Massey Documents by Type
Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294
Browse
14 results
Search Results
Item Characterisation of pseudogene-like EP400NL in chromatin remodelling and transcriptional regulation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Ph.D.) in Biochemistry at Massey University, Manawatū, New Zealand(Massey University, 2023) Li, ZidongEP400 is an ATP-dependent chromatin remodelling enzyme that has been implicated in DNA double-strand break repair and transcription regulation including Myc-dependent gene expression. It was previously shown that the ectopic expression of the N-terminal domain of EP400 increases the efficacy of chemotherapeutic drugs against cancer cells. This prompted the question of whether the EP400 N-terminal-Like (EP400NL) gene, which resides next to the EP400 gene locus, also plays a similar role in epigenetic transcriptional regulation to the full-length EP400 protein. To characterize the function of the EP400NL nuclear complex, a stable cell line expressing TAP-tagged EP400NL was established, and the EP400NL complex was affinity purified and analyzed by mass spectrometry. EP400NL was found to form a human NuA4-like chromatin remodelling complex that lacks both the TIP60 histone acetyltransferase and EP400 ATPase. However, despite no histone acetyltransferase activity being detected, the EP400NL complex displayed H2A.Z deposition activity on a chromatin template comparable to the human NuA4 complex, suggesting another associated ATPase such as BRG1 or RuvBL1/RuvBL2 catalyses the reaction. In addition to a role in H2A.Z deposition, it was also determined that the transcriptional coactivator function of EP400NL is required for serum and IFNγ- mediated transcriptional activation of the immune checkpoint gene PD-L1. EP400NL, cMyc and multiple identified ATPases such as BRG1, RuvBL1/RuvBL2 were shown to be recruited to the promoter region of PD-L1. To further demonstrate the importance of EP400NL in regulating Myc and IFNγ-mediated PD-L1 expression, CRISPR/Cas9 mediated EP400NL indels were introduced in H1299, a human non-small cell lung carcinoma cell line. These EP400NL indel cell lines show compromised gene induction profiles with significantly decreased PD-L1 expression from both Myc and IFNγ stimulation experiments. In contrast to full-length EP400NL, two deletion mutants (Δ246- 260 and Δ360-419) lacked the ability to enhance the expression level of PD-L1 mRNA or protein, indicating that these regions are important for coactivator activity. Collectively, these data show that EP400NL plays a role as a transcription coactivator for cMyc-mediated gene expression and provides a potential target to modulate PD-L1 expression in cancer immunotherapy.Item Natural variation in bacterial gene regulation : a thesis presented in partial fulfilment of the requirements for the degree of Ph.D in Microbiology & Genetics, Massey University College of Science, School of Natural and Computational Sciences(Massey University, 2021) Vlková, MarkétaIt has been over 160 years since Charles Darwin set out the theory of evolution by natural selection. This theory is broadly accepted these days. However, it is still not completely understood how natural selection shapes particular cell mechanisms and behaviours. There is a limited research about selection acting on gene regulation. To address the questions about how selection shapes gene regulation we used a collection of environmental E. coli isolates. We quantified the genetic variability of 605 promoters within this collection of highly diverged strains. We then selected ten promoters (aldA, yhjX, lacZ, aceB, mtr, cdd, dctA, ptsG, purA, and tpiA) which highly differ in their genetic variability to analyse their phenotypic variability. We used fluorescent reporter assays with flow cytometry to measure changes in gene expression with high-throughput and at single cell resolution. In order to discern natural selection acting on gene regulation we compared phenotypes from segregating promoter variants, which have been subject to natural selection and random promoter variants that have never been subject to natural selection. We generated the random variants using PCR random mutagenesis. Beside focusing on the changes in the overall expression (i.e., transcription and translation), we examine selection acting on transcription only. This we achieved be implementing self-cleaving ribozyme insulation. In this thesis we showed that natural selection towards high plasticity and low noise is common among regulated E. coli promoters. We also verify that the