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Heglind, Mikael. "Functional studies of two forkhead genes /." Göteborg : Institute of Biomedicine, Department of Medical and Clinical Genetics, The Sahlgrenska Academy at University of Gothenburg, 2010. http://hdl.handle.net/2077/21481.
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Rice, Kim. "Functional analysis of the HOX11 target genes ALDH1A1 and FHL1." Thesis, Rice, Kim (2004) Functional analysis of the HOX11 target genes ALDH1A1 and FHL1. PhD thesis, Murdoch University, 2004. https://researchrepository.murdoch.edu.au/id/eprint/277/.
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HOX11 is a developmental regulator that plays a crucial role in the normal development of the spleen and is also aberrantly activated by the t(10;14)(q24;q11) and variant t(7;10)(q35;q24) translocations in a subset of T-cell acute lymphoblastic leukaemias (TALLs). The recent finding that HOX11 is deregulated in up to 40% of childhood TALLs when abnormalities not detected by cytogenetics are included, suggests that the over-expression of HOX11 and subsequent deregulation of downstream target genes are critical events in the progression of this tumour type. To date, three candidate HOX11 target genes have been reported, two of which are Aldehyde Dehydrogenase 1a1 (ALDH1A1) and Four and a Half LIM domain Protein 1 (FHL1). This investigation focused on two aspects of HOX11 function, namely its roles as a transcriptional regulator and as a nuclear oncoprotein capable of inducing neoplastic transformation. More specifically, we sought to further understand the role of HOX11 in tumorigenesis by 1) Confirming target gene status of ALDH1A1 and FHL1 by assessing whether their proximal promoter regions are transcriptionally regulated by HOX11, 2) Investigating the regulatory elements/transcriptional complexes involved in the response of ALDH1A1 to HOX11 in both a T-cell and an erythroid cell line in order to gain an insight into the mechanism(s) responsible for mediating a HOX11 activity and 3) Assessing the ability of ALDH1A1 and FHL1 to perturb normal patterns of haematopoiesis, on the basis that the transforming capabilities of HOX11 are thought to derive from its ability to affect haematopoietic cell differentiation.
To confirm ALDH1A1 and FHL1 as target genes, they were both characterised in terms of the ability of their proximal promoters to be transcriptionally regulated by HOX11 using luciferase reporter assays. Significant repression of the proximal promoters of ALDH1A1 and FHL1 by HOX11 was observed in PER-117 T-cells which provided further evidence for their status as target genes. In the case of ALDH1A1, a CCAAT box (-74/-70bp) was identified as the primary cis-regulatory element involved in ALDH1A1 transcription and repression by HOX11 appeared to occur, either directly or indirectly, via interactions at the CCAAT box. Electromobility shift assays (EMSAs) revealed the disruption of a specific complex at this site by HOX11, which also altered the formation of complexes at a non-canonical TATA box (a GATA box at -34/-29bp). Significantly, HOX11 was shown to have the potential to interact with TFIIB, a member of the basal transcriptional complex. This, together with the presence of a TFIIB responsive element immediately 5' of the GATA box, suggested that HOX11 may repress transcription by interfering with members of a preinitiation complex on the ALDH1A1 promoter. The transcriptional repression by HOX11 demonstrated in T-cells was dependent on DNA binding helix 3 of the homeodomain, suggesting that repression may require DNA binding. Alternatively, this region may be required for stable protein-protein interactions. In support of this, the in vitro association of HOX11 with TFIIB was disrupted upon deletion of helix 3, and the HOX11 H3 mutant switched from a transcriptional repressor to a potent activator of transcription. Together, this data supports a model whereby HOX11 represses transcription by interfering with activation complexes at the CCAAT box and at the GATA box possibly via protein-protein interactions involving the homeodomain helix 3, whereas deletion of the region disables repressor-specific interactions, resulting in potent activation by HOX11.
Luciferase reporter gene assays investigating the response of nested deletions of the ALDH1A1 promoter to HOX11 in the HEL900 erythroleukaemic cell line, also identified the CCAAT box (-74/-70bp) as the primary cis-regulatory element involved in ALDH1A1 transcription. However, in stark contrast to the its effect in T-cells, HOX11 was shown to activate transcription in the HEL cell line, both from the empty pGL3Basic luciferase reporter vector and from the ALDH1A1 promoter, in a manner independent of the homeodomain DNA binding helix 3. HOX11 thus appears to be a dichotomous regulator, capable of both transcriptional activation and repression depending on the circumstances. The mechanisms underlying these two functions are also appear to be distinct, with repression but not activation requiring the presence of homeodomain helix 3.
ALDH1A1 encodes an enzyme involved in the irreversible conversion of retinaldehyde to the biologically active metabolite, retinoic acid (RA) and appears to be physiologically regulated by Hox11 in the developing spleen. Since RA is a potent modulator of cellular differentiation, proliferation and apoptosis, the dysregulation of RA synthesis is likely to have severe consequences for the cell and may constitute a mechanism whereby overexpression of HOX11 predisposes T-cells to malignant transformation. FHL1 also appears to have potential relevance to tumorigenesis, given that it encodes protein isoforms with suspected roles in transcriptional regulation. As a starting point to investigate a possible link between these HOX11 target genes and leukaemogenesis, the effect of overexpressing ALDH1A1 and FHL1 on murine haematopoiesis was assessed following reconstitution of lethally irradiated mice with retrovirally-transduced primary murine bone marrow cells. The enforced expression of ALDH1A1 in bone marrow was associated with a marked increase in myelopoiesis and a decrease in B and T-lymphopoiesis. By contrast, overexpression of FHL1 was not associated with perturbations in myelopoiesis or lymphopoiesis, although a slight increase in erythropoiesis was observed in the bone marrow. While further work is required to clarify the possible oncogenic roles of both of these HOX11 target genes, these findings have served to identify ALDH1A1 in particular, as a gene which could potentially be involved in HOX11-mediated tumorigenesis.
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Rice, Kim. "Functional analysis of the HOX11 target genes ALDH1A1 and FHL1 /." Access via Murdoch University Digital Theses Project, 2004. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20051012.93820.
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Fernandes, Alinda. "Lentiviral-mediated gene delivery to investigate the functional role of neuropsychiatric genes." Thesis, King's College London (University of London), 2013. https://kclpure.kcl.ac.uk/portal/en/theses/lentiviralmediated-gene-delivery-to-investigate-the-functional-role-of-neuropsychiatric-genes(b6392e47-e94b-4e64-b343-e7eafd696b26).html.
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Genetic studies have led to the identification of several candidate genes, some novel and others established, that may contribute to the risk of developing neuropsychiatric disorders. For example, dopamine receptor genes are established candidates for a number of psychiatric disorders such as Parkinson’s Disease, alcohol addiction and mood disorders. On the other hand, a gene of unknown function, AUTS2 (Autism susceptibility candidate 2), has recently been associated with alcohol consumption in a GWAS meta-analysis performed by our group. Interestingly, it has been associated with a broad range of neuropsychiatric disorders including autism, epilepsy and schizo-affective disorders. This thesis looked to address two broad aims: to establish lentiviral-mediated gene delivery technique in vivo by delineating the role of two well characterised Dopamine receptors D2R and D3R and to functionally characterise the role of AUTS2. By successfully establishing lentiviral mediated gene manipulation in vitro and in vivo, this thesis presents data for a similar role of nucleus accumbens D2R and D3R in novelty-induced locomotion while these receptors have a differential function in the regulation of light-induced locomotor behaviour in rats. Additionally, using molecular biology and in silica methods, this thesis demonstrates that AUTS2 is a nuclear protein and presents indications of its function as a neurodevelopmental gene with a potential role in neural migration, although its specific role has yet to be corroborated. Collectively, findings from this thesis will increase our understanding of the genetic link with brain function and behavioural traits. This will therefore have implications for overall neuropsychiatric research, as it will help understand molecular mechanisms underlying these conditions and possibly direct in the identification of potential therapeutic targets.
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Kacar, Betül, Eva Garmendia, Nurcan Tuncbag, Dan I. Andersson, and Diarmaid Hughes. "Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs." AMER SOC MICROBIOLOGY, 2017. http://hdl.handle.net/10150/625744.
