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1
Patel, Premal Harshad. "Evolution of DNA polymerase active site /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/6361.
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Nayak, Dhananjaya. "Conformational mechanisms in T7 RNA polymerase transcription a dissertation /." San Antonio : UTHSC, 2008. http://learningobjects.library.uthscsa.edu/cdm4/item_viewer.php?CISOROOT=/theses&CISOPTR=44&CISOBOX=1&REC=11.
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Tuusa, J. (Jussi). "Human DNA polymerase ε:expression, phosphorylation and protein-protein interactions." Doctoral thesis, University of Oulu, 2001. http://urn.fi/urn:isbn:9514265815.
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Abstract
DNA replication is a process in which a cell duplicates its genome before cell division,
and must proceed accurately and in organized manner to guarantee maintenance of the
integrity of the genetic information. DNA polymerases are enzymes that catalyse the
synthesis of the new DNA strand by utilizing the parental strand as a template. In
addition to chromosomal replication, DNA synthesis and therefore DNA polymerases are also
needed in other processes like DNA repair and DNA recombination. The DNA polymerase is
an essential DNA polymerase in eukaryotes and is required for chromosomal DNA
replication. It has also been implicated in DNA repair, recombination, and in
transcriptional and cell cycle control. The regulation of the human enzyme was explored
by analysing its expression, phosphorylation and protein-protein interactions.
Expression of both the A and B subunits of the human DNA polymerase ε was strongly
growth-regulated. After serum-stimulation of quiescent fibroblasts, the steady-state mRNA
levels were up-regulated at least 5-fold. In actively cycling cells, however, the
steady-state mRNA and protein levels fluctuated less than 2-fold, being highest in
G1/S phase.
The promoter of the B subunit gene was analysed in detail. The 75 bp core promoter was
essentially dependent on the Sp1 transcription factor. Furthermore, mitogenic control of
the promoter required an intact E2F binding element, and binding of E2F2, E2F4 and p107
was demonstrated in vitro. A down-regulation element, located
immediately downstream from the core promoter, bound E2F1, NF-1 and pRb transcription
factors. A model of the promoter function is presented.
Topoisomerase IIβ binding protein 1 (TopBP1) was found to be associated with human
DNA polymerase ε. TopBP1 contains eight BRCT domains and is homologous to
Saccharomyces cerevisiae Dpb11, Schizosaccharomyces
pombe Cut5, Drosophila melanogaster Mus101 and the human
Breast Cancer susceptibility protein 1 (BRCA1). TopBP1 is a phosphoprotein, whose
expression is induced at the G1/S border and is required for
chromosomal DNA replication. It co-localizes in S phase with BRCA1 into discrete foci,
which do not represent sites of ongoing DNA replication. However, if DNA is damaged or
replication is blocked in S phase cells, TopBP1 and BRCA1 re-localize into proliferating
cell nuclear antigen (PCNA) containing foci that represent stalled replication forks.
Finally, phosphorylation of DNA polymerase ε was described and at least three
immunologically distinct and differentially phosphorylated forms were shown to exist.
Phosphorylation is on serine and threonine residues and shows a cell cycle dependent
fluctuation, but is not affected by DNA damage or by inhibition of DNA replication. BRCA1
co-immunoprecipitates with a hypophosphorylated form of DNA polymerase ε. In
contrast, TopBP1 was shown to be associated with a hyperphosphorylated form.
4
Talanian, Robert Vincent. "Development of Selective Inhibitors of DNA Polymerase Delta: A Thesis." eScholarship@UMMS, 1989. https://escholarship.umassmed.edu/gsbs_diss/66.
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This thesis is divided into three parts, united by the theme of the development of selective inhibitors of mammalian cell DNA polymerase delta (pol δ). The first part consists of an investigation of the cytotoxic mechanism(s) of certain 2-substituted adenine analogs, selected on the basis of their inhibitory properties towards DNA polymerase alpha (pol α) and mammalian cell DNA synthesis. The second is a direct search for inhibitors of isolated pol δ, and an investigation of inhibitory mechanisms. The third consists of measurement of the effects of a selective pol δ inhibitor on cellular DNA synthesis.
Mechanism of Cytotoxicity of 2-substituted adenine analoqs. The mechanism of inhibition by 2-(p-n-butylanilino)-2'-deoxyadenosine (BuAdA), and related compounds, of Chinese hamster ovary (CHO) cell ([3H]thymidine [3H]TdR) incorporation, was investigated. The potency of the compound could largely be explained by its potency (IC50 = 23 μM) as an inhibitor of CHO cell [3H]TdR uptake. BuAdA inhibited incorporation by CHO cells of [32p]phosphate into DNA relatively weakly, displaying an IC50value of 80 μM.
Differential inhibition of DNA polymerases alpha and delta. Known DNA polymerase inhibitors of a structurally wide range were screened for their ability to inhibit pol δ derived from calf thymus selectively with respect to pol α derived from the same tissue. Pyrophosphate (PPi) and difluoromethanediphosphonate each inhibited pol δ weakly, but with greater potency than pol α. Based on this lead, an expanded series of PPi analogs was screened. Carbonyldiphosphonate (COMDP) inhibited pol δ with a potency (Ki = 1.8 μM) twenty-two times greater than that displayed for pol α. Kinetic studies indicated that COMDP inhibited pol δ competitively with the dNTP specified by the template, but not competitively with the template:primer. Analogous experiments with pol α showed that the compound inhibited that enzyme uncompetitively with the dNTP, and not competitively with the template:primer. COMDP was a weak inhibitor of the 3' → 5' exonuclease activity of pol δ, displaying an IC50value greater than 1 mM.
Inhibition of permeabilized cell DNA synthesis bv a selective pol δ inhibitor. The potency of COMDP as an inhibitor of permeabilized CHO cell DNA synthesis (IC50= 200 μM) did not clearly indicate the participation of pol δ in cellular DNA replication.
Prospectus. The thesis concludes with a prospectus for the development of pol δ inhibitors with improved properties compared to COMDP.
5
Lamble, Sarah. "Directed evolution of Thermus aquaticus DNA polymerase by compartmentalised self-replication." Thesis, University of Bath, 2009. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.507743.
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The thermophilic enzyme, Thermus aquaticus (Taq) DNA polymerase, is an essential tool in molecular biology because of its ability to synthesis DNA in vitro and its inherent thermal stability. Taq DNA polymerase is widely used in the polymerase chain reaction (PCR), an essential technique in a broad range of different fields from academic research to clinical diagnostics. The use of PCR-based tests in diagnostic testing is ever increasing; however, many of the samples being tested contain substances that inhibit PCR and prevent target amplification. Many attempts have been made to engineer polymerases not only to increase resistance to overcome the problem of inhibition, but also to enhance other characteristics such as fidelity, processivity and thermostability. Heparin, found in blood samples, and phytate, found in faecal samples, are two examples from a number of known PCR inhibitors. The mode of action of most PCR inhibitors is not well understood, but inhibition is thought to occur by enzyme binding or through the chelation of Mg2+ ions essential for PCR. In this project, a system of directed evolution by compartmentalised self-replication (CSR) was established and successfully employed to screen a mutant library for Taq DNA polymerase variants with enhanced resistance to the inhibitors heparin and phytate. CSR is a recently-established high-throughput method for the creation of novel polymerases, based on a feedback loop whereby polymerase variants replicate their own encoding gene. A mutant library of 106 variants was produced by random mutagenesis error-prone PCR, in which only the polymerase domain of Taq was mutagenised. Firstly, the CSR system was established and tested by performing a screen in the presence of heparin to select for heparin-resistant variants. Characterisation of selected variants revealed that a single round of CSR had produced a Taq variant (P550S, T588S) with a 4-fold increase in heparin resistance. The IC50 was increased from 0.012U/ml heparin to 0.050U/ml heparin. The study with heparin was followed by a phytate screen, in which two rounds of CSR were performed with an initial round of error-prone PCR followed by re-diversification (recombination) of the mutant library using the staggered extension process (StEP). The two rounds of CSR yielded a Taq variant with a 2-fold increase in phytate-resistance compared to the wild-type, with IC50 increased from 360μM phytate to 700μM phytate. The best phytate mutant (P685S, M761V, A814T) was further characterised and it was found that the catalytic activity, thermostability and fidelity of the mutant were comparable to the wildtype enzyme. The position of resistance-conferring mutations of the novel Taq variants evolved in this study provided some evidence for the inhibitors’ predicted modes of action in the case 2 of both phytate and heparin. As phytate’s mode of action is poorly understood, further investigations were performed to elucidate its role in PCR inhibition. A thorough investigation into the importance of relative phytate and Mg2+ levels on PCR was conducted and revealed for the first time convincing evidence that the primary mode of phytatemediated PCR inhibition is by chelation. Further work led to the successful crystallisation of Taq in the presence of phytate, although subsequent X-ray diffraction data to 2.5Å did not reveal phytate bound within the enzyme structure. Site-directed mutagenesis studies were used to probe cross-over between heparin and phytate-conferring mutations. Thus, in addition to providing valuable information for novel Taq variants with a potential application in fecal-based PCR diagnostic tests, this project has begun to provide insight into the fundamental aspects of the mode of action of phytate as a polymerase and PCR inhibitor.
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Stenbak, Carolyn Rinke. "Foamy virus polymerase : enzymatic activities and assembly /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/11510.
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Maisonneuve, Caroline. "Effets des analogues de la thymidine sur l'oxydation des acides gras chez la souris : implication dans la physiopathologie de la lipoatrophie." Paris 5, 2004. http://www.theses.fr/2004PA05N046.