self-cleaving ribozyme RiboJ activity is highly effective and that this genetic tool can be used to detect changes in transcription alone. Utilizing the RiboJ we were then able to detect both directional and diversifying selection acting on lacZ promoter. This thesis thus broadens the knowledge about natural selection acting on gene regulation and provides a new insights into how promoters are shaped in nature by selection, including some most well-characterized bacterial promoters. This work also demonstrates a new application of RiboJ ribozyme that has not been published before.Item Investigating the molecular basis of histidine catabolism in a human pathogenic bacterium Pseudomonas aeruginosa PAO1 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology & Genetics at Massey University, Auckland, New Zealand(Massey University, 2021) Sreeja Jayan, KiranPseudomonas aeruginosa is an opportunistic and a nosocomial pathogen of significant medical concern, particularly for cystic fibrosis patients. The extensive metabolic flexibility coupled with an array of virulence factors make them ubiquitous and successful in causing persistent multi-drug resistant infections. Pathogens exploit nutrient-rich hosts, and thus nutrients can be considered as signals perceived by bacteria that allow host recognition and coordination of expression of metabolic and virulence genes for successful colonization. A deeper understanding of the metabolic pathways and host perception mechanisms are significant from a therapeutic perspective in the current era of antibiotic resistance. Histidine is an amino acid that can serve as a source of carbon and nitrogen to many bacteria. Histidine catabolism in Pseudomonas spp., is widely known to occur via a 5-step enzymatic pathway, and the genes for histidine utilization (hut) are negatively regulated by HutC protein. The enteric bacteria and some others utilize a 4-step enzymatic pathway for histidine catabolism, which differs from the 5-step pathway in the direct conversion of intermediate formiminoglutamate (FIGLU) to glutamate. However, P. aeruginosa contains an additional operon (dislocated from the hut locus) encoding for formimidoylglutamase enzyme and its regulator, which can break down FIGLU similar to 4-step pathway. Previous studies report the accumulation of histidine metabolites, urocanate and FIGLU, in the mammalian tissues and reduced virulence of P. aeruginosa defective in histidine catabolism towards animal models. But the implications of the presence of two pathways for histidine catabolism or mechanisms associated with virulence remain elusive. This prompted us to examine the hut pathways and mechanisms that link hut with virulence in P. aeruginosa PAO1. First, computational analysis identified a transporter gene (named figT) adjacent to formimidoylglutamase enzyme (FigA) and transcriptional regulator FigR. This led to a new hypothesis that the three genes (figRAT) are responsible for the uptake and utilization of FIGLU, and they are not involved in histidine utilization as previously thought. Genetic analyses utilizing site-directed mutagenesis and lacZ reporter fusions confirmed that figT encodes for a FIGLU-specific transporter whose expression is induced by FIGLU. The figT gene is co-transcribed with figRA, and its expression is activated by FigR. Furthermore, gene expression studies indicate that FIGLU is the physiological inducer of fig operon, while histidine and urocanate are indirect inducers (by virtue of conversion to FIGLU). Growth and fitness assays revealed that histidine is predominantly catabolised via the 5-step hut pathway (not via the FigRAT system). Together, our genetic and phenotypic data show that fig operon is involved in the direct utilization of FIGLU from the environment. Phylogenetic analysis showed that figRAT genes are highly conserved and present in all completely sequenced strains of P. aeruginosa, but we found no evidence for horizontal gene transfer events. Previous work in Zhang’s laboratory suggests that urocanate derived from host tissues could serve as a signalling molecule, eliciting P. aeruginosa infections via interaction with the HutC regulator. Here, we aimed to seek further genetic, biochemical, and phenotypic evidence to improve our understanding of the global regulatory roles for HutC beyond histidine catabolism and determine their potential contribution to the colonization of eukaryotic hosts. Utilizing in silico analysis, we predicted 172 novel HutC-target sites in the genome of P. aeruginosa PAO1 with a P value less than 10-4. Six selected