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Genes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here, we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenous tufA gene in the Escherichia coli genome with its extant and ancestral homologs. The extant homologs represented tuf variants from both near and distant homologous organisms. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 billion years ago (bya). Our results demonstrate that all of the foreign tuf genes are transferable to the E. coli genome, provided that an additional copy of the EF-Tu gene, tufB, remains present in the E. coli genome. However, when the tufB gene was removed, only the variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth which demonstrates the limited functional interchangeability of E. coli tuf with its homologs. Relative bacterial fitness correlated with the evolutionary distance of the extant tuf homologs inserted into the E. coli genome. This reduced fitness was associated with reduced levels of EF-Tu and reduced rates of protein synthesis. Increasing the expression of tuf partially ameliorated these fitness costs. In summary, our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria. IMPORTANCE Horizontal gene transfer (HGT) is a fundamental driving force in bacterial evolution. However, whether essential genes can be acquired by HGT and whether they can be acquired from distant organisms are very poorly understood. By systematically replacing tuf with ancestral homologs and homologs from distantly related organisms, we investigated the constraints on HGT of a highly conserved gene with multiple interaction partners. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 bya. Only variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth, demonstrating the limited functional interchangeability of E. coli tuf with its homologs. Our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.
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Fan, Li. "Map based candidate gene cloning and functional analysis of genes involved in VLCFAs synthesis." [Ames, Iowa : Iowa State University], 2007.
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Ashwood, Lauren M. "Characterising the functional venom profiles of sea anemones." Thesis, Queensland University of Technology, 2022. https://eprints.qut.edu.au/227457/1/Lauren_Ashwood_Thesis.pdf.
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This project examined the relationship between the ecological roles of venom and patterns of venom production in sea anemones. It was found that sea anemones produce multiple venoms and that these distinct venoms are associated with the ecological functions of envenomating structures. Overall, this information provides insights into the ecological context of venom regulation and provides a rational framework for pharmaceutical bioprospecting in sea anemones.
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Kolle, Gabriel Victor. "Functional analysis of vertebrate Crim1 /." [St. Lucia, Qld.], 2002. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16804.pdf.
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Couldrey, Christine. "A gene trap approach to identify the functional role of genes expressed during murine gametogenesis." Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624246.
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Tronnersjö, Susanna. "Functional studies of RNA polymerase II-dependent transcription in yeast Saccharomyces cerevisiae /." Uppsala : Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, 2006. http://epsilon.slu.se/2006109.pdf.
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Smoler, Gunilla Kanter. "Functional characterization of conserved checkpoint genes." Göteborg, Sweden : Dept. of Cellular and Molecular Biology, Göteborg University, 1998. http://books.google.com/books?id=vM5qAAAAMAAJ.
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Swiatek, Magdalena. "Functional analysis of plastid-encoded genes." Diss., lmu, 2002. http://nbn-resolving.de/urn:nbn:de:bvb:19-1680.
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El-Hassi, Mohamed F. "Functional analysis of some yeast genes." Thesis, Durham University, 1997. http://etheses.dur.ac.uk/4767/.
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A series of mutant strains of the yeast Saccharomyces cerevisiae that are sensitive to osmotic stress and also have a defect in vacuolar biogenesis have been isolated (M. Latterich, PhD Thesis 1992). The mutations that cause this pleiotropic phenotype are termed ssv, for salt sensitive vacuolar mutants. Complementation analysis has revealed that ssv mutations fall into one of 18 complementation groups. A MAP kinase related signal transduction pathway, termed the HOG pathway for High Osmolarity Glycerol, has been identified in yeast. This pathway senses osmotic stress and invokes the cellular response, one aspect of which is the accumulation of intracellular glycerol (Brewster et. al, 1993). Mutations in the HOG pathway often cause an osmosensitive phenotype similar to that shown by ssv mutations. This work sets out to characterise several ssv strains for defects in the HOG pathway. These strains were subjected to osmotic stress and the intracellular and extracellular glycerol determined and compared to control strains and conditions. Many of the strains showed reduced, or even elevated in one case, glycerol levels compared to wild-type strains. No correlation could be made between these glycerol levels and the activity of the rate-limiting enzyme, glycerol-3-phosphate dehydrogenase (GPDH) determined in an independent study. Transcription of the GPDH gene is under the control of the HOG pathway. In a separate study, the nucleotide sequence of a short region of yeast chromosome VII was determined. Approximately 11,000 bases of DNA from the right sub-telomeric region was sequenced. Analysis of the DNA sequence showed four potential open reading frames. One of these encoded the YORl gene and another a protein related to PAU1 The remaining two ORFs, termed ORFl and ORF2, encoded potential proteins of unknown function. Disruption cassettes containing the LEU2 selectable marker were constructed for both ORFl and ORF2. Successful disruption of ORFl was achieved, but no viable transformants were ever recovered after attempted disruption of 0RF2..ORFl gene knockouts are viable and show no observable phenotype under a range of growth conditions. Subsequent analysis of ORFl and 0RF2 after the completion of the Yeast Genome Project, shows that both ORFl and 0RF2 are members of different sub- telomeric associated gene families. 0RF2 encodes a putative Y' protein.
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Kanter, Smoler Gunilla. "Functional characterisation of conserved checkpoint genes." Göteborg : [s.n.], 1998. http://catalog.hathitrust.org/api/volumes/oclc/45150869.html.
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Read, Jason Tyler. "Towards conditional gene inactivation in the mouse colon and functional analysis of genes influencing colorectal carcinogenesis." Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/22575.
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Dysregulation of the Wnt signalling pathway is implicated in the carcinogenic transformation of normal cells in a number of human tissues. Mutations in Wnt pathway signalling molecules appear to be particularly important in the development of colorectal neoplasia. This thesis investigates the effects of modulating levels of Wnt pathway components on downstream molecules <i>in vivo</i> and <i>in vitro</i>. To examine the effects of loss of Wnt pathway components on the biology of the mouse large intestine work was undertaken to assemble a tissue specific, inducible Cre (CreER<sup>tm</sup>) transgene construct. This formed part of a group effort using various lengths of upstream sequence from the mouse homeobox gene Cdx1 to drive CreER<sup>tm</sup>. In order to ensure that sufficient Cdxl promoter sequence to achieve adequate expression levels and localization a recently described methodology based on the Red a-b-g recombination system of bacteriophage Lambda was used in an effort to insert the CreER<sup>tm</sup> coding sequence into a Bacterial Artificial Chromosome (BAC). The BAC used in the construction of the transgene was determined to contain previously uncharacterised upstream Cdx1 promoter elements, which may play a role in determining correct tissue expression pattern. In order to further characterise the newly isolated Cdxl promoter elements, portions of the BAC derived sequence were used in the construction of reporter plasmids encoding the Enhanced Green Fluorescent Protein (EGFP). The EGFP based reporter plasmids, which contained various fragments of the Cdx1 promoter containing a number of transcriptional control elements, were microinjected and liposome transfected into murine embryonic stem cells, colorectal cell lines and primary murine colonocytes in order to determine the contribution of the transcriptional elements to levels of expression. The work presented in this thesis contributes to the development of a transgenic mouse line bearing a conditional, inducible intestinal epithelial specific Cre recombinase and to the understanding of regulation of the Wnt signalling pathway in response to genotoxic damage as well as expression of the murine homeobox gene Cdxl.
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Papin, Marine. "Efficiency and impact of recurrent microbial inoculation in soil, a lab to field assessment." Electronic Thesis or Diss., Bourgogne Franche-Comté, 2024. http://www.theses.fr/2024UBFCK051.