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Les inhibiteurs nucléosidiques de la transcriptase inverse (INTIs) sont des médicaments anti-VIH pouvant induire des lipodystrophies. Le but de cette thèse a été d'évaluer les effets mitochondriaux et métaboliques des INTIs (AZT, d4T, ddC,ddI, 3TC chez la souris. Après deux semaines de traitement, seuls les analogues de la thymide (AZT et d4T) augmentaient l'oxydation hépatique des acides gras et les corps cétoniques plasmatiques, sans induire un dysfonctionnement mitochondrial profond. Ces effets étaient reproduits par l'acide β-aminoisobutyrique (BAIBA), un catabolite de la thymine. Après six semaines, seuls le d4T, l'AZT et le BAIBA augmentaient les corps cétoniques et diminuaient la masse grasse corporelle des souris Swiss,sans dépléter l' ADNmt dans le tissu.Nucleoside reverse-transcriptase inhibitors (NRTIs), used in the treatment of HIV infection can induce lipodystrophy. The aim of this work was to better characterize the mitochondrial and metabolic effects induced by NRTIs (AZT, d4T, ddC, ddI, 3TC) in mice. The thymidine analogs AZT and d4T increased fatty acids oxidation in liver and plasma ketone bodies after 2 weeks of treatment, without a profund mitochondrial dysfunction. These effects were reproduced by β-aminoisobutyric acid (BAIBA), a thymine catabolite. The derivatives d4T, AZT and BAIBA still increased ketone bodies and decreased body fat mass after 6 weeks of treatment in Swiss mice. However, mitochondrial DNA was not decreased in epididymal white adipose tissue and glucido-lipidic metabolism was unchanged
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Whiting, Sam H. "Studies into the characteristics and mechanism of strand displacement synthesis by retroviral reverse transcriptase /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/11494.
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Feghoul, Linda. "Mécanismes d'action et de résistance des Adénovirus C au Brincidofovir." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC303/document.
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Les adénovirus humains (HAdV) sont responsables d’une importante morbidité et mortalité chez les patients receveurs de cellules souches hématopoïétiques (CSH), en particulier chez les enfants. Le brincidofovir (BCV) prodrogue du cidofovir est un analogue de la cytosine. Il inhibe l’activité de l’ADN polymérase (ADN pol) des HAdV par arrêt de l’élongation et compétition avec les nucléotides naturels. Les objectifs de ce travail sont d’étudier la diversité génétique de l’ADN pol des HAdV et de mettre en place des outils pour l’analyse de la sensibilité des HAdV C et de résistance aux BCV. Dans une première partie, nous avons réalisé le séquençage complet du gène ADN pol de 60 souches virales cliniques. Les résultats montrent une variabilité nucléotidique de 9,9% et peptidique de 8,7%, variable en fonction du type. La région la plus variable est la région NH2 terminale avec 44,2% des mutations d’acides aminés. Aucune des mutations décrites comme associées à la résistance au CDV ou au BCV n’a été détectée. L’analyse du polymorphisme a aussi montré que les séquences ADN pol permettent d’identifier les sources de transmission au cours d’une épidémie. Enfin nos observations suggèrent la faible probabilité d’évènements de recombinaison comme facteur d’évolution du gène codant pour l’ADN pol. Dans une seconde partie, nous avons développé une stratégie de génétique inverse à partir de deux plasmides, un codant pour le génome entier délété de l’ADN pol (Adv-deltaPol) et un codant uniquement pour l’ADN pol (Pol) (3 kilobase) dans lequel les mutations sont introduites par mutagénèse dirigée (Pol-muté). Après une étape de recombinaison bactérienne en un seul plasmide (Adv-deltaPol-Pol-muté), des cellules 293T sont transfectées afin de produire les virus recombinants. La susceptibilité au BCV des virus mutés sera analysée sur des cellules A549. Au total de 12 plasmides Pol-muté ont été produits, le virus recombinant L677F a été obtenu. Les productions des autres virus recombinants sont en coursHuman adenoviruses (HAdV) are responsible for significant morbidity and mortality in hematopoietic stem cell (HSC) recipients, particularly in children.Brincidofovir (BCV) prodrug of cidofovir is an analogue of cytosine. It inhibits the DNA polymerase (DNA pol) activity of HAdV by stopping elongation and competition with natural nucleotides.The objectives of this work are to study the genetic diversity of the pol DNA of HAdV and to set up tools for the analysis of HAdV C sensitivity and resistance to BCV.In a first part, we carried out the complete sequencing of the DNA pol gene of 60 clinical viral strains. The results show a nucleotide variability of 9.9% and peptide variability of 8.7%, variable according to the type. The most variable region is the NH2 terminal region with 44.2% of the amino acid mutations. None of the mutations described as associated with CDV or BCV resistance were detected. Polymorphism analysis has also shown that DNA pol sequences can identify sources of transmission during an outbreak. Finally, our observations suggest the low probability of recombination events as a factor of evolution of the gene coding for the DNA pol. In a second part, we developed a reverse genetic strategy from two plasmids, one coding for the whole genome deleted from the DNA pol (Adv-deltaPol) and one coding only for the viral DNA pol (Pol). 3 kilobase) in which the mutations are introduced by site-directed mutagenesis (Pol-mutated). After a bacterial recombination step into a single plasmid (Adv-deltaPol-Pol-mutated), 293T cells are transfected to produce the recombinant viruses. BCV susceptibility of mutated viruses will be analyzed on A549 cells. A total of 12 Pol-mutated plasmids were produced, recombinant virus L677F was obtained. Production of other recombinant viruses is ongoing
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Jackson, Constanza. "Synthesis of 2’ Modified Primers to Characterize Extension Events by Mutant Taq DNA Polymerases." Scholarship @ Claremont, 2015. http://scholarship.claremont.edu/scripps_theses/592.
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Oligonucleotides enable many biotechnological applications; however they are easily degraded by nucleases. Many nucleotides modified at the 2’ position are degraded at decreased rates which improves oligonucleotide utility. Most applications of oligonucleotides rely on enzymatic synthesis. Unfortunately, native DNA polymerases do not recognize most useful modified nucleotide substrates. Directed evolution has been used to identify mutants of Taq DNA polymerase I (Taq) that recognize substrates with 2’ modifications. While mutant enzymes capable of modified nucleotide addition have been identified, to date, all of these enzymes are limited by their inability to synthesize full length modified DNA. Despite considerable efforts to evolve new activity there has been little work done to quantitatively characterize these evolved enzymes. This thesis work presents efforts to synthesize modified primers that will help comparatively and quantitatively characterize three enzymes previously evolved to recognize 2’ modified substrates. Using the methods developed in this thesis project, our lab will be able to characterize the relationship between the number of modified nucleotides in the primer terminus and the rate of modified and unmodified nucleotide addition. Future work will identify key enzymatic steps that limit extension in these enzymes with implications for the future design of Taq mutants capable of synthesizing long 2’ modified oligonucleotides.
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Roupioz, Yoann. "La lésion 2-désoxyribonolactone : étude de la réactivité et des conséquences biologiques de ce dommage de l'ADN." Grenoble 1, 2001. http://www.theses.fr/2001GRE10079.
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La lesion 2-desoxyribolactone est un site abasique de l'adn forme par des rayonnements ultra-violet ou des irradiations gamma, par action d'antibiotiques tels que la neocarzinostatine ou de certains complexes metalliques. Ce dommage a la particularite d'etre alcali-labile et de generer des coupures de brins d'adn. Un des buts du travail presente a ete la mise au point d'une voie de synthese generale et quantitative de cette lesion a partir d'un precurseur photoreactif incorpore dans des oligonucleotides de longueur variable. Les oligonucleotides synthetises constituent un outil de choix pour l'etude de la reactivite chimique ou biologique du dommage, ou l'etude du caractere alcali-labile de la 2-desoxyribonolactone. Des durees de demi-vie de la lesion en fonction du ph, de la temperature ou de la longueur du fragment d'adn ont ete mesurees. Ces informations ont permis de mettre au point les conditions experimentales appropriees pour l'etude de la synthese translesionnelle d'adn et de la reparation in vitro de la lesion, ce qui constitue la deuxieme partie de ce travail. Nous avons etudie l'activite de trois adn polymerases et de differentes enzymes, presentes chez e. Coli ou chez l'homme, chargees de la reparation d'une autre lesion abasique : le site 2-desoxyribose. Les resultats obtenus sur l'activite des ap-endonucleases de type ii laissent presager que ces enzymes sont de bonnes candidates a la reparation in vivo de la 2-desoxyribonolactone puisque l'activite detectee in vitro est semblable a celle obtenue sur le substrat de predilection de ces enzymes : le site 2-desoxyribose. D'autre part, la synthese translesionnelle d'adn, catalysee par les enzymes que nous avons testees, met en evidence l'incorporation preferentielle de damp face a la lesion par certaines polymerases. La regle du a decrite pour d'autres lesions, est donc egalement valable pour la lesion 2-desoxyribonolactone.
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Gardella, Thomas James. "A Genetic Analysis of RNA Polymerase-Promoter Interactions: A Thesis." eScholarship@UMMS, 1988. http://escholarship.umassmed.edu/gsbs_diss/200.
