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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
11

Estudos de reconhecimento biomolecular por eletroforese capilar / Capillary electrophoresis-based biomolecular recognition studies

Sandro Hillebrand 09 September 2005 (has links)
Esta tese trata do desenvolvimento de métodos bioanalíticos, baseados na técnica de eletroforese capilar, para duas aplicações distintas: a análise de complexos formados pela ligação entre proteínas e DNA e detecção e monitoramento de hidrólise de GTP catalisada por enzimas. No primeiro capítulo descreve-se uma investigação sobre a viabilidade de ensaios tipo EMSA (?electrophoretic mobility shift assay?) em chips com microcanais para eletroforese. Os fatores de transcrição purificados c-Jun(AP1) e p-50(NFkB) foram usados nos estudos de ligação a sondas de DNA fita dupla contendo as seqüências consenso de ligação dos fatores AP1, NFkB e AP2. As sondas de DNA sintéticas continham como modificação a marcação com o corante fluorescente Cy5 ligado à extremidade 5?, sendo que as mesmas seqüências não marcadas foram usadas para experimentos de competição. Ensaios tipo EMSA em chip puderam ser realizados em cerca de 2 h com baixo consumo de amostra e sem a necessidade de usar marcação com material radioativo. A sondas de DNA e os complexos formados nas reações de ligação foram analisados no Bioanalyzer usando tanto o procedimento padrão para a análise de DNA quanto um protocolo modificado. Nesta modificação não foi usado corante de intercalação mas 4,9 nM de Cy5-dCTP que foi adicionado ao gel, permitindo apenas a detecção de DNA previamente marcado. Apesar da necessidade de ajustes no método para cada proteína testada, foi mostrado o potencial de se substituir o método de EMSA em gel por métodos baseados em eletroforese em chip. Um experimento de competição foi realizado com sucesso mostrando a ligação do fator de transcrição p-50 à sonda contendo a seqüência consenso NFkB. Este experimento foi considerado como prova de princípio para a hipótese estudada. No segundo capítulo relata-se o desenvolvimento de um método para a detecção e monitoramento in vitro de atividade nucleotídeo-trifosfatase. O método que se mostrou robusto e reprodutível foi aplicado para investigar a atividade GTPase de uma proteína recombinante contendo o domínio catalítico de uma septina humana SEPT4 / Bradeiona (GST-rDGTPase). O exemplo de aplicação demonstra que a técnica de eletroforese capilar pode substituir o método tradicionalmente usado com marcação radioativa para detecção de atividade GTPase inclusive em estudos de cinética enzimática. Os parâmetros cinéticos determinados para a GST-rDGTPase foram: vmax = 1.7 μ M min-1 ± 0.1 and Km = 1.0 mM ± 0.3; kcat = 9 x 10-3 SM-1. O efeito de co-fatores como Mg2+ and Mn2+ também foi estudado. O método analítico descrito também se mostrou útil para a análise de di- e trifosfatos de outros nucleotídeos. / This Thesis concerns on the development of capillary electrophoresis-based bioanalytical methods for two distinct applications: DNA-protein binding analysis and monitoring of enzyme-catalyzed GTP hydrolysis. In the first chapter, the feasibility of on-chip electrophoretic mobility-shift assays (EMSA) is investigated. Purified transcription factors c-Jun(AP1) and p-50(NFkB) were used for binding studies to dsDNA probes containing the consensus sequences from AP1, NFkB and AP2 regulatory sequences. DNA probes were modified at the 5?-end with the Cy5 and unlabeled oligos were used for competition experiments. On-chip-EMSA could be carried out within ca. 2 h with low sample consumption and no need to handle radioactive material. Both, the dsDNA probes and the shifted oligos from binding reactions were analyzed on the ?2100 Bioanalyzer? using either, the standard procedure for DNA analysis or a modified protocol, in which no intercalating dye was used. Instead, 4.9 nM Cy5-dCTP was added to the gel matrix allowing the detection of only Cy5-labeled DNA. Despite the need of specific adjustments for each protein, we have shown the potential for replacing slab gel-based EMSA for on-chip methods. A competition experiment to show sequence specific binding of the transcription factor p-50 to the consensus sequence NFkB is presented as a proof of principle. In chapter II, a capillary electrophoresis-based method for in vitro detection and monitoring of nucleotide-triphosphatase activity is described. This robust and reproducible method was used to investigate GTPase activity of a recombinant protein construct containing the catalytic domain of Human SEPT4 /Bradeion ? (GST-rDGTPase). The application example demonstrates that the capillary electrophoresis technique can replace classical radioactive methods for GTPase activity assays and may be used as a routine analytical tool. Enzyme kinetics of GST-rDGTPase was studied and yielded the following kinetic parameters: vmax = 1.7 μM min-1 ± 0.1 and Km = 1.0 mM ± 0.3; kcat = 9 x 10-3 s-1. In addition the effect of co-factors such as Mg2+ and Mn2+ in the catalytic activity was investigated. The described analytical method was also shown to be useful to analyze di- and triphosphated forms of other nucleotides.
12