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Face à l'urgence de mettre en place des pratiques plus durables pour protéger l'environnement tout en maintenant la production agricole, les inoculants microbiens suscitent un intérêt croissant en raison de leur potentiel pour réduire la dépendance aux intrants synthétiques. Cependant, malgré des décennies de recherche sur les inoculants microbiens du sol, leurs bénéfices sur les rendements agricoles restent très variables en fonction des sols, des climats, des génotypes végétaux et des souches inoculées, rendant les résultats difficiles à prédire et fragilisant in fine la confiance des agriculteurs. Ce travail explore la pertinence des inoculations récurrentes comme levier pour atténuer la résistance biotique de la communauté microbienne résidente du sol et favoriser l’établissement de l’inoculant. Ce travail étudie également les impacts de ces inoculations sur la communauté microbienne résidente. Dans une première expérience en microcosme, nous avons montré que l'inoculation récurrente pouvait améliorer, de manière transitoire l'abondance de l'inoculant (Pseudomonas fluorescens) avec un impact mineur sur la communauté bactérienne résidente. Une deuxième expérience en serre a mis en évidence le potentiel inattendu de l'inoculation récurrente, réalisée jusqu’au semis, pour favoriser la croissance du maïs avec un moindre impact sur la communauté bactérienne résidente par rapport à l'inoculation récurrente à partir du semis. La troisième expérience, réalisée au champ, a reflété les défis de transférer les bénéfices observés sur la croissance des plantes des conditions contrôlées vers des conditions non contrôlées. Finalement, ce travail suggère que le moment et la fréquence de l'inoculation doivent être ajustés de manière complémentaire. C’est-à-dire que l'inoculation récurrente peut augmenter temporairement l’abondance de l’inoculant lors des stades précoces critiques de la croissance des plantes. Cela peut soit favoriser la colonisation de l’hôte lorsqu’une dose adéquate est appliquée, soit influencer indirectement la communauté microbienne du sol au moment du semis<br>With the urgent need to adopt more sustainable practices that sustain agricultural production while protecting the environment, microbial inoculants are gaining increasing attention for their potential to reduce reliance on agrochemicals. However, despite decades of research, the benefits of soil microbial inoculants for crop yields remain highly variable across different soils, climates, plant genotypes, and inoculant strains. This variability makes outcomes difficult to predict and may ultimately reduce farmers’ confidence. This work explores the potential of recurrent inoculations as a strategy to overcome the biotic resistance of the resident soil microbial community and promote inoculant establishment. It also examines the effects of these inoculations on the resident microbial community. In a first microcosm experiment, we showed that recurrent inoculation could transiently improve the abundance of the inoculant (Pseudomonas fluorescens) with minimal impact on the resident bacterial community. A second experiment in greenhouse evidenced the unexpected potential of recurrent inoculation carried out until sowing to enhance maize growth while exerting a weaker impact on the bacterial resident community compared to recurrent inoculation starting at sowing. The third experiment conducted under field conditions reflected the challenges of translating the growth benefits observed in controlled environments to uncontrolled field conditions. Overall, this work suggests that both the timing and frequency of inoculation should be adjusted in a complementary way. Specifically, recurrent inoculation may transiently enhance the abundance of the inoculant during the critical early stages of plant growth. This may either promote successful host colonization when an adequate dose is applied, or indirectly influence the soil microbial community at sowing
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Johnston, David Morris. "Functional genomics of plant chitinase-like genes." Thesis, University of British Columbia, 2006. http://hdl.handle.net/2429/64.
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The Arabidopsis chitinase-like1 (Atctl1) mutant, pom1 is compromised in primary cell wall development, resulting in short roots when grown on high sucrose and shortened hypocotyls when grown in darkness. To better understand this phenotype and the evolution of AtCTL1 and its homologue, AtCTL2, we obtained a large number of CTL sequences and determined the phylogenetic relationships among them. Since microarray analysis had suggested a change in auxin response or homeostasis in pom1, I used the auxin reporter DR5::GUS in the pom1 background to assess changes in distribution. To assess whether the biochemical functions of AtCTL1 homologues in Arabidopsis and other plants are conserved, I transformed pom1 with AtCTL2 and CTLs from poplar (Populus trichocarpa x Populus deltoides clone H-11) and from Picea glauca (spruce) and assessed rescue of the pom1 phenotype. To further understand CTL expression and function, Arabidopsis and poplar CTL promoter::GUS fusions were also expressed in Arabidopsis, PopCTL1 overexpressed in Arabidopsis, and CTL expression down regulated in poplar by RNAi. Our results indicate that CTL genes represent an ancient family encoding proteins of conserved biochemical function. In dicots, represented by Arabidopsis and poplar) duplicated CTL genes are differentially expressed in conjunction with primary and secondary cell wall development, respectively. Mutation of these genes results in improperly formed primary walls in certain cell types in the case of AtCTL1, and an impairment in the differentiation of vascular bundles for AtCTL2. Overexpression of PopCTL1 in Arabidopsis seems to over stimulate the differentiation of vascular bundles, and our studies show that auxin distribution is altered in the Atctl1 mutant. Down regulation of PopCTL1 and PopCTL2 in poplar appears to phenocopy aspects of these mutations, resulting in secondary cell walls that appear to have less deposition of lignin and an accelerated production of secondary xylem respectively. While specific biochemical function(s) of CTL genes were not studied, potential functions are discussed.
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Patel, Nrupali. "Functional Analyses of Cyst Nematode Parasitism Genes." NCSU, 2008. http://www.lib.ncsu.edu/theses/available/etd-03102008-221413/.
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Cyst nematodes in the genus Heterodera are sedentary endoparasites that induce elaborate feeding cells within host roots by secreting proteins produced within nematode esophageal glands into plant cells. Functional analyses of selected cyst nematode parasitism genes that encode such secreted proteins was the objective of this dissertation. Homologs of four parasitism genes initially isolated from Heterodera glycines, including Hg4F01 (annexin-like protein), HgSYV46 (CLAVATA3-like plant peptide mimic), Hg4E02 and Hg5D08 (novel proteins with putative host nuclear localization) were isolated from Heterodera schachtii, which can infect Arabidopsis thaliana. Greater than 90% nucleotide and predicted amino acid identity existed between the four parasitism genes homologs of H. glycines and H. schachtii. mRNA in situ hybridization and immunolocalization confirmed the expression of each gene product exclusively within the nematode esophageal gland cells. Since eukaryotic annexins affect many cellular processes involving calcium-dependent membrane association, the potential function of the Hs4F01 secreted into plant cells was analyzed. Similar to annexin mutants in Arabidopsis, transgenic Arabidopsis expressing Hs4F01 produced no observable plant phenotype, but were more susceptible to nematode infection. Hypersensitivity to osmotic stress in an Arabidopsis annAt1 annexin mutant was reduced (complemented) in mutants that expressed Hs4F01, suggesting a functional similarity of nematode and plant annexins within plant cells. Host derived RNA interference (RNAi) to silence Hs4F01 transcripts significantly reduced the number of H. schachtii females developing on roots that express dsRNA to Hs4F01. Expression of Hs4E02 and Hs5D08 in Arabidopsis produced no observable plant phenotype and susceptibility to H. schachtii was not altered in plants that expressed Hs4E02. Silencing of HsSYV46 using host-derived RNAi demonstrated a significant reduction in the development of nematode females on Arabidopsis roots that expressed double-stranded RNA to HsSYV46. Expression of dsRNA to Hs4E02 and Hs5D08 in Arabidopsis roots did not affect nematode susceptibility. In summary, parasitism gene products confirmed to have cellular functions similar to their plant homologs, including Hs4F01 (annexin-like protein) and HsSYV46 (CLAVATA/ESR-like peptide) were demonstrated by RNAi to have a significant biological role in cyst nematode parasitism of host plant roots.
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Thacker, Zubin. "Functional analysis of E. coli specific genes." Thesis, University of Edinburgh, 2004. http://hdl.handle.net/1842/11457.
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The basis of my research has been to identify and characterise the function of ‘<i>E. coli</i> specific’ genes. <i>E. coli</i> specific genes were identified after comparing 33 gamma proteobacterial genomes at the Microbial Genome Database (MBGD). The database was used to investigate the change in the number of <i>E. coli</i> specific genes since the completion of the first <i>E. coli</i> genome in 1997. Forty nine selected <i>E. coli </i>specific genes were deleted in 38 separate deletion events on the genome of the model <i>E. coli </i>K-12 MG1655 using the modified pKO3 deletion procedure. Gene expression levels and patterns in various phases of growth in LB broth were measured and are reported here. Mutant and parent strains were tested for growth on a variety of conditions in agar based media. A mutant of the <i>yigE</i> ORF was found to be sensitive to high concentrations of nickel and cobalt in the growth medium. Mutation of the <i>E. coli</i> specific <i>htrC</i> gene showed no temperature sensitivity as reported in published literature and is shown here to play no part in the heat shock response of <i>E. coli. </i>The chromosomal position of the acetate utilisation gene <i>ackB</i> is experimentally demonstrated to be close to the known acetate kinase gene <i>ackA</i> at 50 minutes on the K-12 chromosome. Mutations in ORFs <i>yceP, ydeK </i>and <i>ygjMN</i> made the mutants sensitive to high levels of the basic dye gentian violet. This study shows that the group of genes specific to <i>E. coli</i> targeted here are expressed, show different patterns of expression and some are phenotypically functional. This study draws attention to a fraction of genes whose functional contribution to <i>E. coli</i> may have been underestimated due to their poor conservation in genomes other than <i>E. coli.</i>
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Waters, A. M. "Genetic and functional dissection of ciliary genes." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1395927/.
