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Characterization of psychrophilic alleles of essential genes as means of generating temperature-sensitive strains of mesophilic organismsPankowski, Jaroslaw 13 April 2016 (has links)
Essential genes are involved in control of the basic metabolism of their host. These genes encode elements involved is such crucial processes as DNA replication, transcription, translation or biosynthesis of important molecules. What makes essential genes unique is the fact that they cannot be lost from the genome. If any of them becomes inactivated it would result in inevitable death of an organism. Because of their role they can be efficiently used to control the survival of genetically modified organisms. Specific regulatory mechanisms can be applied to modulate the activity of essential genes, which prevents an organism from growing at determined conditions. Such mechanisms are called “kill switches” and have been developed in recent years as a response to significant development in the field of molecular biology.
Proteins encoded by psychrophilic organisms are characterized by decreased resistance to thermal denaturation. This is believed to be a result of adaptation to low-temperature environment, where mutations that destabilize the protein structure are not selected against. For these reasons they often cannot perform their functions at moderate temperatures, which are typical for mesophilic organisms. At the same time psychrophilic proteins do not display any inhibition at permissive conditions.
Use of psychrophilic alleles of essential genes has been proposed as a method of rendering modified organisms incapable of surviving at elevated temperatures. This allows generation of attenuated strains of pathogenic bacteria or generally safe versions of laboratory organisms. A temperature-sensitive organism can be created by substituting a single essential gene in mesophilic organism with its psychrophilic homologue. This can be facilitated by using the host’s native recombination system or through the use of plasmid based allele shuffling mechanisms.
The objective of this work was to analyze a number of psychrophilic alleles of various essential genes for their ability to cause temperature-sensitive phenotype in mesophilic bacterium Francisella novicida. The special attention has been placed on investigating psychrophilic alleles of bacterial DNA ligase. Furthermore a selected psychrophilic strain has been characterized as a potential source of multiple temperature-sensitive alleles of essential genes. Finally the secondary focus was to develop a simple and robust mechanism allowing efficient exchange of alleles of essential genes in the mesophilic host. / Graduate
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Biochemical and biophysical studies of MDM2-ligand interactionsWang, Shao-Fang January 2012 (has links)
MDM2, murine double minute 2, is a RING type-E3 ligase protein and also an oncogene. MDM2 plays a critical role in determining the steady levels and activity of p53 in cells using two mechanisms. The N-terminal domain of MDM2 binds to the transactivation domain of p53 and inhibits its transcriptional activity. The RING domain of MDM2 plays a role in the ubiquitination (and degradation) of p53. Several proteins are responsible for the ubiquitination mechanism including the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3). Since the E2-E3 interaction is essential for ubiquitination, the protein-protein recognition site is a potential drug target. Two different MDM2 RING constructs were expressed and purified: MDM2RING (residues 386-491) and MDM2RING△C (residues 386-478). Both constructs were characterised using dynamic light scattering, size exclusion chromatography, mass spectrometry, NMR and electron microscopy. E3 ligase activity in vitro was also studied. Taken together these results showed that the MDM2RING construct formed a concentration-dependent oligomeric structure. In contrast, the MDM2RING△C construct formed a dimer at all concentrations. Both MDM2RING and MDM2RING △ C retain E3 ligase activity. However, the MDM2RING△C construct is less active. Full length E2 enzyme UbcH5a was also purified. Various biophysical techniques were used to study its interaction with MDM2 as well as with potential small molecule inhibitors as in principle, small molecules which disrupt the interaction between MDM2 and UbcH5a, could prevent/promote ubiquitination of p53. The dimerisation of MDM2 is important for its E3 activity and the C8-binding site potentially provides a second druggable site. In this work, peptide 9, which has the same sequence as the C-terminus of MDMX (an MDM2 homologue) was found to inhibit MDM2 E3 activity. Various biological techniques including NMR, fluorescence anisotropy, and electrospray mass spectrometry were used to investigate the interaction between two inhibitory peptides and MDM2. A major part of project involved virtual screening (VS) to search for small molecules which can affect MDM2-dependent ubiquitination. Three potential targets were considered: (1) the C8-binding site of MDM2; (2) the UbcH5a-binding site of MDM2; and (3) the MDM2-binding site of UbcH5a. Several small molecules were identified using our virtual screening database-mining and docking programs that were shown to affect MDM2-dependent ubiquitination of p53. In terms of understanding the complex biochemical mechanism of MDM2 this work provides two interesting and functionally relevant observations: (i) the MDM2 RING△C construct is a dimer as this would not be expected form the existing studies, and has less E3 ligase activity than MDM2RING; (ii) small molecules that bind MDM2 on the E2 binding site enhanced E3 ligase activity. One model to explain these observations is that binding of small molecule activators family to the RING induces a change in the conformation of the Cterminal tail residues which may enhance E2 binding.