candidates were subject to experimental verification for HutC binding by means of gel shift assays (EMSA) and/or DNAse I footprinting assays, and all were able to bind with purified HutChis6 proteins. Further, a hutC deletion mutant was constructed by site-directed mutagenesis and subjected to phenotypic characterization. Phenotypic analyses revealed that hutC is involved in biofilm formation, tobramycin-induced biofilm formation, cell motility, and pyoverdine production. Significantly, we found that mutation of hutC resulted in reduced killing of C. elegans by P. aeruginosa PAO1. Finally, we observed distinct binding patterns for HutC interaction with the hutF promoter DNAs in P. aeruginosa PAO1 and P. fluorescens SBW25 (a model plant-colonizing bacterium used for studies of histidine catabolism). Molecular investigations revealed that the differences were not caused by HutC proteins from either species, but HutC recognized a distinct site proximal to hutFSBW25. This site displayed sequence similarity with the NtrC-binding site and was called the Pntr site. Functional analysis of the significance of Pntr site identified that Pntr site is necessary for the wild-type level production of HutF in P. fluorescens SBW25 during growth on histidine. Overall, the results from this study enhance our understanding of hut catabolism in Pseudomonas and contribute to novel molecular mechanisms associated with the virulence of P. aeruginosa PAO1. The identification of fig operon for the utilization of FIGLU (accumulated in host tissues) and global regulatory role of HutC in gene expression have broader implications from a therapeutic perspective in treating P. aeruginosa PAO1 infections. The ability of HutC to recognise multiple distinct DNA-binding sites suggests complex modes of gene regulation mediated by HutC and promotes further studies to fully understand the functional significance of genes in the HutC regulon.Item Regulation of histidine catabolism in Pseudomonas fluorescens SBW25 : a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology & Genetics at Massey University, Auckland, New Zealand(Massey University, 2019) NarenThe pathway of histidine utilization (hut) has been a model for studying bacterial gene expression, particularly the coordination between cellular carbon and nitrogen metabolisms. Early studies in enteric bacteria led to the concept of catabolite repression, which explains the inhibitory effects of glucose on the utilization of alternative carbon sources such as histidine and lactose. Briefly, transcription of hut genes is activated by the catabolite-activating protein (CAP) charged with cAMP and the NtrBC/NAC cascade when histidine is used as a source of carbon and nitrogen, respectively. However, this well-defined paradigm does not hold for many non-enteric bacteria, including the closely related Pseudomonas. This work aims to define the molecular basis of hut gene expression in Pseudomonas, using the plant growth-promoting bacterium P. fluorescens SBW25 as a model. Previous work identified all hut genes involved in histidine uptake and subsequent enzymatic breakdown, which are organized in three transcriptional units in the hut locus: hutF, hutCD and hutU-G. Like in enteric bacteria, histidine-induced expression of hut operons is mediated by the HutC repressor with urocanate, the first intermediate of the histidine degradation pathway, as the effector molecule. However, the precise interactions between HutC and its hut operator sites remain elusive; more importantly, recent progress suggests a new role of HutC in global gene regulation beyond histidine catabolism. Moreover, two two-component systems CbrAB and NtrBC are involved in hut activation, but it remains unknown whether they act in a direct or indirect manner. In this study, I first examined the molecular interactions between His6-tagged HutC protein and probe DNAs of the PhutU and PhutF promoters. Results of electrophoretic mobility shift assay (EMSA) and DNase I footprinting indicate that HutC binds to a consensus sequence of TGTA-N2-TACA (named Phut site), and involves complex oligomerization in response to varying concentrations of urocanate. A novel weak HutC binding sequence (termed Pntr site) was identified in the PhutF promoter, which may help strengthen the repression of hutF. Significantly, this Pntr site shows no sequence similarity to the previously recognized Phut site, instead it is homologous to the NtrC-binding consensus sequence (GCACCA-N3-TGGTGC). Next, the Phut consensus sequence was used to predict HutC target genes in the genome of