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Ciliopathy disorders are associated with either abnormal formation or function of cilia. Mutations have been described in over 60 ciliary genes to date. With the identification of over 1,000 ciliary polypeptides, other disorders exhibiting ciliopathy features could result from mutations in other ciliary genes. A new ciliopathy disease gene, CENPF, has been identified in a kindred exhibiting midgestation lethality with congenital malformations suggestive of a novel ciliopathy phenotype. Where conventional approaches such as genome-wide linkage analysis and homozygosity mapping had failed, whole exome capture coupled with massive parallel deep sequencing was successful in elucidating the genetic cause in a single affected case. Identification of compound heterozygous mutations in the causative gene was facilitated through analysis of an unfiltered approach for depth of coverage. Utilising a combinatorial approach of comparative genomics and proteomics, a novel ciliogenic role for the causative gene was identified and proposed by modelling cenpf loss of function in D. rerio and supported by interactions found with I ft 88 and Kif3b, key regulators of ciliogenesis. These data support emerging vidence for the existence of acytoplasmic dynein 1-dependent multiprotein complex which has dual roles in mitosis and ciliogenesis.
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Yu, Man. "Functional Analysis of the Cis-Regulatory Elements I56i, I56ii and I12b that Control Dlx Gene Expression in the Developing Forebrain of Mouse and Zebrafish." Thesis, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/20165.
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The vertebrate Dlx gene family consists of multiple convergently transcribed bigene clusters and encodes a group of homeodomain-containing transcription factors crucial for the development of forebrain, branchial arches, sensory organs and limbs. At least four cis-regulatory elements (CREs) are responsible for Dlx expression in the forebrain: URE2 and I12b in the Dlx1/Dlx2 (zebrafish dlx1a/dlx2a) locus, and, I56i and I56ii in the Dlx5/Dlx6 (zebrafish dlx5a/dlx6a) locus. Here, we first show that unlike the other three enhancers, mouse I56ii CRE targets a group of GABAergic projection neurons expressing striatal markers Meis2 and Islet1. Meis2 and Islet1 proteins can activate reporter gene transcription via the I56ii CRE, suggesting that they may be potential upstream regulators of Dlx genes in vivo. To determine whether there exists a dlx-mediated regulatory pathway during zebrafish GABAergic neuron formation, we establish two independent lines of transgenic fish in which the GFP reporter gene is controlled by a 1.4kb dlx5a/dlx6a intergenic sequence (encompassing zebrafish I56i and I56ii) and a 1.1kb fragment containing only I56i CRE, respectively. Our observations reveal that dlx5a/dlx6a regulatory elements exhibit a fairly specific activity in the zebrafish forebrain and may be essential for GABAergic neuron generation, while I56i and I56ii are likely to play distinct roles in modulating this process in different subpopulations of cells. Disruption of dlx1a/dlx2a or dlx5a/dlx6a function leads to a marked decrease of enhancer activity in the diencephalon and midbrain as well as a comparatively lesser extent of reduction in the telencephalon. In order to define the specific contribution of various individual CREs to overall Dlx regulation, we also generate a mutant mouse model in which I12b CRE is selectively deleted. Despite that mice homozygous for I12b loss develop normally and harbor no overt morphological defects in the forebrain, targeted deletion of this enhancer results in a significant reduction of Dlx1/Dlx2 transcript levels and seemingly perturbs cell proliferation in the subpallial telencephalon, particularly in the ventricular and subventricular zones of ganglionic eminences. Taken together, these data illustrate a complex and dynamic Dlx regulation in the early developing forebrain through the implications of multiple Dlx CREs with overlapping and diverse functions.
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Sung, Young Kwan. "Functional analysis of potato yellow mosaic geminivirus genes." Thesis, Imperial College London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321667.
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Stival, Sena Juliana. "Structural and functional evolution of genes in conifers." Doctoral thesis, Université Laval, 2017. http://hdl.handle.net/20.500.11794/27679.
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Le développement de nouvelles techniques a accéléré l'exploration structurale et fonctionnelle des génomes des conifères et contribué à l’étude de leur physiologie et leur adaptation aux conditions environnementales. Cette thèse s’intéresse à l’évolution des gènes chez les conifères et (i) fait le point sur les facteurs génomiques qui ont influencé la structure des gènes et (ii) analyse une grande famille de gènes impliqués dans la tolérance à la sécheresse, les déhydrines. Notre étude de la structure génique s’est fait à partir de diverses séquences de l’épinette blanche (Picea glauca [Moench] Voss) provenant de clones BAC, de l'assemblage du génome et de l’espace génique obtenu à partir de la technologie de «sequence capture». Par le biais d’analyses comparatives, nous avons observé que les conifères présentent plus de séquences introniques par gène que la plupart des plantes à fleurs (angiospermes) et que la longueur moyenne des introns n'était pas directement corrélée à la taille du génome. Nous avons constaté que les éléments répétitifs qui sont responsables de la très grande taille des génomes des conifères affectent également l'évolution des exons et des introns. Dans la deuxième partie de la thèse, nous avons entrepris la première analyse exhaustive de la famille des gènes des déhydrines chez les conifères. Les analyses phylogénétiques ont indiqué l'apparition d'une série de duplications de gènes dont une duplication qui a provoqué l'expansion de la famille génique spécifiquement au sein du genre Picea. L’analyse démontre que les déhydrines ont une structure modulaire et présentent chez les conifères des agencements variés de différents motifs d'acides aminés. Ces structures sont particulièrement diverses chez l'épinette et sont associées à différents patrons d'expression en réponse à la sècheresse. Dans l’ensemble, nos résultats suggèrent que l'évolution de la structure génique est dynamique chez les conifères alors que l'évolution des chromosomes est largement reconnue comme étant lente chez ceux-ci. Ils indiquent aussi que l'expansion et la diversification des familles de gènes liés à l'adaptation, comme les déhydrines, pourraient conférer de la plasticité phénotypique permettant de répondre aux changements environnementaux au cours du long cycle de vie qui est typique de plusieurs conifères.<br>Technical advances have accelerated the structural and functional exploration of conifer genomes and opened up new approaches to study their physiology and adaptation to environmental conditions. This thesis focuses on the evolution of conifer genes and explores (i) the genomic factors that have impacted the evolution of gene structure and (ii) the evolution of a large gene family involved in drought tolerance, the dehydrins. The analysis of gene structure was based on white spruce (Picea glauca [Moench] Voss) sequence data from BAC clones, the genome assembly and the gene space obtained from sequence capture. Through comparative analyses, we found that conifers presented more intronic sequence per gene than most flowering plants (angiosperms) and that the average intron length was not directly correlated to genome size. We found that repetitive elements, which are responsible for the very large size of conifer genomes, also affect the evolution of exons and introns. In the second part of the thesis, we undertook the first exhaustive analysis of the dehydrin gene family in conifers. The phylogenetic analyses indicated the occurrence of a series of gene duplications in conifers and a major lineage duplication, which caused the expansion of the dehydrin family in the genus Picea. Conifer dehydrins have an array of modular amino acid structures, and in spruce, these structures are particularly diverse and are associated with different expression patterns in response to dehydration stress. Taken together, our findings suggest that the evolution of gene structure is dynamic in conifers, which contrast with a widely accepted slow rate of chromosome evolution. They further indicate that the expansion and diversification of adaptation-related genes, like the dehydrins in spruce, may confer the phenotypic plasticity to respond to the environmental changes during their long life span.
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Wu, Yu. "Identification and Functional Characterization of Adipogenesis-related Genes." University of Toledo Health Science Campus / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=mco1229546422.
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Contos, James John Achilles. "Genomic and functional characterization of lysophospholipid receptor genes /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1999. http://wwwlib.umi.com/cr/ucsd/fullcit?p9945772.