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Transcription initiation is a key step at which gene expression can be regulated. The sigma subunit of RNA polymerase provides the enzyme with the ability to recognize promoter sequences and initiate transcription at specific sites on the chromosome. The molecular basis of sigma function is not well known. It has been suggested that sigma factors confer promoter specificty by making direct contacts to the promoter DNA (Losick and Pero, 1981). To test this idea, suppressors of promoter down mutations were sought that affected the promoter recogniton properties of the σ70 subunit of E. coli RNA polymerase. Four such sigma mutants were obtained, two of which are allele-specific. One of these mutants has a change at a position in the predicted helix-turn-helix DNA binding structure which lies in a conserved region of the protein (region 4). This mutant specifically suppresses promoter down mutations in the -35 region of the promoter. The other mutant has a change at a residue that lies in a predicted α-helix of conserved region 2. This mutant specifically suppresses promoter mutations in the -10 region of the promoter. These data support the idea that regions 2 and 4 of sigma interact with the -10 and -35 regions of the promoter, respectively.
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Butler, Michelle Marie. "Probing the dNTP Binding Region of Bacillus subtilis: DNA Polymerase III with Site-Directed Inhibitors: A Dissertation." eScholarship@UMMS, 1992. https://escholarship.umassmed.edu/gsbs_diss/132.
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6-(p-Hydroxyphenylhydrazino) uracil (H2-HPUra) is a selective and potent inhibitor of the replication-specific DNA polymerase III (pol III) of Gram+ bacteria such as Bacillus subtilis. Although a pyrimidine, H2-HPUra derives its inhibitory activity from its specific capacity to mimic the purine nucleotide, dGTP. The project described in this thesis dissertation involves the use of H2-HPUra-like inhibitors to probe the structure and function of the pol III active site. It consists of two separate problems which are summarized below.
Production of a potent bona fide dGTP form of inhibitor. A method was devised to successfully convert the H2-HPUra inhibitor prototype to a bona fide purine, using N2-benzyl guanine as the basis. Structure-activity relationships of benzyl guanines carrying a variety of substituents on the aryl ring identified N2-(3,4-dichlorobenzyl) guanine (DCBG) as a compound equivalent to H2-HPUra with respect to potency and inhibitor mechanism. DCBdGTP, the 2'-deoxyribonucleoside 5'-triphosphate form of DCBG, was synthesized and characterized with respect to its action on wild-type and mutant forms of pol III. DCBdGTP acted on pol III by the characteristic inhibitor mechanism and formally occupied the dNTP binding site with a fit which permitted its polymerization. The latter experiment identified the site for the binding of the inhibitor's aryl moiety as a distinct site located at a distance of approximately 6-7 Å from the base-paired 2-NH group of a bound dGTP.
Attempt to covalently label amino acid residue 1175, a putative participant in inhibitor binding. Azp-12, a point mutation of serine 1175, yields a form of pol III whose inhibitior sensitivity varies specifically as a function of the composition of the para substituent of the inhibitor's aryl ring. On the basis of the latter behavior, residue 1175 was hypothesized to be a residue directly involved in the binding of the inhibitor's aryl moiety. To test this hypothesis, residue 1175 was specifically mutated to either cysteine or lysine, each of which presents a side chain amenable to covalent bond formation with appropriately reactive inhibitor forms. Of the two mutant pol III forms, only the cysteine form (pol III-cys) was catalytically active. The kinetic properties and inhibitor sensitivity profile of pol III-cys identified it as a target suitable for potentially irreversible inhibitor forms containing the following groups in the meta position of the aryl ring: -CH2Br, -CH2C1, and -CH2SH. None of the several inhibitors tested selectively or irreversibly inactivated pol III-cys. Possible bases for the failure of this group of inhibitors and for the redesign of more useful covalently reactive inhibitor forms are considered.
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Pessoa-Brandão, Luis. "Genetic and molecular studies of Saccharomyces cerevisiae Cdc7-Dbf4 kinase function in DNA damage-induced mutagenesis /." Connect to full text via ProQuest. IP filtered, 2005.
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Halvas, Elias Konstantine. "Structural determinants of murine leukemia virus (MLV) reverse transcriptase (RT) important for fidelity and drug-resistance in vivo." Morgantown, W. Va. : [West Virginia University Libraries], 2000. http://etd.wvu.edu/templates/showETD.cfm?recnum=1518.
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Thesis (Ph. D.)--West Virginia University, 2000.Title from document title page. Document formatted into pages; contains x, 231 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 188-203).
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Koskiniemi, Sanna. "Dynamics of the bacterial genome rates and mechanisms of mutation /." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-111428.
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Nordvarg, Helena. "Functional Significance of Multiple Poly(A) Polymerases (PAPs)." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2002. http://publications.uu.se/theses/91-554-5285-X/.
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Riviere-Marciacq, Florence. "Synthèse d'analogues structuraux des nucléosides destinés au séquençage d'ADN." Grenoble 1, 1999. http://www.theses.fr/1999GRE10138.
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La methode la plus repandue pour analyser la sequence des acides nucleiques est la technique enzymatique dite de terminaison de chaine developpee par sanger et col. Le principal inconvenient de cette methode est son cout, du notamment a la forte valeur ajoutee des terminateurs de chaine utilises. De nombreuses ameliorations ont ete apportees, mais peu concernent la preparation de terminateurs de chaine simples a synthetiser et donc moins onereux. Une partie de ce manuscrit et consacree a la synthese de familles de terminateurs de chaine potentiels appeles morpholinonucleotides. Trois types de molecules ont ainsi ete preparees de facon simple a partir de reactifs commerciaux. Tous les produits purifies ont ete caracterises par differentes techniques analytiques (rmn, spectrometrie de masse). L'etude des proprietes biochimiques de ces composes a ete realisee dans une deuxieme partie. Une serie d'essais d'incorporation enzymatique avec des adn polymerases et une transferase terminale a ete effectuee, montrant que ces composes sont des substrats interessants. Des reactions de sequencage ont ete egalement mises en uvre. Les resultats obtenus indiquent que les morpholinonucleotides sont des terminateurs de chaine incorpores de maniere base-specifique.
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Kelleher, Colleen Diane. "Characterization of polymerase and RNase H activities of Moloney murine leukemia virus reverse transcriptase in relation to models for retroviral plus-strand synthesis /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/11519.
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CELIA, HERVE. "Etudes structurales de la sous-unite b de la gyrase d'escherichia coli et de l'arn polymerase a de saccharomyces cerevisiae. Microscopie electronique et analyse d'images de cristaux bidimensionnels." Strasbourg 1, 1993. http://www.theses.fr/1993STR13175.
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Des cristaux bidimensionnels de sous-unite b de la gyrase avaient ete obtenus par interaction specifique de la proteine avec un film de lipides greffes covalament a la novobiocine, un inhibiteur des fonctions atpasiques de l'enzyme. Nous avons calcule un modele tridimensionnel de la sous-unite b a partir d'images de cristaux de la proteine colores negativement. Ce modele montre a 2,5-3,0 nm de resolution deux bras allonges formant une structure en v, la base du v contenant le site de liaison a la novobiocine. L'analyse d'images effectuee sur les memes cristaux hydrates congeles a permis de determiner une carte de projection de la proteine a 1,0 nm de resolution, mettant en evidence des details structuraux non resolus en coloration negative. Nous avons compare nos donnees de microscopie electronique avec la structure cristallographique d'un fragment n-terminal de la proteine contenant le site de liaison a l'atp. La localisation du fragment dans la structure de la proteine entiere suggere que les sites de liaison a l'atp et a la novobiocine sont proches l'un de l'autre. Enfin les resultats que nous avons obtenus en diffraction electronique montrent qu'une etude structurale de la proteine a haute resolution peut etre envisagee a partir des cristaux bidimensionnels. Par l'utilisation de lipides charges positivement et etales en monocouche, nous avons pu obtenir les premiers cristaux bidimensionnels d'une arn polymerase eucaryote. Le modele tridimensionnel calcule a partir des cristaux colores negativement montre a 3,0 nm de resolution une structure de forme irreguliere de 111115 nm. Cette structure est caracterisee par un sillon de 10 nm de long, et une protrusion en forme de doigt definissant un canal a l'entree du sillon. Le canal et le sillon ont les dimensions requises pour lier une molecule d'adn double brin et contiennent probablement le site catalytique de polymerisation de l'arn
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Trujillo, Joshua T., Mark A. Beilstein, and Rebecca A. Mosher. "The Argonaute-binding platform of NRPE1 evolves through modulation of intrinsically disordered repeats." WILEY-BLACKWELL, 2016. http://hdl.handle.net/10150/622374.
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• Argonaute proteins are important effectors in RNA silencing pathways, but they must interact with other machinery to trigger silencing. Ago hooks have emerged as a conserved motif responsible for interaction with Argonaute proteins, but little is know about the sequence surrounding Ago hooks that must restrict or enable interaction with specific Argonautes.
• Here we investigated the evolutionary dynamics of an Argonaute-binding platform in NRPE1, the largest subunit of RNA Polymerase V. We compared NRPE1 sequences from more than 50 species, including dense sampling of two plant lineages.
• This study demonstrates that the Argonaute-binding platform of NRPE1 retains Ago-hooks, intrinsic disorder, and repetitive character while being highly labile at the sequence level. We reveal that loss of sequence conservation is due to relaxed selection and frequent expansions and contractions of tandem repeat arrays. These factors allow a complete restructuring of the Ago-binding platform over 50-60 million years. This evolutionary pattern is also detected in a second Ago-binding platform, suggesting it is a general mechanism.