P. fluorescens SBW25. This led to the identification of 88 candidate promoters, eight of which were subject to experimental verification by EMSA and DNase I footprinting. Phenotypic analysis of the hutC deletion mutant showed that hutC is involved in cell motilities. The data is consistent with the predicted global regulatory role of HutC. Histidine utilization poses a significant challenge as it produces excess nitrogen over carbon. The rate of histidine utilization (hut) thus must be carefully regulated. Here we show, for the first time, that expression of hut genes is positively regulated by two global regulators CbrAB and NtrBC in a direct manner, while subjecting to histidine concentration-dependent negative control of the HutC repressor. hut expression is further regulated at the post-transcriptional level by the CbrAB-CrcYZ-Crc/Hfq cascade in response to the presence of succinate (the most preferred carbon source for Pseudomonas). When growing in nutrient-complex conditions such as a minimal salts medium supplemented with succinate and histidine wherein histidine is the sole nitrogen but less-preferred carbon source, CbrAB is involved in directly activating hut transcription but indirectly repressing hut translation. Under this condition, NtrBC plays the dominant role in transcriptional activation of hut genes, but it requires assistance from the HutC repressor. A combination of genetic and biochemical analyses show that HutC acts as a governor to monitor and control the histidine catabolic rate, preventing production of excess ammonium and consequent inactivation of the NtrBC system. HutC additionally recognizes the NtrC binding site responsible for ntrBC expression, which provides a negative feedback for NtrBC autoregulation. Together, data presented in this thesis extend our understanding of carbon catabolite repression to the cellular nitrogen catabolism of Pseudomonas: carbon/nitrogen metabolic balance is maintained by the interplay of CbrAB and NtrBC at the hut operator site, and it requires the local regulator HutC to prevent hut expression from exceeding a critical upper limit. The finding that the HutC regulator is capable of recognizing two distinct DNA binding motifs (Phut and Pntr) has broader implications in gene regulation. Further biochemical analysis is required to unravel the molecular basis of the observed dual site recognition.Item Transcriptional regulation of human topoisomerase II beta : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry(Massey University, 2006) Mawson, ClaireTopoisomerase II has an essential role in maintaining the DNA in the correct topological state required for various cellular processes. Its mechanism of action involves the introduction of a double-Dstranded break into the DNA, passage of a different piece of DNA through the break, followed by the religation of the DNA. Topoisomerase II, in humans, exists as two different isoforms: topoisomerase II alpha, which is cell cycle-Dregulated and highly expressed in rapidly proliferating cells, and topoisomerase II beta, which is ubiquitously expressed and it is not under the influence of the cell cycle. Several chemotherapeutic drugs have been designed to interfere with the catalytic mechanism of the topoisomerase II enzyme. By either stabilising the DNA cleavage complex or interfering with another step of the mechanism, these topoisomerase II targeted drugs promote the entry of the cell into cell death pathways. An increasing problem in the treatment of cancer with these drugs is the rising number of patients with inherited or developed drug-resistance. It has been shown that drug-resistance, at least in part, results from the down-regulation of topoisomerase II expression. The expression of a gene is a highly regulated process and the initiation of transcription represents a major point of regulation. Prior to this study little was known regarding the regulation of transcription of topoisomerase II beta. Understanding the processes surrounding the regulation of this enzyme would provide some insight as to how it is down regulated in drug-resistance. The focus of this study was to examine the role of three elements in the topoisomerase II beta promoter, GCI, ICB1, and ICB2 and the transcription factors that bind to them. Electrophoretic mobility shifts assays revealed that Sp1, Sp3, NF-Y and two uncharacterised proteins are capable of binding to the promoter in vitro. Transient transfection assays showed in vivo that Sp1 was able to activate transcription and that Sp3 inhibited transcription driven by the topoisomerase II beta promoter. In