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Xue, Song. "Functional analysis of sucrose synthase genes in maize." [Gainesville, Fla.] : University of Florida, 2004. http://purl.fcla.edu/fcla/etd/UFE0007380.
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Thacker, G. "Functional analysis of C.jejuni genes encoding putative peptidases." Thesis, London School of Hygiene and Tropical Medicine (University of London), 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.536843.
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Guo, Xiumei. "Regulatory and functional study of human cytoglobin." Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/hkuto/record/B3860145X.
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Zhang, Hao. "Functional characterization of ORF slr0813 in cyanobacterium synechocystis PCC 6803 /." View Abstract or Full-Text, 2002. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202002%20ZHANG.
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Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002.<br>Includes bibliographical references (leaves 196-212). Also available in electronic version. Access restricted to campus users.
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Lestari, Retno. "Functional analysis in Hevea brasiliensis of the HbERF-IXc4 and HbERF-IXc5 genes, two potential orthologs Arabidopsis ERF1 gene." Thesis, Montpellier, SupAgro, 2016. http://www.theses.fr/2016NSAM0042/document.
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Le caoutchouc naturel (CN), a cis-1,4-polyisoprene, est produit principalement par Hevea brasiliensis. Le CN est un matériau très important pour l’industrie du transport et médicale. La demande en CN augmente d’année en année. Le CN est obtenu à partir du latex. Le latex s’écoule des laticifères après saignée de l’écorce des hévéas. L’éthéphon, un libérateur d’éthylène, peut être appliqué sur certains clones d’hévéa pour stimuler la production de latex. La saignée et la stimulation à l’éthéphon sont des stress de récolte conduisant à la production de métabolites secondaires et par conséquence au caoutchouc. La biosynthèse et la signalisation de l’éthylène (ET) et de l’acide jasmonique (JA) jouent un rôle crucial dans la réponse aux stress de récolte. Deux gènes codant des facteurs de réponse à l’éthylène (ethylene response factor, ERF), HbERF-IXc4 et HbERF-IXc5, ont été prédits être orthologue à ERF1 d’Arabidopsis. ERF1 est considéré comme un facteur clé de la réponse de défense à travers l’intégration des voies de signalisation de l’éthylène et du jasmonate. Les transcrits de HbERF-IXc4 et HbERF-IXc5 s’accumulent dramatiquement en réponse à des traitements combinant la blessure, le méthyl jasmonate, et l’éthylène. Ces facteurs sont ainsi supposés être des régulateurs clés au croisement des voies de signalisation de l’éthylène et du jasmonate dans les laticifères. HbERF-IXc4 et HbERF-IXc5 ont plusieurs caractéristiques des facteurs de transcription révélés respectivement lors des expériences de trans-activation et de localisation subcellulaire : ils peuvent activer des éléments GCC agissant en cis des promoteurs des gènes cibles et ils sont présents au niveau du noyau.Dans cette étude, l’analyse fonctionnelle des gènes HbERF-IXc4 et HbERF-IXc5 a été effectuées par sur-expression de ces gènes sous le contrôle de deux promoteurs, 35S CaMV et HEV2.1 dans des lignées transgéniques d’Hevea obtenues par transformation génétique via Agrobacterium tumefaciens. Cette sur-expression a conduit à augmenter les effets des gènes natifs HbERF-IXc4 et HbERF-IXc5. Vingt-neuf lignées à activité GFP ont été sélectionnées sur un milieu contenant de la paromomycine. Douze lignées ont permis régénérées des plantes mais seulement dix ont produit un nombre suffisant de plantes pour réaliser les observations de phénotypage avec au total 1622 plantes transgéniques acclimatées en serre. Ces dix lignées transgéniques ont été confirmées par hybridation moléculaire de type Southern. L’observation morphologique des plants jusqu’à un an montre que les deux gènes (HbERF-IXc4 and HbERF-IXc5) favorisent une meilleure croissance, en termes de hauteur des plants, du diamètre des tiges, et du poids frais et sec des parties aériennes et racinaires, avec une plus forte vigueur et tolérance aux stress abiotiques. Les plants sur-exprimant HbERF-IXc5 ont aussi une meilleure performance que ceux sur-exprimant HbERF-IXc4. Ces résultats montrent aussi un système racinaire plus vigoureux et bien équilibré par rapport à la plante entière. Les analyses de RT-PCR en temps réel révèlent que l’expression des gènes HbERF-IXc4 et HbERF-IXc5 est plus forte dans les lignées transgéniques que la lignée sauvage. L’analyse fine des lignées HbERF-IXc5 montre aussi des modifications anatomiques (activité cambiale, nombre de cellules laticifères, amidon, et largeur du xylème).Ce travail est la première analyse fonctionnelle de facteurs de transcription chez Hevea. Des différences ont été observées entre les lignées HbERF-IXc4 et HbERF-IXc5. Comme ERF1, HbERF-IXc4 et HbERF-IXc5 doivent diriger la réponse à certains stress. HbERF-IXc5 serait un régulateur de la différentiation des laticifers. Cette étude pourrait être complétée par des analyses dans des lignées éteintes pour ces gènes, une comparaison des transcriptomes et métabolome de lignées sauvages et transgéniques, et l’identification des gènes cibles contrôlés par HbERF-IXc4 et HbERF-IXc5<br>Natural rubber (NR) (cis-1,4-polyisoprene) is the main production from Hevea brasiliensis. NR is a very important industrial material for transportation, consumer, and medical. The demand for NR is increasing from year to year. NR is obtained from latex. The latex flows out from laticifers after tapping the bark. Ethephon, an ethylene releaser, can be applied on certain clones to stimulate the latex production. Tapping and ethephon stimulation are sources of harvesting stresses conducing to the production of secondary metabolites and consequent rubber. Ethylene (ET) and jasmonic acid (JA) biosynthesis and signalling pathways play a crucial role in the response to latex harvesting stress. Two Hevea ethylene response factor genes, HbERF-IXc4 and HbERF-IXc5, were predicted to be orthologue to ERF1 from Arabidopsis. ERF1 was suggested to be a key component of defence responses through the integration of ethylene and jasmonic acid signalling pathways. Transcripts of HbERF-IXc4 and HbERF-IXc5 were dramatically accumulated by combining wounding, methyl jasmonate, and ethylene treatment. These factors were assumed to be a key regulator at the crosstalk of ethylene and jasmonate signalling pathways in latex cells. HbERF-IXc4 and HbERF-IXc5 have several features of transcription factor revealed by transactivation experiment and subcellular localization, respectively: they can activate the GCC cis-acting element of promoters of target genes and are localized in nucleus, In this study, functional analysis of HbERF-IXc4 and HbERF-IXc5 genes have been carried out by overexpression under control of two promoters, 35S CaMV and HEV2.1 in transgenic Hevea lines obtained by Agrobacterium tumefaciens-mediated genetic transformation. This overexpression led to emphasize the effect of native HbERF-IXc4 and HbERF-IXc5 genes. Twenty-nine GFP-positive lines were established on paromomycin selection medium. Twelve lines regenerated plants but only ten led to produce a sufficient number of plants for further phenotyping with totally 1,622 transgenic plants in greenhouse. These ten ines were confirmed as transgenic by Southern blot hybridization. Observation of morphology until one year showed both genes (HbERF-IXc4 and HbERF-IXc5) promoted a better growth in terms of plant height, stem diameter, and weight of aerial and root system with higher vigour and better tolerance to some abiotic stresses. Plants overexpressing HbERF-IXc5 have also a better performance than HbERF-IXc4. Data also showed a vigorous root system well balanced with regard to the whole plant. Real-time RT-PCR analyses revealed that expression of HbERF-IXc4 and HbERF-IXc5 genes was higher in transgenic lines compared to wild-type . Analysis in details of HbERF-IXc5 lines also showed some changes in anatomy (cambium activity, number of latex cells, starch, and width of xylem).This work is the first successful functional analysis of transcription factors in Hevea. Some differences have been observed between HbERF-IXc4 and HbERF-IXc5. As ERF1, HbERF-IXc4 and HbERF-IXc5 should drive the response to some stresses. HbERF-IXc5 might be a regulator of laticifer differentiation. This study could be completed with analysis of silenced transgenic lines, comparison of transcriptome, metabolome of wild-type and transgenic lines, and identification of target genes controlled by HbERF-IXc4 and HbERF-IXc5
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Sawers, Ruairidh J. H. "Functional analysis of bundle sheath defective2." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342541.