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Modulation of Base Excision Repair by NucleosomesOdell, Ian 18 November 2010 (has links)
DNA in eukaryotes is packaged into nucleosomes, which present steric impediments to many of the factors and enzymes that act on DNA, including DNA repair enzymes. Within the nucleosome, DNA remains vulnerable to oxidative damage that can result from normal cellular metabolism, ionizing radiation, and various chemical agents. Oxidatively damaged DNA is repaired in a stepwise fashion via the base excision repair (BER) pathway. Other DNA repair pathways, including Nucleotide Excision Repair (NER), Mismatch Repair (MMR), Homologous Recombination (HR), and Non-homologous End-Joining (NHEJ) are all thought to require nucleosome remodeling or disruption. In contrast, it was reported that the first step of BER does not require or induce nucleosome disruption. For example, the human DNA glycosylase hNTH1 (human Endonuclease III) was discovered to excise thymine glycol lesions from nucleosomes without nucleosome disruption, and could excise optimally oriented lesions with an efficiency approaching that seen for naked DNA (Prasad, Wallace, and Pederson 2007). To determine if the properties of hNTH1 are shared by other human DNA glycosylases, we compared hNTH1 with NEIL1, a human DNA glycoylase that also excises thymine glycol from DNA, with respect to their activities on nucleosome substrates. We found that the cellular concentrations and apparent kcat/KM ratios for hNTH1 and NEIL1 are similar. However, NEIL1 and hNTH1 differ in that NEIL1 binds undamaged DNA far more avidly than hNTH1. After adjustment for non-specific DNA binding, hNTH1 and NEIL1 proved to have similar intrinsic activities towards nucleosome substrates. We next wanted to examine the effects of nucleosomes on enzymes that catalyze the remaining steps in BER. We therefore assembled the entire four-step BER reaction with model, lesion-containing nucleosomes. The rates of substrate processing during the first three steps in BER, catalyzed by a DNA glycosylase, AP endonuclease, and DNA Polymerase Pol), varied with the helical orientation of the substrate relative to the underlying histone octamer. In contrast, the rate of action by DNA Ligase III- (in association with XRCC1) was independent of lesion orientation. These results are consistent with structural studies of BER enzymes and the previously proposed DNA unwrapping model for how BER enzymes gain access to lesions in nucleosomes (Prasad, Wallace, and Pederson 2007). During these investigations, we also discovered a synergistic interaction between Pol and Ligase III- complexed with XRCC1 that enhances the repair of lesions in nucleosomes. Together, our results support the hypothesis that DNA glycosylases have evolved to function in specific cellular environments (e.g. NEIL1 may function exclusively during DNA replication), but also possess DNA binding motifs and mechanisms of substrate recognition that impart a similar intrinsic activity on nucleosomes. In addition to hNTH1 and NEIL1, we have discovered that lesion orientation is also an important factor to the activities of APE and Pol and that the complete BER reaction can occur without requiring or inducing nucleosome disruption. Finally, protein-protein interactions between XRCC1 and Pol may be important for the efficient in vivo repair of lesions in nucleosomes.
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Expression exogène du récepteur du facteur autocrine de motilité (AMF-R) dans les cellules COS-7Registre, Marilyn January 2004 (has links)
Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.