• The presence of labile repeat arrays in all analyzed NRPE1 Ago-binding platforms indicates that selection maintains repetitive character, potentially to retain the ability to rapidly restructure the Ago-binding platform.
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Privat, Isabelle. "Obtention et caractérisation de transformants d'Arabidopsis thaliana affectés dans l'expression du facteur de transcription SIG1." Université Joseph Fourier (Grenoble), 2000. http://www.theses.fr/2000GRE10182.
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La transcription plastidiale au cours du developpement est realisee par deux types d'arn polymerase. La pep (plastid-encoded plastid rna polymerase) est de type procaryotique et ses sous-unites sont codees par les genes plastidiaux rpo. L'autre, la nep (nuclear-encoded plastid rna polymerase) est monomerique, de type phagique et codee par le genome nucleaire. L'existence de proteines presentant de fortes homologies avec le facteur 870 d'e. Coli avait depuis longtemps ete suggeree. Trois adnc codant pour trois facteurs sigma potentiels ont ete isoles chez arabidopsis thaliana. Ils sont nommes sig1, sig2 et sig3. Pour determiner le role de ces trois facteurs dans la transcription plastidiale, une strategie antisens a ete utilisee. Seules les lignees transformees avec la construction antisens sig1 ont un phenotype mutant : cotyledons blancs et premiere paire de feuilles vertes. Le phenotype observe est lie a une forte diminution de la quantite d'arnm sig1. La quantite de proteine sig1 est egalement diminuee. L'analyse de l'expression des genes plastidiaux psba, rbcl et des operons rrn et atpi a ete entreprise. Seule l'expression du gene psba est diminuee dans les lignees antisens mutantes. L'expression de rbcl n'est pas affectee alors qu'in vitro sig1 est capable de transcrire ce promoteur. Il est possible d'envisager que certains promoteurs plastidiaux soient reconnus par differents facteurs sigma, comme par exemple rbcl. Le facteur sig3 pourrait palier l'absence du facteur sig1 dans les lignees antisens au niveau de certains promoteurs plastidiaux. Par contre, le promoteur psba semble etre strictement lie au facteur sig1, au moins dans les cotyledons.
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Svarovskaia, Evguenia S. "Structural determinants of murine leukemia virus reverse transcriptase that are important for template switching, fidelity, and drug-resistance." Morgantown, W. Va. : [West Virginia University Libraries], 2000. http://etd.wvu.edu/templates/showETD.cfm?recnum=1538.
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Thesis (Ph. D.)--West Virginia University, 2000.Title from document title page. Document formatted into pages; contains xi, 185 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.
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Bligny, Muriel. "Caractérisation d'une ARN polymérase d'origine nuléaire (NEP) dans les plastes d'épinard." Université Joseph Fourier (Grenoble), 1999. http://www.theses.fr/1999GRE10055.
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Nous avons aborde la caracterisation du systeme transcriptionnel plastidial nep grace a une approche in vitro utilisant le promoteur de l'operon plastidial rrn, pc, et des extraits plastidiaux contenant la nep. Dans un premier temps, nous avons verifie que le promoteur pc, utilise dans les chloroplastes d'epinard, est un promoteur nep. Nous avons ensuite separe et caracterise trois activites transcriptionnelles a partir d'extraits chloroplastiques d'epinard partiellement purifies, et en grande partie grace a la mise au point d'un systeme de transcription in vitro. La premiere correspond a la pep. La seconde, appelee nep-2, reconnait le promoteur pc in vitro et la troisieme, ou nep-1, pourrait etre une arnp de 110 kda de type phagique codee par un gene rpot. En ce qui concerne la nep-2, nous avons observe qu'elle n'est pas inhibee par la tagetitoxine tandis qu'elle l'est partiellement par la rifampicine a une concentration elevee d'une part, et d'autre part, qu'elle semble reconnaitre le promoteur t7. Nous en avons deduit que la nep-2 est probablement une arnp de type phagique. Le fait que la nep-1 pourrait elle aussi etre une arnp de type phagique resulte des observations suivantes : elle est reconnue par un anticorps dirige contre une proteine deduite d'un gene rpot, son poids moleculaire apparent est de 110-120 kda et le test alpa montre qu'elle a les proprietes d'une arnp. Ainsi, non pas une, mais deux arnps de type phagique pourraient partager la transcription du genome plastidial avec la pep ! enfin, nous avons demontre que cdf2 est un facteur d'initiation de la transcription conferant a la nep-2 la capacite de reconnaitre specifiquement le promoteur pc.
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Palmer, Matthew T. "The influence of retroviral codon usage on the acquisition of the tRNA used to prime reverse transcription." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2006. https://www.mhsl.uab.edu/dt/2007r/palmer.pdf.
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Casse, Céline. "Etude de l'activation paradoxale de la transcription a partir du promoteur du ltr-vih-1 par les inhibiteurs de la transcription." Paris 5, 2001. http://www.theses.fr/2001PA05S001.
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L'actinomycine d (actd) et -amanitine sont des inhibiteurs frequemment utilises pour inhiber la transcription. De facon inattendue, la transcription a partir du promoteur ltr du virus de l'immunodeficience humaine acquise de type 1 (vih-1) est activee au niveau de l'elongation dans des cellules humaines et murines traitees avec ces drogues, alors que les promoteurs du cytomegalovirus humain et du gene de choc thermique hsp70 sont reprimes. L'activation du ltr-vih-1 ne fait pas intervenir les sequences b et tar presentes dans le promoteur et coincide avec une phosphorylation du domaine c-terminal (ctd) de la plus grande sous-unite de l'arn polymerase ii, rpb1. L'activation du ltr-vih-1 et la phosphorylation du ctd sont toutes deux inhibees par des inhibiteurs de ctd-kinases et en particulier par le 5,6-dichloro-1--d-ribofuranosyl-benzimidazole. L'efficacite avec laquelle ces composes bloquent la phosphorylation du ctd et la transcription in vivo est correlee avec leur capacite d'inhiber la kinase cdk9/pitalre in vitro. Ainsi le facteur positif de l'elongation de la transcription p-tefb contribuerait-il a la phosphorylation du ctd in vivo et a l'activation du ltr-vih-1 induite par l'actinomycine d.
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Delelis, Charlotte Fanien. "Mise en évidence d'un rôle spécifique de la protéine HSP70 dans le contrôle de la transcription par l'ARN polymérase II dans l'ovocyte d'amphibien." Paris 5, 1998. http://www.theses.fr/1998PA05S024.
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Parmi les protéines de choc thermique (HSP), les protéines HSP/HSC70 sont extrêmement bien conservées au cours de l'évolution, de la bactérie à l'homme. Dans les cellules somatiques, HSP70 ET HSC70, en tant que molécules chaperons, participent à de nombreux phénomènes fondamentaux qui contrôlent la physiologie normale de la cellule. Cependant, au cours du développement embryonnaire et notamment dans les ovocytes, leur fonction reste encore mal définie. L'objet de cette thèse a pour but d'analyser le rôle spécifique des protéines HSP70 ET HSC70 dans l'ovocyte de pleurodèles waltl (amphibien, urodèle). Dans le but d'obtenir des outils moléculaires spécifiques de l'une ou l'autre des deux protéines HSP70 ET HSC70 de l'ovocyte de pleurodèle, nous avons cherché à isoler le gène constitutif HSC70, car seul le gène inductible HSP70 avait été précédemment isolé. Afin d'étudier le rôle spécifique de la protéine HSP70 et de la protéine HSC70 au sein de l'ovocyte, nous avons alors fait appel à la stratégie anti sens qui permet d'étudier le rôle précis d'une protéine par l'inactivation de son arnm. Nos résultats montrent que dans l'ovocyte, la protéine HSP70 stockée et la protéine HSP70 neosynthetisee ont des propriétés fonctionnelles différentes : en effet, seule la protéine HSP70 neosynthetisee est impliquée dans la décondensation des chromosomes nécessaire à la transcription par l'arn polymérase ii, alors que la protéine HSC70 ne l'est pas. Nous avons également démontré que ni la protéine HSP70, ni la protéine HSC70 ne sont impliquées dans le contrôle de l'arn polymérase i. Ainsi, bien que les protéines HSP/HSC70 possèdent un degré d'homologie de 80 à 90%, ce travail met en évidence que ces protéines ont des fonctions différentes : leur identité de séquence ne représente donc pas une identité fonctionnelle.
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Lerner, Leticia Koch. "Papel das proteínas XPD e DNA polimerase eta nas respostas de células humanas a danos no genoma." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-20102014-103732/.