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Gyoergy, Andras. "Functional modularity in gene networks." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/103726.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2016.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (pages 141-150).<br>This thesis addresses two sources of context-dependence in both systems and synthetic biology: retroactivity and competition for shared cellular resources. The contribution is the development of simple-to-use computational tools that aide the analysis and design of multi-module genetic systems. These tools are a result of combining mathematical modeling and theoretical analysis with experiments performed in Escherichia coli. While current approaches most often neglect to account for context-dependence in living systems, experimental evidence demonstrates that such effects have profound influence on system behavior. As a result, modules developed separately are likely to behave differently from predicted, so that they need to be redesigned through a lengthy and ad hoc process every time they are inserted into a different system. To overcome this major limitation, in this thesis I expand the description of gene circuits. First, the description of modules is appended by quantities similar to input and output impedance in electrical networks theory. Second, the description of each protein is appended by a quantity characterizing the amount of resources that are sequestered for its production. As a result, the behavior of modules upon interconnection becomes predictable, facilitating both the rational design of synthetic circuits and furthering our understanding of natural systems. Application examples are considered, which include the design of oscillators and toggle switches, network identification problems, and standard metabolic optimization problems, such as maximizing reaction rates catalyzed by multiple enzymes and maximizing the steady state concentration of heteromultimer complexes.<br>by Andras Gyoergy.<br>Ph. D.
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RIGAMONTI, AURORA. "Functional characterization of SMARCA2 gene." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2016. http://hdl.handle.net/10281/130275.
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My PhD project is based on the characterization of the SMARCA2 gene. On one hand, I focused my attention on the role of SMARCA2 product, the protein brahma (BRM), in the regulation of alternative pre-mRNA splicing. On the other hand, I investigated the transcriptional regulation of SMARCA2 expression.
My first project derived from a comparative evaluation and validation of microarray data from two mitochondrial stress models. The first model represented by an acute mitochondrial stress is constituted by human SH-SY5Y neuroblastoma cells treated with Paraquat (PQ), while the second model is a chronic model constituted from the same cell line stably overexpressing the Superoxide Dismutase 1 carrying the most common mutation found in familiar ALS (SOD1 G93A). The merge of these microarrays data showed that oxidative stress affects the choice of specific alternative last exons (ALEs) increasing the production of transcripts variants terminating at a more proximal ALE. Moreover, oxidative stress induces the transcriptional downregulation of the SMARCA2 gene product BRM, one of the two alternative ATPase subunits of the SWI/SNF complex. I found that in normal condition BRM is enriched on the proximal ALE. In addition, I observed the accumulation of BARD1, a protein that forms a functional heterodimer with BRCA1, which has E3 ubiquitin-ligase activity and interacts with the 50 kDa subunit of CstF inhibiting 3’ end processing. Consistent with these observations, I detected an ubiquitinated pool of CstF50 and showed that ubiquitination is mediated by BARD1/BRCA1. Taken together, these results suggested that the presence of BRM on the proximal exon leads to the BARD1/BRCA1-mediated ubiquitination of CstF50 and the inhibition of 3’ end processing at the proximal poly(A). This in turn allows transcription to proceed to the distal terminal exon.
In the same microarray data used as a starting point for my first project we detected a shift in SMARCA2 expression towards shorter mRNA isoforms upon oxidative stress. Thus, my second project dealt with the characterization of these transcripts. Bioinformatic analysis revealed that the shorter mRNA variants are evolutionarily conserved and are most likely generated from an internal promoter. Interestingly, in zebrafish the short isoform is produced as an independent gene on the same chromosome of the long isoform but in its reverse strand. This peculiar genomic organization hints to a potentially relevant function for this alternative isoform of the BRM protein. Bioinformatic analyses revealed that the short isoform encode a protein that lacks the N-terminal, catalytic ATP-ase domain but shares the C-terminal region that contains a Bromodomain, a protein motif that is known to bind to acetylated histones.
First, I identified the potential alternative promoter region using bioinformatic tools and cloned this region. Using a luciferase reporter system I demonstrated the existence of the alternative promoter. Next, I cloned the short, most conserved isoform and I tested it for interaction with known partners of the full-length BRM protein by Co-Immunoprecipitation (Co-IP). I discovered that BRM-s interacts with histone H3 but not with core component of the SWI/SNF complex. Considering that short isoform does not display an ATPase domain, the Co-IP suggests a possible “dominant negative” role for this protein. If short isoforms works as negative dominant of Brm, the ratio between long and short expressed proteins could become very important for BRM target genes in development and differentiation. In particular, this alteration of ratio could have a negative effect on the cells since several tumor cell lines show a very low level of the long protein. (i.e. human lung tumor cell lines).
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Svingen, Terje, and n/a. "Hox Transcription Factors: Their Involvement in Human Cancer Cells and In Vitro Functional Specificity." Griffith University. School of Biomolecular and Biomedical Science, 2005. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20050830.135356.
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Hox genes are regulatory genes encoding small proteins containing a highly conserved 61-amino acid motif, the homeodomain, that enables Hox proteins to bind to DNA at specifically recognised binding sites and transcriptionally activate their target genes. In mammalian species there are 39 Hox genes and they are structural and functional homologs of the Drosophila homeotic complex (Horn-C). During embryogenesis and early development the Hox genes are expressed in a spatiotemporal fashion, where they operate as master transcriptional regulators. Hox genes are further expressed in fully differentiated adult cells, potentially in a tissue-specific manner involving maintenance of the normal phenotype. In selected oncogenic transformations, dysregulated Hox gene expression has been observed, indicating an involvement of these transcriptional regulators in carcinogenesis and metastasis. Utilising quantitative real-time PCR assays, these studies investigated the expression patterns of 20 Hox genes and two wellcharacterised Hox cofactors (Pbx and Meis) in malignant and non-malignant human breast and skin cancer cells. Dysregulated Hox expression was observed for all malignancies tested, of which some misexpressed Hox genes seemed random, whereas other Hox transcripts showed altered levels potentially corresponding with the invasive capacity of the cells. Also, the Hox cofactors Pbx and Meis showed no marked changes in expression levels from the non-malignant to the malignant phenotypes, indicating that it is dysregulated Hox gene expression rather than dysregulated gene expression of Hox cofactors that potentially commit the cell to redifferentiate and undergo oncogenic transformation. Although the Hox proteins are known to be key transcriptional regulators of development, the mechanisms by which they gain their in vivo functional specificity is still largely unknown. They all show strikingly similar transcriptional specificity in vitro, yet show unique specificity in their in vivo environment. This paradox has been the subject of intense scrutiny, however very few direct Hox target genes have been identified, making it a difficult task to decipher the exact manner in which Hox proteins exert their functional potential. Therefore, the studies presented herein were aimed at identifying further Hox target genes in the human system. Utilising differential display approaches, several potential downstream target genes were isolated. Substantiated with real-time PCR assays, one of these potential targets was selected as a likely direct Hox gene target, and as such subjected to further studies. By the combination of bioinformatic analyses, transfection protocols and luciferase assays, a gene encoding the SR-related protein SRrpl3O was shown to be trans-activated in vitro by HOXD4 via a putative Hox binding element within its promoter region. This is the first reported link between Hox transcription factors and the SR and SR-related family of pre-mRNA splicing proteins, offering a new and exciting insight into the complex nature of Hox functional specificity. Finally, this thesis also puts forward new ideas regarding how the Hox proteins gain their transcriptional and functional specificity. Utilising bioinformatic tools in conjunction with performing an extensive review of the disparate catalogue of Hox-related research reports, work herein offers the first comprehensive analysis of the mammalian Hox gene targets in relation to their promoter structures, as well as with respect to the expanded Hox DNA-binding elements. This work reports that identified Hox targets generally contain TATA-less core promoters, many of which have several GC-box elements. The Hox binding elements show no apparent preference regarding their location relative to the transcription start site (TSS), as they are found both upstream and downstream of the TSS, as well as being located close to proximal core promoter elements for some genes and at more distant positions in other gene promoters. Finally, the core Hox binding element TAAT/ATTA contains only part of the necessary recognition sequence involved in Hox-DNA binding, and the notion that flanking base pairs dictate trans-regulatory potential is further explored with the hypothesis that the immediate 3' base pair dictates an activator/repressor-switch of the Hox trans-regulatory effect.