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Mechanismus des pre-tRNA-Spleißens : Struktur und Funktion pflanzlicher und animaler RNA-Ligasen / Mechanism of tRNA splicing: structure and function of plant and animal RNA ligasesEnglert, Markus January 2005 (has links) (PDF)
Transfer Ribonukleinsäuren werden von der RNA Polymerase III als Vorläufer tRNA transkribiert und durchlaufen eine Vielzahl von Reifungsschritten hin zur maturen tRNA. Neben der Hydrolyse der 5´- und 3´-Flanke durch die RNase P und die tRNase Z, sowie einer Vielzahl von Basenmodifizierungen, wird bei einigen pre-tRNAs das Intron herausgespleißt. Die ersten intronhaltigen tRNA Gene wurden in der Hefe Saccharomyces cerevisiae nachgewiesen und folglich wurde der Spleißmechanismus in diesem Organismus als erstes untersucht. Eine tetramere tRNA Spleißendonuklease spaltet das Intron an den Exongrenzen heraus und eine tRNA Ligase ligiert die entstandenen tRNA Hälften zur gespleißten tRNA. Einzig in der Hefe und anderen Pilzen konnten bisher die Gene für die tRNA Ligase identifiziert werden. Weder molekularbiologische Ansätze – wie z.B. DNA Hybridisierung, Expressions-“Screening“ und funktionelle Komplementationsstudien mit einem tRNA Ligase-defizienten Hefestamm – noch Datenbanksuchen mit der bekannten Hefe tRNA Ligasesequenz haben in den vergangenen Jahren zur Identifizierung eines pflanzlichen oder animalen tRNA Ligase Gens geführt. In dieser Arbeit ist es erstmals gelungen, das tRNA Ligase Protein aus Weizenkeimen bis zur Homogenität zu isolieren und mit Hilfe erhaltener Peptidsequenzen die entsprechenden Kern-codierten Gene in höheren und niederer Pflanzen zu identifizieren. Die Ligaseaktivität wurde für das klonierte, rekombinant überexprimierte tRNA Ligaseprotein bestätigt. Weiterhin wurde zum ersten Mal das Ligaseprotein aus Schweineleber aufgereinigt und das zugehörige Gen im humanen Genom identifiziert. / Transfer ribonucleic acids (tRNAs) are produced by RNA polymerase III and undergo multiple maturation steps until to the mature tRNA. Besides the endonucleolytic removal of 5´- and 3´-flanks by RNase P and tRNase Z and a multitude of base modifications, the introns of some pre-tRNA is spliced out. The first intron-containing tRNA genes have been identified in Saccharomyces cerevisae and consequently the splicing mechanism has been studied in this organism first. A tetrameric splicing endonuclease cleaves the intron at the exon borders and a tRNA ligase ligates the resulting tRNA halves to the spliced tRNA. The gene for this tRNA ligase has up to now only been identified in yeast and in other fungi. Neither molecular biological approaches – as, e.g., DNA hybridisation, expression screening and functional complementation studies with a tRNA ligase-deficient yeast strain – nor data bank searches with the known yeast tRNA ligase sequence have led to the identification of a plant or animal tRNA ligase gene. In this work a purification to homogeneity has been achieved for the wheat germ tRNA ligase protein for the first time, followed by the identification of the corresponding nuclear-encoded genes in higher and lower plants with the help of resulting peptide sequences. The ligase activity was confirmed for the cloned, recombinant overexpressed tRNA ligase protein. Moreover, the ligase protein from pig liver was purified and the corresponding gene identified in the human genome.
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Mechanisms of molecular switching in the Wnt signal transduction pathwayFlack, Joshua Edwin January 2018 (has links)
Wnt signalling is a critical cellular communication pathway controlling cell fate in all metazoan organisms. Timely activation of this pathway is crucial to coordinate development, control homeostasis of adult tissues, and to avoid cancer. Wnt signal transduction depends primarily on the activities of three multiprotein complexes; the 'degradasome', which targets the central effector β-catenin for degradation in the absence of Wnt; the 'signalosome', which is assembled by Dishevelled upon Wnt-receptor binding to inactivate the degradasome, thus allowing β-catenin to accumulate; and the 'enhanceosome', which captures β-catenin, granting it access to target genes and relieving their transcriptional repression by Gro/TLE. Many of the components of these complexes have now been identified, but details of their regulation, and in particular the mechanisms by which they are switched on and off, remain poorly understood. The majority of this thesis is concerned with the mechanism by which β-catenin relieves the transcriptional repression imposed upon Wnt target genes, and thereby activates the Wnt 'transcriptional switch'. In Chapter 2, I present data showing that apposition of Gro/TLE and UBR5, a HECT E3 ubiquitin ligase, by β-catenin promotes Gro/TLE ubiquitylation, earmarking it for extraction by the VCP/p97 ATPase and ultimately leading to inactivation of its repressive function. In Chapter 3, I present the results of a different, ongoing study to identify the mechanism by which a cytoplasmic negative regulator, Naked, acts to interfere with the function of Dishevelled, promoting the switching of signalosomes and the termination of canonical Wnt signalling. These findings advance our understanding of the mechanisms by which the Wnt signalling pathway is switched on and off, and suggest new targets for therapeutic intervention in Wnt- driven cancers.