Reaktionsmechanismus der Typ III Restriktionsendonuklease EcoP15I und eine Anwendungsmöglichkeit in der molekularen Diagnostik

Reich, Stefanie 01 September 2004 (has links)
EcoP15I ist ein Vertreter der multifunktionalen, heterooligomeren Typ III Restriktionsendonukleasen. Typ III Restriktionsendonukleasen sind wegen der Lage ihres Spaltortes, ca. 25 bp vom Erkennungsort entfernt, von besonderem Interesse für Anwendungen in der Medizin und funktionellen Genomanalyse. EcoP15I erkennt die DNA-Sequenz 5''-CAGCAG und benötigt für eine effektive DNA-Spaltung zwei invers orientierte Erkennungsorte auf einem DNA-Molekül. Nach dem bisherigen DNA-Translokations-Modell bindet je ein EcoP15I-Protein an je einen Erkennungsort und startet dann durch ATP-Hydrolyse vermittelte DNA-Translokation. Die Kollision der beiden EcoP15I-DNA-Komplexe initiiert die DNA-Doppelstrang-Spaltung. Experimente zur Erkennungsort-Suche von EcoP15I zeigen, dass über längere Distanzen offenbar nicht das "Sliding", sondern ein dreidimensionaler Prozess die bevorzugte Bewegung von EcoP15I an der DNA ist. Eine erhöhte Anzahl von Wiederholungen von CAG-Trinukleotiden (CAG-Repeats) im Exon 1 des Gens für Chorea Huntington (Huntington Disease - HD) führt zur Manifestation dieser neurodegenerativen Erkrankung. Für die Diagnostik der Erkrankung ist die exakte Bestimmung der Anzahl der CAG-Repeats von Bedeutung. Diese Arbeit zeigt die Spaltung von HD Gen Exon 1 DNA durch EcoP15I. Die halbautomatische, hoch-sensitive Analyse dieses Spaltmusters ermöglicht die exakte Bestimmung der Anzahl der CAG-Repeats. Diese Arbeit liefert den ersten Nachweis für die DNA-Translokation durch eine Typ III-Restriktionsendonuklease. Die postulierten EcoP15I-DNA-Schlaufen wurden mit Hilfe der Rasterkraftmikroskopie (SFM) abgebildet. Dadurch wird das DNA-Translokations-Modell der DNA-Spaltung durch EcoP15I bestätigt. Es werden Gemeinsamkeiten und Unterschiede des gesamten DNA-Spaltvorganges der Typ III Restriktionsendonuklease EcoP15I in bezug auf andere Restriktionsendonukleasen diskutiert. / EcoP15I is a multifunctional, hetero-oligomeric Type III restriction enzyme. Type III restriction enzymes are of general interest in medicine and functional genome analysis because they cut DNA 25 bp downstream of their recognition site. EcoP15I recognises the DNA sequence 5`-CAGCAG and needs two inverse oriented recognition sites for effective DNA cleavage. According to the present translocation collision model DNA cleavage was proposed to result from ATP dependent DNA translocation, which is expected to induce DNA loop formation, and collision of two enzyme-DNA complexes. Experiments show that EcoP15 moves rather in a three-dimensional than in a "sliding" process in search for its recognition site. Huntington''s disease (HD) is a progressive neurodegenerative disorder with autosomal-dominant inheritance. The disease is caused by a CAG trinucleotide repeat expansion located in the first exon of the HD gene. To diagnose the illness the exact determination of the number of CAG repeats is necessary. This study shows that the number of CAG repeats in the HD gene can be determined by restriction of the DNA with the endonuclease EcoP15I and subsequent high-resolution analysis of the restriction fragment pattern using the ALFexpress DNA Analysis System. Here, for the first time DNA translocation by the Type III restriction enzyme EcoP15I is demonstrated. The postulated EcoP15-DNA loops are visualised using scanning force microscopy. This confirms the translocation-collision model for DNA cleavage by EcoP15. Similarities and differences between the DNA cleavage processes of the Type III restriction enzyme EcoP15I and other restriction enzymes are discussed.
13