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A via de Reparo por Excisão de Nucleotídeos (NER) é responsável pelo reparo das lesões causadas pela luz ultravioleta (UV) e de outras lesões capazes de distorcer a dupla hélice, bloqueando a replicação e a transcrição. Os pacientes que apresentam as síndromes recessivas raras Xeroderma Pigmentosum (XP), tricotiodistrofia (TTD) e síndrome de Cockayne (CS) possuem mutações em algum dos 11 genes relacionados ao NER e à transcrição basal. Mutações na proteína XPD levam ao surgimento de diferentes fenótipos: XP, TTD, XP/CS ou COFS (Cerebro-Oculo-Facio-Skeletal Syndrome), uma forma rara de CS. Os pacientes XP apresentam alta incidência de câncer de pele, o que não ocorre com os pacientes TTD e CS, além de poderem apresentar perda neuronal progressiva, enquanto todos os CS e TTD apresentam uma diminuição na mielinização do cérebro. As neuropatologias são provavelmente associadas a problemas no reparo de danos endógenos no DNA das células nervosas. Diversos trabalhos mostraram o envolvimento do NER no reparo desses danos, os quais pensava-se serem reparados apenas por outro mecanismo, o Reparo por Excisão de Base. Neste trabalho mostramos que fibroblastos de pacientes XP-D, XP-D/CS e TTD, portadores de mutações em XPD, são sensíveis ao estresse oxidativo induzido pelo tratamento com azul de metileno fotoativado, apresentando bloqueio prolongado no ciclo celular e permanência da sinalização de danos ao DNA. A complementação das diferentes linhagens com o gene XPD/ERCC2 foi capaz de restaurar a sobrevivência celular. Foram detectadas diferenças importantes na capacidade de reparo/retomada da transcrição após danos gerados por estresse oxidativo em DNA plasmidial, além da ativação de vias diferentes de morte celular: fibroblastos XP-D apresentam maior capacidade de reparo e apresentam morte por apoptose após estresse oxidativo, enquanto os fibroblastos XP-D/CS e TTD apresentam menor capacidade de reparo ativação de mais de uma via de morte celular (apoptose e necrose), diferenças que podem estar ligadas ao fenótipo dos pacientes. Mutações no gene codificante para a DNA polimerase n, POLH, estão associadas à forma variante de XP (XP-V). Pol n é uma polimerase especializada na síntese translesão (TLS) de fotoprodutos, além de estar implicada na TLS de outros tipos de lesões como bases oxidadas, e em vias não relacionadas à TLS como a hipermutação somática e à replicação de regiões de DNA com arquiteturas não-canônicas. Neste trabalho mostramos que os fibroblastos de pacientes XP-V apresentam sensibilidade ao estresse oxidativo. Mostramos uma indução da proteína pol n em fibroblastos primários após danos genotóxicos, associada ao aumento da capacidade de lidar com a parada na forquilha de replicação, possibilitando a continuidade da replicação do DNA e ao aumento da sobrevivência celular. Mostramos uma diferença na estabilidade genômica nos genes das imunoglobulinas dos pacientes XP-V idosos em comparação com os pacientes jovens e controles de idade pareada, mostrando que a ausência dessa polimerase pode estar ligada ao aumento da instabilidade genômica nesses genesThe Nucleotide Excision Repair (NER) pathway is responsible for the repair of UV photoproducts and other bulky lesions that block both replication and transcription. Patients with the rare recessive disorders Xeroderma Pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne Syndrome (CS) carry mutations in one of the 11 NER genes, linked to repair and basal transcription. Mutations in XPD lead to different phenotypes: XP, TTD, XP/CS or COFS (Cerebro-Oculo-Facio-Skeletal Syndrome), a rare form of CS. XP patients have high incidence of skin cancer, which does not occur in TTD or CS patients, although ther may present neurodegeneration, while all CS and TTD patients have neurodevelopmental symptoms linked to dysmielynation. The pathology of these neurological diseases is probably associated with deficient repair of DNA lesions in nervous cells, generated by endogenous processes. Many groups including ours have demonstrated the involvement of NER in the repair of these lesions, previously thought to be exclusively repaired by Base Excision Repair. In this work we show high sensitivity of both primary and transformed XP-D, XP-D/CS and TTD human fibroblasts in response to oxidative stress generated by photoactivated methylene blue, with prolonged cell cycle arrest and DNA damage signaling. The complementation of the three different cell lines with the XPD/ERCC2 gene was able to restore cell survival. We detected important differences in repair capacity/transcription resumption after damage generated by oxidative stress in plasmid DNA, besides the activation of different cell death pathways: XP-D cells have higher repair capacity and die by apoptosis, while XP-D/CS and TTD cells have little repair capacity and activate more than one death pathway (apoptosis and necrosis). We believe these differences can be related to the patients\' phenotypes. Mutations in DNA polymerase n coding gene, POLH, are associated with the variant form of XP (XP-V). Pol n is a translesion synthesis (TLS) polymerase specialized in the TLS past CPD photoproducts, besides other lesions like oxidized bases, and in other processes like somatic hypermutation and DNA replication in structured regions. In this work we show XP-V human fibroblasts are sensitive to oxidative stress. We detected an induction of pol n after genotoxic stress in primary cells, associated with increased ability to deal with the stalled replication fork, and consequently to DNA replication restart and cell survival. In addition, we detected a difference in genomic stability in immunoglobulin genes in aged XP-V patients in comparison to both young patients and age-matched controls, showing the absence of this polymerase may be linked to increased genomic instability in these genes
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Ong, Jennifer Lee. "Directed evolution of DNA polymerases with altered substrate specificities." Thesis, University of Cambridge, 2004. https://www.repository.cam.ac.uk/handle/1810/284037.
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Ostler, Jeffery Brent Jr. "Characterization of Pol IV and Pol V-Dependent Non-Coding RNAs Derived from aGeminivirus Genome." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1492698361649423.
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Angius, Federica. "Molecular basis of membrane protein production and intracellular membranes proliferation in E. coli." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC217/document.
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Le système d’expression le plus utilisé pour la production des protéines membranaires, est le système basé sur l’ARN polymérase T7 (ARNpol T7) (Hattab et al., 2015). L'inconvénient de ce système est néanmoins que la vitesse de transcription de l’ARNpol T7 est dix fois plus rapide que celle de l’enzyme bactérienne. Depuis l’isolement de mutants spontanés, notamment C41 (DE3) et C43 (DE3) (Miroux et Walker, 1996) et l’identification de leurs mutations dans le génome, il apparaît clairement que la toxicité provoquée par la surproduction des protéines membranaires est liée à la quantité trop élevée d’ARNpol T7 dans la cellule (Wagner et al., 2008 ; Kwon et al., 2015). Les protéines membranaires ont besoin d’une vitesse de transcription/traduction plus basse pour se replier correctement dans la membrane de la bactérie. Le premier objectif de ma thèse était d’étendre l’amplitude du promoteur du système T7 sur laquelle est basée l’expression des protéines. Pour cela, nous avons isolé et caractérisé de nouvelles souches bactériennes dans lesquelles le niveau d’ARNpol T7 était efficacement régulé par un mécanisme non transcriptionnel très favorable à l’expression des protéines membranaires (Angius et al., 2016). Le deuxième objectif était de comprendre la prolifération des membranes intracellulaires chez E. coli suite à la surexpression de la protéine AtpF, une sous unité membranaire du complexe de l’ATP synthétase (Arechaga et al., 2000). Pour mieux comprendre les voies métaboliques impliquées dans la biogenèse, la prolifération et l’organisation des membranes, nous avons utilisé une approche de séquençage d’ARN à haut débit à différents temps après induction de la surexpression de la sous-unité AtpF dans la souche C43 (DE3). Ensuite, et en collaboration avec Gerardo Carranza and Ignacio Arechaga (Université de Cantabria, Espagne), nous avons construit et étudié des mutants de C43 (DE3) déficients pour les trois gènes codants pour des enzymes de la biosynthèse des cardiolipides afin d’évaluer leur participation dans la biogénèse des membranes intracellulairesThe most successful expression system used to produce membrane proteins for structural studies is the one based on the T7 RNA polymerase (T7 RNAP) (Hattab et al., 2015). However, the major drawback of this system is the overtranscription of the target gene due to the T7 RNAP transcription activity that is over ten times faster than the E. coli enzyme. Since the isolation of spontaneous mutants, namely C41(DE3) and C43(DE3) (Miroux and Walker, 1996) and the identification of their mutation in the genome, it becomes clear that reducing the amount of the T7 RNAP level removes the toxicity associated with the expression of some membrane proteins (Wagner et al., 2008; Kwon et al., 2015). Also, some membrane proteins require a very low rate of transcription to be correctly folded at the E. coli membrane. The first objective of my PhD was to extend the promoter strength coverage of the T7 based expression system. We used genetic and genomic approaches to isolate and characterize new bacterial strains (Angius et al., 2016) in which the level of T7 RNAP is differently regulated than in existing hosts. A second objective was to understand intracellular membrane proliferation in E. coli. Indeed it has been shown that over-expression of membrane proteins, like overexpression of AtpF of E. coli F1Fo ATP synthase is accompanied by the proliferation of intracellular membranes enriched in cardiolipids (Arechaga et al., 2000). To understand metabolic pathways involved in membrane biogenesis, proliferation and organization, we used a RNA sequencing approach at several time point upon over-expression of the F-ATPase b subunit in C43(DE3) host. On the other hand, in collaboration with Gerardo Carranza and Ignacio Arechaga (University of Cantabria, Spain) we studied C43(DE3) cls mutants, in which the cardiolipids genes A, B and C are deleted, to test how they participate to intracellular membranes structuration
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Das, Prolay. "Long-Range Charge Transfer in Plasmid DNA Condensates and DNA-Directed Assembly of Conducting Polymers." Diss., Georgia Institute of Technology, 2007. http://hdl.handle.net/1853/19856.
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Long-distance radical cation transport was studied in DNA condensates where linearized pUC19 plasmid was ligated to an oligomer and transformed into DNA condensates with spermidine. DNA condensates were detected by Dynamic Light Scattering and observed by Transmission Electron Microscopy. Introduction of charge into the condensates causes long-distance charge migration, which is detected by reaction at the remote guanines. The efficiency of charge migration in the condensate is significantly less than it is for the corresponding oligomer in solution. This result is attributed to a lower mobility for the migrating radical cation in the condensate, caused by inhibited formation of charge-transfer-effective states. Radical cation transport was also studied in DNA condensates made from an oligomer sandwiched between two linearized plasmids by double ligation. Unlike the single ligated plasmid condensates, the efficiency of charge migration in the double ligated plasmid-condensates is high, indicative of local structural and conformational transformation of the DNA duplexes.