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Molecular Methods for Campylobacter and Arcobacter DetectionAbu-Halaweh, Marwan, n/a January 2005 (has links)
Twenty species and six subspecies of the genera Arcobacter and Campylobacter have been described to date. All are Gram-negative, microaerophilic, curved, spiral or S-shaped cells, and are members of the order Campylobacterales, class Epsilonproteobacteria phylum Proteobacteria. Though most members are pathogenic, C. jejuni, C. coli and A. butzleri are the most frequently isolated species from patients suffering from gastrointestinal illness. The current methods for their detection, identification, and differentiation are cumbersome, time consuming and lack specificity. DNA based molecular techniques including real-time Polymerase Chain Reaction (PCR) and Fingerprinting methods Terminal Restriction Fragments Length Polymorphism (T-RFLP) and Ligase Detection Reaction (LDR) have been used in this project to develop rapid detection and identification methods for Campylobacter and Arcobacter species. Five real-time PCR methods were developed which include: (a) rapid detection and identification of Campylobacter species using real-time PCR adjacent hybridisation probes, (b) rapid identification of C. jejuni using SYBR Green I, (c) rapid detection and differentiation of Arcobacter species using adjacent hybridisation probes, (d) rapid detection and differentiation of Arcobacter species and the Campylobacter group (C. coli, C. jejuni, C. lari, C. hyoilei, C. helviticus, C. hyointestinalis, C. insulaenigrae, C lanienae) using melting temperature (Tm) of adjacent hybridisation probes, and (e) a one tube real-time PCR multiplex for the rapid detection and identification of Campylobacter species, C. coli and C. jejuni using a TaqMan Probe, in an iCycler iQTM (BioRad, USA) and Light CyclerTM (Idaho Technology, USA). [Continued ...]
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Characterization of the E3 Ubiquitin Ligase Pirh2Tai, Elizabeth 01 September 2010 (has links)
The p53 tumour suppressor gene is inactivated by mutation in over 50% of all human cancers. The p53 protein is activated and stabilized through several post-translational modifications in response to various stresses and promotes cell cycle arrest and apoptosis. Thus, regulation of p53 is critical for normal cellular function. Pirh2 is a p53-regulated gene recently identified in our laboratory which encodes an E3 RING-finger ubiquitin ligase that binds to p53 and negatively regulates p53 by targeting it for ubiquitin-mediated proteolysis. Pirh2 is similar to another well-characterized E3 RING finger ubiquitin ligase, Mdm2, which also participates in a similar negative feedback loop with p53. At least seven E3 ubiquitin ligases are known to target p53 for degradation and the reason for this functional redundancy is unclear. The purpose of this study is to characterize Pirh2 activity.
This study has two aims the first is to identify additional interacting proteins for Pirh2, and the second is to delineate Pirh2 regulation of p53. Using several tandem affinity purification strategies and a GST-pull down approach, we have identified PKC delta as a candidate interacting protein. The second aim is to further characterize Pirh2 regulation of p53. Splenocytes and thymocytes from Pirh2-/- mice demonstrate a subtle increase in total p53 levels after irradiation when compared to wild-type controls. Phosphoserine 15 p53 levels are significantly higher in splenocytes and thymocytes from Pirh2 -/- mice relative to wild-type counterparts. Cells stably transfected with Pirh2 have decreased levels of phosphoserine 15 p53 and decreased induction of p21 relative to vector control and Mdm2 expressing cells.
The stability of the p53 protein is primarily regulated through ubiquitin mediated proteolysis, and there are multiple ubiquitin ligases targeting p53 for degradation. Here we are able to address the question of functional redundancy by indicating that Pirh2 can target serine 15 phosphorylated p53 which is reported to not be regulated by Mdm2.
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Characterization of the E3 Ubiquitin Ligase Pirh2Tai, Elizabeth 01 September 2010 (has links)
The p53 tumour suppressor gene is inactivated by mutation in over 50% of all human cancers. The p53 protein is activated and stabilized through several post-translational modifications in response to various stresses and promotes cell cycle arrest and apoptosis. Thus, regulation of p53 is critical for normal cellular function. Pirh2 is a p53-regulated gene recently identified in our laboratory which encodes an E3 RING-finger ubiquitin ligase that binds to p53 and negatively regulates p53 by targeting it for ubiquitin-mediated proteolysis. Pirh2 is similar to another well-characterized E3 RING finger ubiquitin ligase, Mdm2, which also participates in a similar negative feedback loop with p53. At least seven E3 ubiquitin ligases are known to target p53 for degradation and the reason for this functional redundancy is unclear. The purpose of this study is to characterize Pirh2 activity.