Die Typ III Restriktionsendonuklease EcoP15I

Wagenführ, Katja 13 March 2009 (has links)
EcoP15I gehört zu den heterooligomeren Typ III Restriktionsendonukleasen. Der multifunktionale Enzymkomplex ist aus zwei Modifikations- und zwei Restriktions-Untereinheiten aufgebaut und katalysiert sowohl die Spaltung als auch die Methylierung der DNA. Für die effektive Spaltung der doppelsträngigen DNA benötigt EcoP15I zwei invers orientierte Erkennungsorte mit der DNA-Sequenz 5’-CAGCAG. Die Spaltung erfolgt im oberen Strang 24 bis 26 Basen in 3’-Richtung nach dem Erkennungsort und im unteren Strang 26 bis 28 Basen in 5’-Richtung nach dem Erkennungsort. Aufgrund des bislang größten definierten Abstandes zwischen Erkennungs- und Spaltort ist EcoP15I ein wichtiges Werkzeug in der funktionellen Genomanalyse. Die Aufklärung der Domänenstruktur beider EcoP15I-Untereinheiten durch limitierte Proteolyse zeigte, dass die Restriktions-Untereinheit modular aufgebaut ist. Sie besteht aus zwei stabil gefalteten Domänen, der N-terminalen Translokase- und der C-terminalen Endo-Domäne. Beide Domänen sind durch einen flexiblen Linker verbunden. In der Modifikations-Untereinheit dagegen wurden keine Domänen identifiziert. Durch Insertion von Aminosäuren in und um den Linkerbereich konnten Enzymmutanten hergestellt werden, die bevorzugt die Positionen mit größten Abstand zum Erkennungsort spalteten. Wurden dagegen in dieser Region Aminosäuren deletiert, verloren die Enzymmutanten ihre DNA-Spaltaktivität. Die photochemische Vernetzung von EcoP15I mit spezifischer DNA ergab, dass EcoP15I drei Kontakte zum Phosphatrückgrat des ersten Adenins im Erkennungsort ausbildet. Ein Kontakt wird dabei über die Aminosäure S635 im C-terminalen Teil der Modifikations-Untereinheit hergestellt, zwei weitere entstehen durch die Aminosäuren Y248 und K421 der Restriktions-Untereinheit. Die transmissionselektronenmikroskopische Abbildung des negativ kontrastierten EcoP15I-Enzym zeigte einen symmetrischen Aufbau und stellt somit eine Grundlage für die Entwicklung eines dreidimensionalen Modells dar. / EcoP15I belongs to the hetero-oligemeric type III restriction endonucleases. The multifunctional enzyme complex consists of two modification and two restriction subunits and catalyses both the cleavage and methylation of the DNA. For effective cleavage of the double stranded DNA EcoP15I needs two inversely oriented recognition sites with the DNA sequence 5’-CAGCAG. The cleavage occurs 24 to 25 bases in 3’-direction from the recognition sequence in the top strand and 26 to 28 bases in 5’-direction from the recognition sequence in the bottom strand. Because of the largest known distance between recognition and cleavage site so far EcoP15I is an important tool in functional genomics. The elucidation of the domain structure of EcoP15I restriction as well as the modification subunit by limited proteolysis showed that the restriction subunit has a modular structure. It consists of two stable folded domains, the N-terminal translocase domain and the C-terminal endonuclease domain. Both domains are connected by a flexible linker. In contrast to the restriction subunit no domains could be detected in the modification subunit. Enzyme mutants that were constructed by insertion of amino acids in and around the linker region cleaved preferentially the position with the largest distance between recognition and cleavage site. The enzyme mutants lost their DNA cleavage activity when the amino acids in this region were deleted. The photochemical crosslinking of EcoP15I with specific DNA showed that EcoP15I forms three contacts to the phosphate backbone of the first adenine of the recognition site. One contact is made by amino acid S635 in the C-terminal part of the modification subunit. Two others are made by amino acids Y248 and K421 of the restriction subunit. The transmission electron microscope picture of the negatively stained EcoP15I enzyme showed a symmetric form and therefore it constitutes a basis for the development of a three dimensional model.
14

Besonderheiten der DNA-Erkennung und Spaltung durch die Restriktionsendonuklease EcoRII