Organic monomer units having extended ð-conjugation as part of a long conducting polymer was synthesized and characterized. The monomer units were covalently attached to particular positions in DNA oligonucleotides by either the convertible nucleotide approach or by phosphoramidite chemistry. Successful attachment of the monomer units to DNA were confirmed by mass spectral analysis. The DNA-conjoined monomer units can self assemble in the presence of complementary sequences which act as templates that can control polymer formation and structure. By this method the para-direction of the polymer formation can be enforced and may be used to generate materials having nonrecurring, irregular structures.
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D'Abbadie, Marc François. "Directed evolution of polymerases with altered substrate specificities : the paradigm of ancient DNA." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613870.
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Zahoransky, Viktor Wendelin. "Information Transmission Across Generations : Thermodynamics and Evolutionary Implications." Electronic Thesis or Diss., Université Paris sciences et lettres, 2023. http://www.theses.fr/2023UPSLS012.
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La Phi29 ADN polymérase (DNAP) dérive du bactériophage Phi29 et réplique l'ADN dans des conditions isothermes par amplification en cercle roulant. Il s'agit d'une enzyme particulièrement intéressante en raison de son exceptionnelle processivité et de son faible taux d'erreur, de l'ordre d'une paire de bases mésapparente pour chaque 10^(-5) à 10^(-6) nucléotides incorporés. La Phi29 DNAP atteint cette haute fidélité grâce à une fonction catalytique supplémentaire: La capacité à corriger les erreurs d'incorporation de bases par excision de nucléotides. Malgré les nombreuses études qui ont déjà été menées sur cette enzyme, la coordination entre ses principales fonctions catalytiques, la synthèse de l'ADN et la correction des erreurs, n'est pas entièrement comprise. Dans ce travail, nous développons plusieurs essais massivement parallélisés et à très haut débit, basés sur de grandes bibliothèques de gènes (10^(6)), afin de tester et de cribler les variants de la Phi29 DNAP dans un contexte évolutif. Pour la première fois, une technique d'émulsification membranaire est adaptée aux réactions d'auto-réplication compartimentée isotherme in vitro (iviCSR) facilitant le criblage simultané de variants dans différentes conditions environnementales. Nous avons trouvé des preuves que le variant R223T de la Phi29 DNAP peut répliquer l'ADN de manière plus progressive que l'enzyme WT dans des conditions éprouvantes et que la position de l'acide aminé 223 contribue à la coordination du compromis activité-fidélité de l'enzymePhi29 DNA polymerase (DNAP) derives from bacteriophage Phi29 and replicates DNA under isothermal conditions by rolling circle amplification. It is a particularly interesting enzyme due to its outstanding processivity and low error rates in the range of one base-pair mismatch for every 10^(-5) to 10^(-6) nucleotides incorporated. Phi29 DNAP achieves such high fidelity by means of an additional catalytic function: The ability to correct for base misincorporations by nucleotide excision. Despite the many studies that have already been conducted on this enzyme, the coordination between its main catalytic functions, DNA synthesis and error correction, is not fully understood.In this work we develop several massively parallelised, ultra-high-throughput assays, based on large (10^(6)) gene libraries, to challenge and screen for Phi29 DNAP variants in an evolutionary setting. For the first time, a membrane emulsification technique is adapted to in vitro isothermal compartmentalised self-replication (iviCSR) reactions facilitating simultaneous screenings of variants in different environmental conditions. We found evidence that Phi29 DNAP variant R223T can replicate DNA more processively than the WT enzyme under challenging conditions and that amino acid position 223 contributes to the coordination of the enzyme's activity-fidelity trade-off
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Ho, Wan Kévin. "Etude de l'initiation du processus de réparation de l'ADN couplée à la transcription par pinces magnétiques." Paris 7, 2011. http://www.theses.fr/2011PA077191.
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La réparation de l'ADN couplée à la transcription (TCR), qui cible les lésions de l'ADN perturbant la progression du complexe d'élongation au cours de la transcription, est un mécanisme important dans le maintien de l'intégrité du génome. La TCR est retrouvé aussi bien chez les procaryotes que chez les eucaryotes. Certaines maladies héréditaires humaines, comme le syndrome de Cockayne, sont associées à des anomalies dans cette voie de réparation. Chez la bactérie Escherichia coli, la translocase TRCF, produit du gène mfd, joue un rôle central dans cette réponse cellulaire: elle reconnaît une ARN polymérase bloquée en élongation, dissocie le complexe d'élongation et recrute la machinerie de réparation UvrABC. Nous avons étudié le mécanisme de la TCR par micromanipulation de molécules d'ADN individuelles à l'aide d'une pince magnétique. Cette approcha nous a permis de suivre la dissociation d'une ARN polymérase par TRCF en temps réel. L'analyse de la distribution des temps de dissociation du complexe d'élongation à différentes concentrations de TRCF nous a permis de mettre en évidence et de caractériser les étapes cinétiquement limitantes du recrutement de TRCF et de son activité catalytique au cours de la TCR. Ainsi, notre étude montre que la dissociation de TARN polymérase par TRCF se fait en 3 étapes: (i) recrutement de TRCF sur la polymérase bloquée, (ii) activation de TRCF et initiation de la dissociation par réenroulement de 2/3 de la bulle de transcription et (iii) dissociation complète de l'ARN polymérase. Nous proposons que cette dernière étape, dont la durée est relativement longue, permettrait à TRCF de recruter les enzymes de réparation par interaction directeTranscription coupled repair (TCR), a sub-pathway of the nucleotide excision repair mechanism, is activated when a RNA polymerase (RNAP) is arrested during transcription by DNA damage. TCR is a ubiquitous cellular response important for maintenance of DNA integrity. Some human genetic disorders are associated with defect on TCR, like the Cockayne syndrom for instance. In the bacterium Escherichia coli, TRCF, the product of the mfd gene, is the DNA translocase that couples transcription and DNA repair: it recognizes a stalled ternary elongation complex, dissociates it, and recruits the UvrABC repair machinery. We used a single molecule approach, by rneans of magnetic tweezers, to study the initation of TCR. This approach allowed us to monitor the dissociation of a single RNAP by a single TRCF in real time. Statistical analysis of the time required to dissociate the stalled RNAP at different concentrations of TRCF has shown that the displacement of the ternary elongation complex consists is a three steps process: (i) recruitment of TRCF to the stalled RNAP, (ii) activation of TRCF and initiation of the dissociation of RNAP by rewinding of 2/3 of the transcription bubble, and (iii) complete dissociation of the elongation complex. The complex present during this last step is characterized by a long lifetime, which suggests that it would behave as a temporally reliable marker of DNA damage, favorizing the recruitment of the UvrA
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Gouge, Jérôme. "Etudes biophysique et structurale d'ADN polymérases." Paris 7, 2011. http://www.theses.fr/2011PA077087.
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La réplication du génome est réalisée par des ADN polymérases qui vont dupliquer l'information génétique en insérant un nucléotide dans le brin amorce de façon complémentaire au brin matrice. La sélection du nucléotide est réalisée lors de sa liaison dans le site catalytique. Le taux d'erreur des polymérases est d'environ 10⁻⁶ erreurs/base répliquée. Cette fidélité est assurée parce que les réplicases peuvent dégrader l'amorce en cas de mésappariement. Cependant, l'ADN est soumis à des agressions constantes de l'environnement si bien que Les réplicases doivent parfois faire face à des bases lésées ou des cassures double brin. Dans certains cas, elles peuvent insérer un nucléotide dans le brin amorce alors que l'instruction n'est pas canonique. Dans d'autres cas, elles s'arrêtent pour faire intervenir la machinerie de réparation, où d'autres ADN polymérases sont impliquées. Nous avons entrepris des études structurales d'une réplicase d'archée, P. Abyssi, pour comprendre son interaction avec un ADN canonique mais aussi avec des bases désaminées. A l'aide d'une dizaine de structures à résolution atomique, nous avons pu proposer un modèle rendant compte de l'arrêt de la polymérase lorsqu'elle reconnaît une base désaminée en amont du site catalytique. Un second volet porte sur des polymérases impliquées dans la réparation de l'ADN au travers de l'étude de polymérases X eucaryotes et bactériennes. Nous avons en effet résolu à 2,5 À la structure du complexe ternaire d'une polymérase X eucaryote. L'étude cristallographique des polymérases X bactériennes a conduit à enregistrer des données à 3,4 À. Les données SAXS suggèrent un arrangement des domaines inédits et très ouvertIn all cells, the replication step is achieved by DNA polymerases that duplicate genetic information by adding nucleotides at the 3' end of a primer using complementarity to a template strand. The correct nucleotide is chosen after binding in the catalytic site. To get an error rate as low as 10"6 errors/replicated base, polymerases have the ability to degrade the primer strand if a mismatch is incorporated. However DNA is subjected to various exogenous and endogenous attacks so that polymerases are often facing damaged bases or double strand breaks. Depending on the organism they are able to replicate genetic information even if it has been corrupted. In some other cases they stop until DNA reparation pathways come into play. We have conducted structural studies on an archaeal (P. Abyssi) DNA polymerase to order to better understand the discrimination between canonical and deaminated bases. Using a dozen crystallographic structures we have proposed a model that describes how the protein recognizes damaged bases before they enter in the catalytic site. A second part of the work deals with studies of eukaryotic and bacterial polymerases involved in DNA repair. We solved at 2,5 À the structure of a eukaryotic ternary complex that shows new interactions between the protein and DNA. Crystallographic studies two bacterial polymerases recently led to a 3,4 À data set that allowed molecular replacement solution to be found. The SAXS data indicate a new arrangement of the different domains in an open and extended way
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Siegmund, Vanessa [Verfasser]. "DNA and RNA Polymerases with Expanded Substrate Scope : Synthesis of Modified Nucleic Acids Using Engineered Polymerases Generated by Directed Evolution / Vanessa Siegmund." Konstanz : Bibliothek der Universität Konstanz, 2013. http://d-nb.info/1043443320/34.