This study has two aims the first is to identify additional interacting proteins for Pirh2, and the second is to delineate Pirh2 regulation of p53. Using several tandem affinity purification strategies and a GST-pull down approach, we have identified PKC delta as a candidate interacting protein. The second aim is to further characterize Pirh2 regulation of p53. Splenocytes and thymocytes from Pirh2-/- mice demonstrate a subtle increase in total p53 levels after irradiation when compared to wild-type controls. Phosphoserine 15 p53 levels are significantly higher in splenocytes and thymocytes from Pirh2 -/- mice relative to wild-type counterparts. Cells stably transfected with Pirh2 have decreased levels of phosphoserine 15 p53 and decreased induction of p21 relative to vector control and Mdm2 expressing cells.
The stability of the p53 protein is primarily regulated through ubiquitin mediated proteolysis, and there are multiple ubiquitin ligases targeting p53 for degradation. Here we are able to address the question of functional redundancy by indicating that Pirh2 can target serine 15 phosphorylated p53 which is reported to not be regulated by Mdm2.
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Localisation et caractérisation d'un élément de réponse à la triiodothyronine (T3) dans la partie promotrice du gène de la synthétase des acides gras (FAS) chez l'oieMartel, Caroline January 2006 (has links) (PDF)
L'obésité est maintenant perçue comme un problème de santé majeur dans notre société. C'est pourquoi la compréhension des mécanismes moléculaires liés au développement de cette maladie est primordiale. Des études comparatives entre des poulets de lignées grasses et des poulets de lignées maigres ont montré que la lipogénèse hépatique était plus élevée chez les animaux gras. La Synthétase des Acides Gras (FAS) est connue comme étant responsable de la synthèse des lipides saturés à longues chaînes. Des études antérieures ont démontré que ce gène est régulé à la fois par l'état nutritionnel et hormonal. Dans ce contexte, l'objectif de la présente étude était d'examiner le rôle de l'hormone thyroïdienne (T3), et aussi d'un mélange de T3 et d'insuline, sur l'activité transcriptionnelle du gène aviaire de la FAS. Certains des éléments agissant au niveau de la réponse à l'insuline ont déjà été identifiés chez l'humain, le poulet, la souris et le rat. Afin d'identifier l'effet de la T3 et d'un mélange de T3 et d'insuline, un fragment d'ADN de 1,6 Kpb de la partie promotrice de FAS a été isolé, séquencé et cloné dans un vecteur contenant le gène rapporteur codant pour la chloramphénicol acétyl transférase (CAT). Cette construction nommée 1.6FAS-pJFCAT1 a ensuite été transfectée de façon transitoire dans les cellules HepG2 et les hépatocytes embryonnaires de poulet. Les analyses d'expression ont démontré une augmentation de l'activité du gène rapporteur codant pour CAT de 2.5 fois, suite à une stimulation de 48 heures avec 1.6µM de T3. Ce promoteur posséderait donc un élément de réponse nécessaire à la modulation par cette hormone. Des constructions contenant des délétions successives de ce grand fragment d'ADN et des alignements de séquences ont permis l'identification de la région interne localisée entre
-902/ -577 pb où se retrouve l'élément de réponse à la T3, appelé TRE pour 'Thyroïd Hormone Response Element'. Des analyses de retard sur gel (EMSA) utilisant comme TRE un oligonucléotide contenant les bases -732 à -692 ont ensuite permis d'identifier des protéines se fixant sur ce fragment d'ADN en réponse à la T3, renforçant ainsi la présence possible d'un TRE. Une augmentation de l'activité CAT de 2.3 fois a d'ailleurs aussi été obtenue après 48 heures de stimulation à la T3 lors d'expériences de transfections transitoires avec la construction contenant le fragment -765/-663 placé devant le promoteur TK. Le TRE correspondrait donc à la séquence CGCCCTgtggTAACCT, séquence très différente du consensus établi et très différente des T3RE retrouvés dans la partie promotrice du gène de l'enzyme malique. Il a toutefois une forte homologie avec un TRE retrouvé dans le gène FAS chez l'humain, le poulet, la souris et le rat. Ceci semble démontrer la forte variation de ces éléments de réponse. L'analyse par retard sur gel révèle qu'en présence de T3 seulement il y a fixation du récepteur TR alors que lorsque l'insuline est aussi présente, il semble que le facteur de transcription RXR se fixe aussi. Ceci suggérerait l'idée que la formation d'hétérodimères TR/RXR serait grandement favorisée en présence d'insuline. Ces informations, en combinaison avec les résultats récents obtenus par d'autres équipes sur la régulation de la FAS, serviront à la compréhension des mécanismes de régulation de la transcription d'un gène clé de la lipogenèse hépatique. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Hormone thyroïdienne, TRE, Synthétase des acides gras, FAS, Lipogenèse.
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