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Svingen, Terje. "Hox Transcription Factors: Their Involvement in Human Cancer Cells and In Vitro Functional Specificity." Thesis, Griffith University, 2005. http://hdl.handle.net/10072/365774.
Full textAbstract:
Hox genes are regulatory genes encoding small proteins containing a highly conserved 61-amino acid motif, the homeodomain, that enables Hox proteins to bind to DNA at specifically recognised binding sites and transcriptionally activate their target genes. In mammalian species there are 39 Hox genes and they are structural and functional homologs of the Drosophila homeotic complex (Horn-C). During embryogenesis and early development the Hox genes are expressed in a spatiotemporal fashion, where they operate as master transcriptional regulators. Hox genes are further expressed in fully differentiated adult cells, potentially in a tissue-specific manner involving maintenance of the normal phenotype. In selected oncogenic transformations, dysregulated Hox gene expression has been observed, indicating an involvement of these transcriptional regulators in carcinogenesis and metastasis. Utilising quantitative real-time PCR assays, these studies investigated the expression patterns of 20 Hox genes and two wellcharacterised Hox cofactors (Pbx and Meis) in malignant and non-malignant human breast and skin cancer cells. Dysregulated Hox expression was observed for all malignancies tested, of which some misexpressed Hox genes seemed random, whereas other Hox transcripts showed altered levels potentially corresponding with the invasive capacity of the cells. Also, the Hox cofactors Pbx and Meis showed no marked changes in expression levels from the non-malignant to the malignant phenotypes, indicating that it is dysregulated Hox gene expression rather than dysregulated gene expression of Hox cofactors that potentially commit the cell to redifferentiate and undergo oncogenic transformation. Although the Hox proteins are known to be key transcriptional regulators of development, the mechanisms by which they gain their in vivo functional specificity is still largely unknown. They all show strikingly similar transcriptional specificity in vitro, yet show unique specificity in their in vivo environment. This paradox has been the subject of intense scrutiny, however very few direct Hox target genes have been identified, making it a difficult task to decipher the exact manner in which Hox proteins exert their functional potential. Therefore, the studies presented herein were aimed at identifying further Hox target genes in the human system. Utilising differential display approaches, several potential downstream target genes were isolated. Substantiated with real-time PCR assays, one of these potential targets was selected as a likely direct Hox gene target, and as such subjected to further studies. By the combination of bioinformatic analyses, transfection protocols and luciferase assays, a gene encoding the SR-related protein SRrpl3O was shown to be trans-activated in vitro by HOXD4 via a putative Hox binding element within its promoter region. This is the first reported link between Hox transcription factors and the SR and SR-related family of pre-mRNA splicing proteins, offering a new and exciting insight into the complex nature of Hox functional specificity. Finally, this thesis also puts forward new ideas regarding how the Hox proteins gain their transcriptional and functional specificity. Utilising bioinformatic tools in conjunction with performing an extensive review of the disparate catalogue of Hox-related research reports, work herein offers the first comprehensive analysis of the mammalian Hox gene targets in relation to their promoter structures, as well as with respect to the expanded Hox DNA-binding elements. This work reports that identified Hox targets generally contain TATA-less core promoters, many of which have several GC-box elements. The Hox binding elements show no apparent preference regarding their location relative to the transcription start site (TSS), as they are found both upstream and downstream of the TSS, as well as being located close to proximal core promoter elements for some genes and at more distant positions in other gene promoters. Finally, the core Hox binding element TAAT/ATTA contains only part of the necessary recognition sequence involved in Hox-DNA binding, and the notion that flanking base pairs dictate trans-regulatory potential is further explored with the hypothesis that the immediate 3' base pair dictates an activator/repressor-switch of the Hox trans-regulatory effect.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Biomolecular and Physical Sciences<br>Full Text
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Hoffmann, Eva R. "Functional analysis of MLH1." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.249558.
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Man, Kwun-nok Mimi. "Functional implications of cytoglobin, a novel protein, in liver fibrosis." Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/hkuto/record/B38820730.
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Linnenbrink, Miriam [Verfasser]. "Population genetic and functional analysis of the B4galnt2 gene in the genus Mus (Rodentia; Muridae) / Miriam Linnenbrink." Kiel : Universitätsbibliothek Kiel, 2012. http://d-nb.info/1053683189/34.
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Garrett, Christine. "Functional mapping of pea legumin upstream regulatory elements using TI plasmid vectors." Thesis, Durham University, 1991. http://etheses.dur.ac.uk/6041/.
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The leg A gene from Pisum sativum L. has been extensively characterised and a distinct pattern of developmental and organ-specific gene expression demonstrated. Homology between legumin genes from other species has given some indication of those sequences which may be responsible for the regulation at the level of transcription. This study was designed to provide a functional analysis of the upstream sequences. A number of plasmid vectors containing a maximum of 1.2 kb of upstream sequence from the leg A gene of Pisum sativum I., ligated to the coding region of the nopaline synthase (nos) gene, were constructed. The use of smaller promoter fragments and the insertion of spacer DNA within the promoter region was employed in an effort to localise the regions of 5' flanking sequence which may play a role in tissue specific expression. In a minority of tumours derived from tissue transformed with the vector containing the ' full-length ' leg A promoter, low levels of nopaline were detected, but not with those containing a shorter promoter fragment. Results from the analysis of Seed tissue indicates that 800 bp of the leg A promoter was insufficient to direct tissue-specific expression of the fused nopaline synthase gene in transgenic Nicotiana tabacum, although one individual plant showed a constitutive pattern of nopaline synthesis. However, published results obtained with legumin and other storage protein gene promoters would suggest that this promoter fragment should have been sufficient to confer seed-specific expression. This suggests that there may have been undesirable secondary structures, or small undetected rearrangements, introduced during the construction of the transcriptional fusions between leg A and nos. Alternatively the marker gene may be inadequately sensitive to permit detection of low levels of expression.
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Nganvongpanit, Korakot. "Functional analysis of genes during bovine preimplantation embryo development." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=980875153.
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Lee, Heon-Jin. "Functional analysis of the murine genes, MOCS1 and Sox15." [S.l.] : [s.n.], 2003. http://webdoc.sub.gwdg.de/diss/2003/lee/lee.pdf.
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Akhavantabasi, Shiva. "Functional Characterization Of Two Potential Breast Cancer Related Genes." Phd thesis, METU, 2012. http://etd.lib.metu.edu.tr/upload/12614275/index.pdf.
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Cancer may arise as a result of deregulation of oncogenes and/or tumor suppressors. Although much progress has been made for the identification of such cancer related genes, our understanding of the complex tumorigenesis pathways is still not complete. Therefore, to improve our understanding of how certain basic mechanisms work in normal and in cancer cells, we aimed to characterize two different breast cancer related genes. First part of the study focused on subcellular localization USP32 (Ubiquitin Specific Protease 32) to help understand the function of this uncharacterized gene. USP32 is a member of deubiquitinating enzymes (DUBs) and the gene maps to a gene rich region on 17q23. Genes on 17q23 are known to undergo amplification and overexpression in a subset of breast cancer cells and tumors. DUBs are known to be implicated in a variety of cellular functions including protein degradation, receptor endocytosis and vesicle trafficking. Therefore to elucidate the function of USP32, we localized the full length USP32 protein fused to GFP, in HeLa cells, using Fluorescence Protease Protection (FPP) assay and confocal microscopy. Results suggested a Golgi localization for USP32 as confirmed by co-localization study via BODIPY-TR, a Golgi specific marker. Additional investigations to find the role of USP32 in Golgi will further clarify the function of this candidate oncogene. Second part of the study focused on a potential tumor suppressor. For this purpose, we functionally characterized miR-125b, a microRNA gene as a potential tumor suppressor in breast cancer. microRNAs are regulators of gene expression and their deregulation is detected in cancer cells. miR-125b is reported as a down regulated microRNA in breast cancers. In this study, we investigated the expression, function and possible targets of miR-125b in breast cancer cell lines (BCCLs). Our results revealed a dramatic down regulation of miR-125b in a panel of BCCLs. Restoring the expression of miR-125b in low miR-125b expressing cells decreased the cell proliferation and migration as well as cytoplasmic protrusions, detected by staining of actin filaments. While connection of miR-125b and cell motility based on ERBB2 targeting has been reported earlier, here we present data on ERBB2 independent effects of miR-125b on cell migration in non-ERBB2 overexpressing breast cancer cells. Our results showed involvement of a miR-125b target, ARID3B, in cell motility and migration. Our findings showed miR-125b to be an important regulator of cell proliferation and migration in ERBB2 negative breast cancer cells, possibly through regulating multiple targets.