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Amiott, Elizabeth Anne. "A model for the carbon source regulation of yeast mitochondrial transcription /." Connect to full text via ProQuest. IP filtered, 2005.
Find full textAbstract:
Thesis (Ph.D. in Molecular Biology) -- University of Colorado, 2005.Typescript. Includes bibliographical references (leaves 100-113). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
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Chadsey, Meggen Shepherd. "Regulation of the flagellar specific sigma factor, sigma28, of Salmonella typhimurium by the anti-sigma factor FlgM /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/11490.
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Kardashliev, Tsvetan Dinkov Verfasser], Ulrich [Akademischer Betreuer] [Schwaneberg, and Lothar [Akademischer Betreuer] Elling. "Directed Evolution of DNA Polymerases for Advancement of the SeSaM Mutagenesis Method and Biotransformations with P450 BM3 Monooxygenase / Tsvetan Dinkov Kardashliev ; Ulrich Schwaneberg, Lothar Elling." Aachen : Universitätsbibliothek der RWTH Aachen, 2015. http://d-nb.info/1126124001/34.
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Kardashliev, Tsvetan Dinkov [Verfasser], Ulrich [Akademischer Betreuer] Schwaneberg, and Lothar [Akademischer Betreuer] Elling. "Directed Evolution of DNA Polymerases for Advancement of the SeSaM Mutagenesis Method and Biotransformations with P450 BM3 Monooxygenase / Tsvetan Dinkov Kardashliev ; Ulrich Schwaneberg, Lothar Elling." Aachen : Universitätsbibliothek der RWTH Aachen, 2015. http://d-nb.info/1126124001/34.
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Vasale, Jessica J. "Roles of Cellular RNA-Dependent RNA Polymerases in Endogenous Small RNA Pathways in Caenorhabditis elegans: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/481.
Full textAbstract:
The RNA interference (RNAi) pathway in Caenorhabditis elegans is a two-step, small RNA-mediated silencing pathway. Unlike in other organisms, Dicer processing of double-stranded RNA into small interfering (si) RNAs is not sufficient in worms to induce gene silencing. The activity of cellular RNA-dependent RNA polymerase (RdRP) is necessary to synthesize a secondary pool of siRNAs, which interact with a unique class of Argonaute proteins to form the functional effector complexes that mediate silencing. The aims of this thesis were to: 1) characterize the role of RdRP family members in endogenous small RNA biogenesis; 2) identify the Argonaute proteins that interact with RdRP-dependent small RNAs; and 3) investigate the biological function of RdRP-dependent small RNA pathways in C. elegans.
In this thesis, I describe genetic, deep sequencing, and molecular studies, which identify 22G-RNAs as the most abundant class of endogenous small RNA in C. elegans. The 22G-RNAs resemble RdRP-dependent secondary siRNAs produced during exogenous RNAi, in that they possess a triphosphorylated 5’ guanine residue and exhibit a remarkable strand bias at target loci. Indeed, I show that 22G-RNAs are dependent on the activity of the RdRPs RRF-1 and EGO-1 and function in multiple distinct endogenous small RNA pathways. Interestingly, I have found that RRF-1 and EGO-1 function redundantly in the germline to generate 22G-RNAs that are dependent on and interact with members of an expanded family of worm-specific Argonaute (WAGO) proteins. The WAGO/22G-RNA pathway appears to be a transcriptome surveillance pathway that silences coding genes, pseudogenes, transposons, and non-annotated, or cryptic, transcripts. In contrast, I have found that EGO-1 alone is required for the biogenesis of a distinct class of 22G-RNAs that interact with the Argonaute CSR-1. Surprisingly, the CSR-1/22G-RNA pathway does not appear to silence its targets transcripts. Instead, the CSR-1/22G-RNA pathway is essential for the proper assembly of holocentric kinetochores and chromosome segregation.
Lastly, I show that a third endogenous small RNA pathway, the ERI pathway, is a two-step silencing pathway that requires the sequential activity of distinct RdRPs and Argonautes. In the first step of this pathway, the RdRP, RRF- 3, is required for the biogenesis of 26G-RNAs that associate with the Argonaute, ERGO-1. In the second step, RRF-1 and EGO-1 generate 22G-RNAs that associate with the WAGO Argonautes.
This work demonstrates how several C. elegans small RNAs pathways utilize RdRPs to generate abundant populations of small RNAs. These distinct categories of small RNAs function together with specific Argonaute proteins to affect gene expression, to play essential roles in development, and in the maintenance of genome and transcriptome integrity.
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Sharma, Vikas. "Approaches to detect and classify Megavirales." Thesis, Aix-Marseille, 2015. http://www.theses.fr/2015AIXM5031.
Full textAbstract:
Les Megavirales appartiennent à des familles de virus géants infectant un grand nombre d'hôtes eucaryotes. Leurs génomes ont des tailles variant de 100 kb to 2.5 mb et leur composition a montré des caractéristiques surprenantes qui ont soulevées diverses questions sur l’origine et l’évolution de ces virus. Les études de métagénomique environnementale ont montré qu’il existe une «matière noire», composée de séquences reliées à aucun organisme connu. Cependant, l'identification des séquences a été principalement réalisée en utilisant les séquences ADN ribosomal (ADNr), ce qui conduit à ignorer les virus. D’autres gènes informationnels « cœur », incluant la DNA-dependant RNA polymerase (RNAP) constituent d'autres marqueurs qui apparaissent comme plus appropriés pour une classification plus exhaustive des séquences, puisqu’ils apparaissent conservés dans les organismes cellulaires ainsi que les mégavirus. Nous avons utilisé un petit ensemble de gènes universels conservés incluant la RNAP et avons reconstruit des séquences ancestrales pour rechercher des séquences reliées aux mégavirus dans les bases de données. Cela a permis d’identifier trois nouvelles séquences de megavirus qui avaient été mal annotées comme correspondant à des organismes cellulaires, ainsi que de nouveaux clades viraux dans les bases métagénomiques environnementales. De plus, nous avons montré que l’ordre Megavirales constituait une quatrième branche monophylogénétique ou « TRUC » (pour Things Resisting Uncompleted Classification). Nos analyses montrent également que la RNAP ainsi que quelques autres gènes utilisés dans nos études permettent de considérer un répertoire plus complet d’organismes que l’ADNrNucleocytoplasmic large DNA viruses (NCLDVs), or representatives of order Megavirales, belong to families of giant viruses that infect a large number of eukaryotic hosts. These viruses genomes size ranges from 100 kb to 2.5 mb and compose surprising features, which raised various questions about their origin and evolution. Environmental metagenomic studies showed that there is a “dark matter”, composed of sequences not linked to any known organism. However sequence identification was mainly determined using ribosomal DNA (rDNA) sequences, which led therefore to ignore viruses, because they are devoid of such genes. Informational genes, including DNA-dependant RNA polymerase (RNAP), are other markers that appear as more appropriate for a comprehensive classification as they are conserved in cellular organisms (Bacteria, Archaea and Eukarya) and in Megavirales. We used a small set of universally conserved genes that included RNAP and reconstructed ancestral sequences to search for megavirus relatives in sequence databases and to perform phylogeny reconstructions. This allowed identified three megaviral sequences that were misannotated as cellular orgainsms, and new viral clades in environmental databases. In addition, we delineated Megavirales as a fourth monophylogenetic TRUC (things resisting uncompleted classification) aside cellular organisms. Moreover, we classified by phylogenetic and phyletic analyses based on informational genes new giant viruses as new bona fide members of the fourth TRUC. Our analyses shows that RNAP as well as a few other genes used in our studies allow a more comprehensive overview and classification of the biological diversity than rDNA
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Kosarek, Jayme Nicole. "Characterization of the in vivo functions of Y-Family DNA polymerases kappa and Rev1." 2008. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=402.
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"The development of a rapid detection method for mycobacterium tuberculosis in clinical specimens using DNA amplification." Chinese University of Hong Kong, 1995. http://library.cuhk.edu.hk/record=b5888502.
Full textAbstract:
by Au Lai Yin, Cathy.Thesis (M.Phil.)--Chinese University of Hong Kong, 1995.
Includes bibliographical references (leaves 50-66).