addition the key activating elements appear to be ICB2 and GC1, while ICB1 is inhibitory.Item Regulation of Topoisomerase IIa expression in humans : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand(Massey University, 2006) Senior, KellyIn mammalian cells, the loss or down-regulation of tumour-suppressor genes and/or the mutation or overexpression of proto-oncogenes, whose products promote unregulated proliferation in cells, characterise the process of malignant transformation. This generates mitogenic signals that promote abnormal cell growth resulting in tumour progression. Topoisomerase IIα (topo IIα) is an enzyme present in elevated concentrations in highly proliferating cells due to the requirement for untwisting and unknotting of the DNA which is essential for replication. Because of this requirement, a number of anti-cancer drugs have been designed with topo IIα as their primary target. The effectiveness of these drugs however is limited by the development of resistance. One factor linked to drug resistance is the down-regulation of topo IIα at the transcription level. Expression of topo IIα appears to be regulated through various transcription factors with members of the Spl family having a major contribution. Previous work has shown down regulation of topo IIα can occur at the level of transcription. Nucleotide sequencing of the topo IIα promoter in drug-resistant cell lines has not revealed any mutations thus far. Three known proteins and one uncharacterised protein are capable of interacting with the proximal topo IIα promoter region. The uncharacterised protein may act as a co-activator or a co-repressor depending on the complement of transcription factors associated with the DNA in this region. Because drug resistant cell lines showed modulated expression of these transcription factors, it is important to identify the unknown protein and characterise its role in regulating topo IIα expression. This research aimed to identify the minimal binding site and DNA elements required for the uncharacterised protein to bind, as well as introduce mutations into this proximal region and examine their functional significance. The results of this study could provide insights into the molecular mechanisms responsible for the development of drug resistance, contributing to more efficient and effective methods for the treatment of cancer.Item Functional analysis of plant Mei2-like proteins : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand(Massey University, 2003) Leung, Susanna Chui-ShanMolecular techniques were used to analyse the function of a novel class of RNA-bindmg proteins in plants, termed Mei2-like. The biochemical function of this class of proteins is unclear Although the conserved presence of three RNA recognition motifs (RRMs) in all members of the family suggests the importance of an RNA binding activity, the precise biochemical mechanism by which these proteins act is unknown. Genetic and molecular analyses of the founding member of the family. Schizosaccharomyces pombe Mei2p, provide of a conceptual framework for the studies of the plant Mei2-like proteins presented here. Therefore, the aims of this thesis were to 1) study the cellular localisation of Mei2p in plant cells, and 2) deduce the functions of plant Mei2-like genes by identifying the protein(s) that physically interact(s) with Mei2-like proteins. Transient expression of GFP-fused Mei2p in onion epidermal cells was performed to show that Mei2p localised into the nucleus in the presence of meiRNA, a non-coding mRNA. Thus plants seem to share the capacity with S. pombe for meiRNA-dependent nuclear localisation of Mei2p. Moreover, intracellular localisation of one of the plant Mei2-like proteins, TERMINAL EAR-Like 2 (TEL2), was studied in onion epidermal cells. The GFP-fused TEL2 localised into the nucleus without co-expression of any special RNA, suggesting that either some RNA species that assist nuclear localisation of TEL2 are already present in onion epidermal cells, or the mechanism of intracellular localisation of TEL2 is different from Mei2p. The yeast two-hybrid system was utilised to identify protein interactors with TEL2. Six proteins were identified, including the well-studied KORRIGAN (KOR) protein. Based on the proteins identified, speculation is offered on how these proteins interact with TEL2. Since TEL genes are expressed in the central zone (CZ) of meristems, and mitotic activity of cells in the CZ is low, TEL2 may be involved in controlling cell division in the CZ via interactions with these proteins.Item Identification of genetic regulators of longevity in dark-held detached Arabidopsis inflorescences : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology, Massey University, Palmerston North, New Zealand.