Studies on the conformational influences of injected DNA revealed that linear molecules were more efficient at forming concatemers and persisting through development. They, therefore, resulted in high levels of expression, and perhaps greater rates of integration. The concatemers generated with circular injected molecules appeared to consist of head to tail arrangements of transgene copies, whereas those generated with linear injected molecules appeared to consist of random arrangements of transgene copies.