Chapter I. --- ABSTRACT --- p.i
Chapter II. --- ACKNOWLEDGMENTS --- p.iii
Chapter III. --- TABLE OF CONTENTS --- p.iv
Chapter IV. --- LIST OF TABLES --- p.viii
Chapter V. --- LIST OF FIGURES --- p.x
Chapter VI. --- INTRODUCTION --- p.1
Chapter VII. --- LITERATURE REVIEW --- p.3
Chapter A. --- Mycobacterial tuberculosis Infections --- p.3
Chapter B. --- Diagnostic Criteria forM .tuberculosis Infections --- p.3
Chapter C. --- Mycobacteriological Laboratory Investigations for M. tuberculosis --- p.4
Chapter 1. --- Conventional methods --- p.4
Chapter 2. --- Rapid methods --- p.4
Chapter D. --- Polymerase chain reaction (PCR) - the Principle --- p.5
Chapter E. --- Application of PCR for Detection of M. tuberculosis --- p.6
Chapter 1. --- Choice of target sequences --- p.6
Chapter 2. --- Choice of method for the detection & identification of PCR-amplified product --- p.7
Chapter 3. --- Studies on pure cultures --- p.9
Chapter a. --- Detection limit - target DNA --- p.9
Chapter b. --- Detection limit - Colony forming units --- p.9
Chapter c. --- Detection limit - Number of cells --- p.10
Chapter 4. --- Studies on clinical specimens --- p.10
Chapter 5. --- Problems --- p.12
Chapter a. --- Availability of target DNA --- p.13
Chapter (i) --- Cell breakage efficiency --- p.13
Chapter (ii) --- Target sequence --- p.14
Chapter b. --- Inhibitory factors for Taq polymerase --- p.14
Chapter c. --- Contamination --- p.15
Chapter VIII. --- MATERIALS AND METHODS --- p.16
Chapter A. --- Bacterial Strains and Strain Maintenance --- p.16
Chapter 1. --- Reference Strains --- p.16
Chapter 2. --- Clinical isolates --- p.16
Chapter B. --- Growth media and culture conditions --- p.17
Chapter C. --- Restriction Fragment Length Polymorphism (RFLP) --- p.17
Chapter 1. --- Extraction of chromosomal DNA from M. tuberculosis --- p.18
Chapter 2. --- Digestion of chromosomal DNA by PVU II --- p.19
Chapter 3. --- Separation of digested DNA fragment by electrophoresis --- p.19
Chapter 4. --- Southern Blotting --- p.19
Chapter 5. --- Preparation of DNA probes by Polymerase Chain Reaction --- p.20
Chapter 6. --- Hybridization --- p.21
Chapter 7. --- Detection --- p.21
Chapter D. --- Assessment of number of organisms --- p.22
Chapter 1. --- Viable cell count --- p.22
Chapter 2. --- Direct cell count --- p.22
Chapter E. --- Assessment of the presence of IS6110/986 in M. tuberculosis isolates --- p.23
Chapter F. --- Human leukaemic monocytic cell line (THP-1) --- p.23
Chapter 1. --- Growth media and maintenance --- p.23
Chapter 2. --- Culture Conditions --- p.24
Chapter 3. --- Uptake of M. tuberculosis --- p.24
Chapter G. --- Cell breakage and DNA extraction methodologies --- p.25
Chapter H. --- Polymerase chain reaction (PCR) methodologies --- p.28
Chapter 1. --- Primer and probe --- p.28
Chapter 2. --- PCR conditions --- p.28
Chapter 3. --- Detection --- p.29
Chapter I. --- Patients and Clinical specimens --- p.30
Chapter 1. --- Patients recruitment --- p.30
Chapter 2. --- Clinical specimens --- p.30
Chapter IX. --- RESULTS --- p.32
Chapter A. --- "Development or Selection of a ""Standardized"" PCR Protocol for the Detection of M. tuberculosis Using Pure Cultures In Vitro" --- p.32
Chapter 1. --- Selection of organisms for verification of the PCR protocol --- p.32
Chapter 2. --- Optimization of the PCR conditions --- p.32
Chapter 3. --- Detection limit of target DNA using the PCR procedure --- p.33
Chapter B. --- Initial Screening of Six Different Cell Breakage Procedures Using Pure Cultures of M. tuberculosis Isolates TB19 &22a Based on Detection Limits of Colony Forming Units and Number of Cells --- p.34
Chapter C. --- Comparison of Method 1 and Method 2 Based on Detection Limits of Colony Forming Units and Number of Cells Using Pure Cultures of the Eight Clinical Isolates of M. tuberculosis with variable copies of IS6110/986 --- p.34
Chapter D. --- Detection of M. tuberculosis Isolates Within Macrophages --- p.35
Chapter 1. --- Uptake of M. tuberculosis cells by THP-1 --- p.35
Chapter 2. --- Comparison of the Six Different Cell Breakage Procedures Using Pure Cultures of M. tuberculosis Isolates TB19 & 22a Phagocytized by Activated THP-1 Macrophages --- p.35
Chapter 3. --- Comparison of Method 1 and Method 2 Using Pure Cultures of the Eight Clinical Isolates of M. tuberculosis Phagocytized by Activated THP-1 Macrophages --- p.36
Chapter E. --- Analysis of Clinical Specimens Using Method 1 & 2 with the Optimized PCR Protocol --- p.36
Chapter 1. --- Bronchial Aspirate & Bronchoaveolar Lavage Fluid --- p.36
Chapter 2. --- Pleural Fluid --- p.37
Chapter 3. --- Tissue --- p.37
Chapter 4. --- Sputum --- p.38
Chapter 5. --- Cerebrospinal Fluid --- p.38
Chapter X. --- DISCUSSION --- p.39
Chapter A. --- Selection of IS6110/986 for DNA amplification --- p.39
Chapter B. --- Optimization of PCR conditions reflected by detection limit of target DNA --- p.40
Chapter C. --- Selection of cell breakage methods based on detection limits of CFU and/or number of mycobacterial cells --- p.41
Chapter D. --- Application of Methods 1 & 2 and the optimized PCR protocol for clinical specimens --- p.43
Chapter 1. --- Bronchial aspirates and bronchoaveolar lavage fluids --- p.43
Chapter 2. --- Pleural fluids --- p.44
Chapter 3. --- Tissues --- p.45
Chapter 4. --- Sputa --- p.46
Chapter 5. --- Cerebrospinal fluids --- p.46
Chapter XI. --- CONCLUSION --- p.48
Chapter XII. --- LITERATURE CITED --- p.50
Chapter XIII --- TABLES --- p.67
Chapter XIV. --- FIGURES --- p.85
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Kranaster, Ramon [Verfasser]. "DNA polymerase activity on solid support : from diagnostics to directed enzyme evolution / vorgelegt von Ramon Kranaster." 2010. http://d-nb.info/1007476508/34.
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"Structural analysis of influenza A virus nucleoprotein and its interaction with RNA and polymerase subunit PB2." Thesis, 2011. http://library.cuhk.edu.hk/record=b6075218.
Full textAbstract:
The poultry-to-human transmission of the influenza virus and the recent H1Nl influenza pandemic have become major concerns worldwide. The nucleoprotein (NP) of influenza virus binds the RNA genome and plays essential role in transcription and replication during the virus life cycle.The study leads to a better understanding towards the RNP organization of influenza virus and provides information for the future design of anti-influenza agents.
We have also shown, by RNP reconstitution assay and co-immunoprecipitation, that the interaction between NP and PB2 is crucial for the proper functioning of the RNP. The functional association of NP and PB2 requires either the PB2 host-determining residue lysine-627 or arginine-630 with the latter involving NP arginine-150 also. Using SPR, we have demonstrated that both residues take part in the direct protein-protein interaction, without the involvement of RNA. These results suggest a dual interaction mechanism between NP and PB2. This may confer replication advantages to the virus, as either one can give an active RNP and explains the increased virulence of avian influenza viruses carrying the E627K mutation in mammalian cells. In addition, our findings identify the NP-PB2 interacting surface, with the PB2 627/630 region facing the RNA binding groove of NP.
We have determined the 3.3 A crystal structure of H5N1 NP, which is composed of head and body domains and a tail loop. Using surface plasmon resonance (SPR), we found the basic loop (residues 73-91) and arginine-rich groove, but mostly a protruding element centering at R174 and R175, to be important in RNA binding. Ribonucleoprotein (RNP) reconstitution assay with these multiple-point and deletion mutants indicate their functional importance towards the transcription-replication activities of the virus polymerase. Single-point mutations at these concerned regions do not have a significant effect on their RNP activities, suggesting that NP mediates RNA-binding through multiple residues.
Ng, Ka Leung.
Adviser: Pang Chui Shaw.
Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 121-136).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
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Čermák, Vojtěch. "Studium mechanismu posttranskripčního a transkripčního umlčování transgenů v buněčné linii tabáku BY-2." Master's thesis, 2012. http://www.nusl.cz/ntk/nusl-306690.
Full textAbstract:
The RNA interference is a mechanism, which allows cells to regulate their genes functions, to establish and maintain heterochromatin and to defend them against invasive nucleic acids. In plants, RNA interference is initiated by double-stranded RNA, which is processed by Dicer into small RNAs, usually 20-24nt long. These small RNAs form a complex with Argonaut protein that participates in different processes based on sequence complementarity. This complex can guide mRNA cleavage, translation blocking and chromatin modifications, resulting either into posttranscriptional silencing (by preventing translation of already existing mRNA, PTGS) or transcriptional silencing (by preventing transcription of mRNA, TGS). The first step of this thesis was to establish different ways of triggering PTGS and to evaluate their functionality and efficiency. The next step was a preparation of a system which would allow to study the transition from posttrancriptional to transcriptional silencing. These so called "indicator lines" should allow to observe the timing and dynamics of this process by utilizing fluorescent proteins. This system is also going to enable to evaluate, how different factors are involved in this process - one of the factors is RNA-dependent RNA polymerase 6 (RDR6) which plays an essential role in...