(Massey University, 2014) Jibran, RubinaHarvested green plant tissues experience a number of stresses including energy deprivation, water disruption, and changes in hormone levels. These stresses accelerate the senescence of the tissues, which causes their deterioration. A comprehensive understanding of how these stresses cause senescence is essential if this unwanted deterioration is to be minimised. In this thesis, I used detached dark-held immature inflorescences of Arabidopsis thaliana (Arabidopsis) to investigate the regulatory programme responsible for the senescence of harvested energy-deprived tissue. Detached dark-held Arabidopsis inflorescences completely degreened at day 5 when held in the dark at 21°C. The degreening was accelerated by exogenously applying ACC, ethrel, MeJA, and ABA that have previously been shown to accelerate senescence in detached dark-held leaves. Higher MeJA concentrations unexpectedly delayed rather than accelerated degreening of the detached dark-held inflorescences and this was associated with reductions in transcripts for the senescence-associated genes SEN4, ANAC029, NAC3, and SAG12. To identify key genetic regulators of inflorescence senescence an untargeted forward genetics approach was utilized. This involved detaching the immature inflorescences grown from ~20,000 ethyl methane-sulfonate-treated (EMS-treated) Arabidopsis (Landsberg erecta) seeds, holding them in the dark at 21°C and visually identifying those that showed a different timing of degreening to wild type. This approach successfully identified inflorescences that were completely degreened at day 3 of dark incubation (two days earlier than wild type) that were designated accelerated inflorescence senescence (ais) and inflorescences that were more green than wild type at day 5 that were designated delayed inflorescences senescence (dis). A total of 10 ais and 20 dis mutants were identified. Interestingly, most of the dis mutants were specific for inflorescence senescence as they did not show delayed senescence in detached dark-held leaves. By utilizing a traditional map-based cloning approach, five dis mutants were mapped to particular chromosomal regions. dis9 was mapped to the top arm of chromosome 3, dis15 was to the bottom of chromosome 2, and dis1, dis34, and dis58 were mapped to chromosome 4. Whole genome sequencing of dis15 and 58 identified the EMS-induced lesions as G to A transitions in the eukaryotic ASPARTYL PROTEASE (AT2G28030) and NON-CODING RNA (AT4G13495), respectively. Transformation of the AT4G13495 DNA fragment into dis58 reverted the dis58 phenotype to wild-type confirming that the non-coding RNA is involved in regulating inflorescence senescence. In addition to these fertile mutants, a sterile agamouslike mutant that had a sepal-petal-petal phenotype was identified. The mutant showed delayed degreening of detached dark-held inflorescences. This prompted me to investigate the mechanism behind the delayed senescence of the sterile homeotic ag-1 mutant. The sepals of the ag-1 inflorescences were found to have both delayed in planta and detached dark-induced senescence. They were also found to be devoid of JA and like wild-type senesced when treated with MeJA. The delayed in planta sepal senescence appeared to be due to the lack of produced JA as the dde2 mutant (defective in JA biosynthesis and devoid of JA) also showed delayed in planta sepal senescence. However, the dde2 mutant did not show delayed darkinduced senescence suggesting that the delayed dark-induced senescence of ag-1 may be through a mechanism that is unrelated to the JA hormone. Taken together, in addition to identifying common regulators of inflorescence and leaf senescence, this screen has also identified novel regulators specific to inflorescence senescence that traditional screens based on leaf senescence would have missed. This suggests that there are both similarities and differences in the genetic pathways regulating leaf and inflorescence senescence. The identification of a range of mutants, some of which appear to be novel, also indicates that the immature detached Arabidopsis inflorescences are a useful system for studying energydeprivation driven senescence. Understanding the role of the dis58 non coding RNA and the other regulators in the mutant collection offers a new and exciting opportunity for ascertaining the regulatory genetic network