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Thorsteinsdottir, Unnur. "Functional analysis of selected Hox homeobox genes in hematopoiesis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq25175.pdf.
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Feiz, Leila. "Functional analysis of puroindoline genes in wheat (Triticum aestivum)." Thesis, Montana State University, 2008. http://etd.lib.montana.edu/etd/2008/feiz/FeizL1208.pdf.
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Grain hardness variation has large effects upon many different end-use properties of wheat (Triticum aestivum). The Hardness (Ha) locus consisting of the Puroindoline a and b genes (Pina and Pinb) controls the majority of grain hardness variation. Starch production is a growing end-use of wheat. The first objective of this study was to estimate the differences in starch yield due to natural and transgenically conditioned grain hardness differences. To accomplish this goal, a small scale wet milling protocol was used to characterize the wet milling properties of two independent groups of isogenic materials varying in grain hardness and in Pin expression level. The results of the first study demonstrate that the Ha locus and puroindoline expression are both linked to wet-milling starch yield and that selection for increased Ha function increases starch yield via enhanced separation of starch granules and the protein matrix during wet milling. The lack of Pin allelic diversity is a major factor limiting Ha functional analyses and wheat quality improvement. So the second objective of this study was to create new Ha alleles in the soft white spring cultivar Alpowa using ethylmethane sulfonate (EMS) mutagenesis. The M 2 population was screened to identify new alleles of Pina and Pinb. One hundred and forty eight new Pin alleles, including 68 missense alleles, were identified. F 2 populations for 49 of the new Pin alleles including 43 unique missense ones were developed after crossing each back to non-mutant Alpowa. Grain hardness was then measured on F 2:3 seeds and the impact of each allele on grain hardness was quantified. The tested mutations comprised a range of functionality from neutral to function abolishing mutations. Seed weight and vigor of all mutation lines was restored among all of the F 2 populations. The new alleles have the potential to improve end use properties of soft and hard wheats.
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Carter, D. P. F. "Long-range functional interactions between genes and regulatory elements." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597329.
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The mouse b-globin locus contains four genes that are expressed throughout development in the same order they are arranged along chromosome 7. Upstream of the genes is a powerful regulatory element called the locus control region (LCR). The LCR is absolutely required for high-level expression of the b-globin genes. The models to explain how LCRs (and enhancers in general) are able to activate expression of the genes fall into two categories: contact and non-contact. The contact models propose that the LCR and the gene form a physical interaction that leads to active transcription. The non-contact models propose that the LCR alters the chromatin topology or nuclear organisation of the locus, or acts as a nucleation point for factors that polymerise along the chromatin fibre to activate the gene. A contact model has been inferred from indirect observations, <i>in vitro</i> experiments, and oversimplified assumptions. In the absence of a physical assay that is able to determine the three-dimensional arrangements of the b-globin locus the mechanisms of LCR activation remains open to speculation. In this thesis I describe the development of a novel technique called RNA tagging and recovery of associated proteins (RNA TRAP). RNA TRAP uses the power of RNA FISH to target an enzyme to the site of a specific actively transcribing gene. The enzyme catalayses the deposition of a biotin tag onto the chromatin in the vicinity of transcription, which acts as a handle to isolate and identify chromatin elements in proximity to the actively transcribed gene. Using RNA TRAP I have shown that elements of the LCR are in significantly physical proximity to the genes they activate <i>in vivo.</i> This is the first physical demonstration of the interaction of the LCR with a gene, and gives insight into the regulation of genes by enhancers.
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Beswick, Richard William. "Functional characterisation of the genes mutated in dyskeratosis congenita." Thesis, Queen Mary, University of London, 2013. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8705.
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Dyskeratosis congenita (DC) is a multi system disorder that exhibits considerable clinical and genetic heterogeneity. It is characterised by mucocutaneous features, bone marrow failure and a predisposition to cancer. Research has identified mutations affecting several telomerase components and patients often have short telomeres, implicating defective telomere maintenance in this disease. Affected components include dyskerin, NOP10 and NHP2, which together with GAR1 form a protein core common to telomerase and all other H/ACA ribonucleoprotein complexes (H/ACA RNPs). Initially characterised as H/ACA RNP components important for pseudouridylation and rRNA processing, their role in the functionally distinct telomerase complex and telomere maintenance is less defined. In order to better understand their implications in DC, this study investigated the importance of these core proteins for the integrity and function of telomerase in human cells. RNAi knockdown studies demonstrated that dyskerin, NOP10 and NHP2 are necessary for the accumulation of TERC (telomerase RNA component); dyskerin and NOP10 for telomerase activity. Moreover, dyskerin was found to be important for maintaining telomere length over time. The impact of NOP10 and NHP2 missense mutations was also analysed in vitro, which indicated that they impair TERC accumulation. The potential effect on pseudouridylation was also considered in this study; the analysis of other H/ACA RNA levels in these knockdown experiments and in a cohort of patients with DKC1 mutations revealed an irregular and inconsistent impact compared to that observed on TERC. Finally, defective telomere maintenance is heavily implicated as the primary cause of DC and very short telomeres have been proposed as a diagnostic marker. This study investigated telomere length in a patient cohort of unprecedented size. It demonstrated the prevalence of the telomere length defect, but telomere length was not found to correlate with either genetic subtype or disease severity, implicating the rate of telomere shortening as the correlating factor instead.
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Alvarez-Saavedra, Ezequiel (Ezequiel Andrès). "Functional analysis of the microRNA genes of C. elegans." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/42948.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.<br>This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.<br>Includes bibliographical references (p. 231-252).<br>MicroRNAs (miRNAs) were discovered in C. elegans during studies of the control of developmental timing. MicroRNAs are a large class of short non-coding RNAs found in many viruses, plants and animals that regulate gene expression through sequence-specific base-pairing with target mRNAs. Initial studies since the identification of many miRNAs only six years ago, have revealed their very diverse roles in biology. Yet, few miRNAs have been studied using loss-of-function mutations. We have generated deletion mutations in 87 miRNA genes in C. elegans, and performed an initial characterization of the 95 miRNA mutants available (86% of known C. elegans miRNAs). We found that the majority of miRNAs are not essential for the viability or development of C. elegans, and mutations in most miRNA genes do not result in grossly abnormal phenotypes. Within species, many miRNAs can be grouped into families according to their sequence similarities. We generated a collection of 12 multiply mutant C. elegans strains that each lacks an entire miRNA family. We found that at least four families display synthetic abnormalities, indicating that miRNAs within a family can have redundant functions. While single mutants are superficially wild-type, mutants deleted for all members of the mir-35 or the mir-51 families show embryonic or early larval lethality, mutants deleted for all members of the mir-58 family show an egglaying defect, and mutants deleted for some members of the let-7 family show defects in developmental timing. We developed a microarray technology suitable for detecting microRNAs and used this microarray to determine the profile of microRNAs expressed in the developing mouse brain. We observed a temporal wave of expression of microRNAs, suggesting that microRNAs play important roles in the development of the mammalian brain.<br>(cont.) We also performed a systematic expression analysis of 334 samples covering diverse human cancers, using a bead-based flow cytometric miRNA expression profiling method we developed. The miRNA profiles reflect the developmental lineage and differentiation state of the tumors, and reveal a general down-regulation of miRNAs in tumors compared to normal tissues.<br>by Ezequiel Alvarez-Saavedra.<br>Ph.D.
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Fairclough, Victoria Ruth. "Functional Analysis of Novel Essential Genes of Staphylococcus aureus." Thesis, University of Sheffield, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.522041.
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Bebek, Gurkan. "Functional Characteristics of Cancer Driver Genes in Colorectal Cancer." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1495012693440067.
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Challa, Anil Kumar. "Identification and functional analysis of Zebrafish orthologs of genes." Connect to this title online, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1061302731.
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Thesis (Ph. D.)--Ohio State University, 2003.<br>Document formatted into pages; contains 119 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2005 Aug. 19.