initiated in energy-deprived tissues that control the deterioration of harvested produce.Item MicroRNA and mRNA analysis of two species of New Zealand Pachycladon : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Manawatu, New Zealand(Massey University, 2014) Carr, Louise MichelleMicroRNAs (miRNAs) are small, non-coding RNAs important in post-transcriptional regulation. In this study, potential miRNAs from two New Zealand Pachycladon species, P. cheesemanii and P. fastigiatum, are identified and compared. Sixteen miRNAs were differentially expressed between the species, most of which have roles in flower and leaf development. Potential targets for 15 miRNAs were located in expressed sequence tag (EST) libraries for P. cheesemanii and/or P. fastigiatum, including a new potential relationship in P. cheesemanii between miR825 and MYB29 (AT5G07690), a transcription factor involved in the synthesis of methionine-derived glucosinolates. From the results of the differential expression analysis and target identification, 27 miRNAs from 21 miRNA families were chosen for pre-miRNA sequencing. Sequences of 15 P. cheesemanii miRNA hairpins and 13 P. fastigiatum miRNA hairpins were validated experimentally. Additionally, mRNA-Seq data obtained at the same time as the miRNAs were analysed. A gene ontology analysis indicated enriched terms for defence responses and miRNAs in P. fastigiatum. This study is the first investigation of the miRNAs present in Pachycladon and how their differential expression contributes to the adaptive divergence between the species.Item Does DNA topography coordinate intra- & inter-chromosomal Galactose gene expression? : this thesis is presented in partial fulfilment of the requirements for the degree of Masters of Science in Molecular Biology at Massey University, Albany, New Zealand(Massey University, 2008) Yu, ShuqiaoFor a long time, DNA had been considered as a stabilized, rigid, and “linear” structure, which acts as a platform for molecular regulators to function. However, genome structure in living cells is far more complex than the linear representation of the primary DNA sequence implies. This thesis aims to investigate whether the position of a gene within the genome plays a role in the regulation of its activity. The galactose (GAL) gene family of Saccharomyces Cerevisiae is used as a model. This gene family enables yeast cells to utilize galactose as an alternative carbon source; and it consists of structural and regulatory genes. Structural genes GAL1, GAL10 and GAL7 exist in a cluster on yeast chromosome II. The products of the regulatory genes, GAL3, GAL4, and GAL80, regulate the expression of the GAL structural genes, depending on the availability of carbon sources. Specifically, GAL gene expression is repressed by glucose, paused for induction (noninduced) by glycerol/lactate, and fully induced by galactose. The aim of this project was to study the relative position of the GAL structural genes within the nucleus, and whether any chromosomal interactions at the GAL locus help to regulate their activation. These were tested in accordance with the expression status of the GAL genes (i.e. repressed, noninduced or induced). Followed confirmation of the existence of any chromosomal interactions, protein/protein complexes that mediate these interactions were attempted to identify. The methods applied in this project were Chromosome Conformation Capture (3C) and Circular Chromosome Conformation Capture (4C), which applied in combination to map the positions of the GAL genes in the context of the overall genome structure. The results indicated that the GAL locus on chromosome II was divided into two “interaction zones”. DNA loops formed around these interaction zones to form an S-shape structure in a carbon source-independent manner. Two novel inter-chromosomal interactions between chromosomes II and XVI, i.e. SVL3-GAL7 and HOS1-GAL10, were also identified. Although these interactions occurred regardless of the GAL gene activities, it was suggested by real-time PCR that the interaction frequency for SVL3-GAL7 declined as the GAL genes being activated. Unfortunately no protein/protein complexes were identified to play an important role in mediating either intra- or inter-chromosomal interactions. Future work will be needed to identify the protein/protein complexes that play a role in mediating the S-Shape structure at the GAL locus and the two inter-chromosomal interactions. Additional works could also focus on the understanding of the functional implication of the interactions between chromosomes II and XVI.