Mücke, Merlind 09 October 2002 (has links)
Die homodimere Typ IIE Restriktionsendonuklease EcoRII erfordert im Gegensatz zu den orthodoxen Typ II Restriktionsendonukleasen die simultane Wechselwirkung mit zwei Kopien ihrer DNA-Erkennungssequenz 5'CCWGG, um die spezifische endonukleolytisch Spaltung der DNA zu katalysieren. In der vorliegenden Arbeit wurde mittels Transmissionselektronenmikroskopie bewiesen, daß EcoRII die Bildung von DNA-Schlaufen an einem linearen DNA-Substrat mit zwei DNA-Erkennungsorten induziert - ähnlich wie andere DNA prozessierende Enzyme und Transkriptionsfaktoren. Kinetische Untersuchungen der DNA-Spaltreaktion von EcoRII mit superhelikaler Plasmid-DNA, die entweder einen oder zwei DNA-Erkennungsorte für EcoRII enthielt, zeigten, daß EcoRII pro Spaltereignis nur an einem der beiden involvierten doppelsträngigen DNA-Erkennungsorte spaltet. Die Studie, in der EcoRII photochemisch mit den Basen der DNA-Erkennungssequenz vernetzt wurde, ergab ein asymmetrisches Vernetzungsmuster, das durch die partielle Asymmetrie an der A/T-Position der ansonsten palindromischen Erkennungssequenz hervorgerufen wird. Wir konnten zeigen, daß die Aminosäure Tyr41 von EcoRII das 5'C des 5'CCAGG-Stranges der Erkennungssequenz kontaktiert. Durch Aufklärung der Domänenorganisation von EcoRII konnten wir das Modell der EcoRII-DNA-Interaktion verbessern. Wir zeigten, daß für die simultane Interaktion des Enzyms EcoRII mit zwei Kopien der Erkennungssequenz zwei verschiedene Domänen verantwortlich sind. Die C-terminale Domäne ist eine neue Restriktionsendonuklease, die effizienter als das vollständige EcoRII an einzelnen Erkennungsorten spaltet. Die N-terminale Domäne bindet spezifisch an die DNA und reduziert die Aktivität des vollständigen Enzyms, indem sie die Spaltung von einem zweiten Erkennungsort abhängig macht. Daher nehmen wir an, daß EcoRII in der Evolution in Form der N-terminalen Domäne eine zusätzliche DNA-Bindungsfunktion akquiriert hat, um eine neue Proteinfunktion zu entwickeln, die die Spaltung von DNA und die Interaktion mit zwei DNA-Erkennungsorten einschließt. Solche Interaktionen sind z.B. Voraussetzung für die DNA-Rekombination oder Transposition. Daher könnte die gegenwärtige EcoRII Restriktionsendonuklease eine evolutionärer Übergang von ortsspezifischen Endonukleasen zu einem neuen Protein sein, das spezifisch mit zwei DNA-Orten interagiert. / The homodimeric type IIE restriction endonuclease EcoRII requires the cooperative interaction with two copies of the recognition sequence 5'CCWGG for DNA cleavage. This is in contrast to the orthodox type II restriction endonucleases which interact with single recognition sequences. We have proven by transmission electron microscopy that EcoRII simultaneously binds two recognition sites on a linear DNA-substrate by looping out the intervening DNA. This DNA-loop formation is similar to that of other DNA processing enzymes and transcription factors. Kinetic investigations of the DNA cleavage of supercoiled DNA-plasmids containing either one or two recognition sites for EcoRII showed that EcoRII cleaves only at one of the two involved double-stranded DNA recognition sites. Photocross-linking of EcoRII to the bases of the recognition sequence revealed an asymmetric cross-linking pattern. This asymmetry is due to the partial asymmetry of the recognition sequence at the central A/T position. Furthermore, we found that amino acid Tyr41 of EcoRII specifically contacts the 5'C of the 5'CCAGG strand of the recognition sequence. We found by limited proteolysis that a two-domain structure enables EcoRII to interact cooperatively with two recognition sites. The C-terminal domain is a new restriction endonuclease that, in contrast to the complete EcoRII, specifically cleaves at single 5'CCWGG recognition sites. Moreover, this new restriction endonuclease cleaves much more efficiently than EcoRII. The N-terminal domain specifically binds the DNA-substrate and reduces the activity of EcoRII by making the enzyme dependent on a second recognition site. Therefore, we assume that a precursor EcoRII enzyme acquired another DNA binding domain to develop a new protein function that includes DNA cleavage and specific interaction with two DNA sites. The current EcoRII protein could be an evolutionary intermediate between a site-specific endonuclease and a protein that functions specifically with two DNA sites such as DNA recombinases and transposases.
15

DNA Oligomers - From Protein Binding to Probabilistic Modelling

Andrade, Helena 26 January 2017 (has links)
This dissertation focuses on rationalised DNA design as a tool for the discovery and development of new therapeutic entities, as well as understanding the biological function of DNA beyond the storage of genetic information. The study is comprised of two main areas of study: (i) the use of DNA as a coding unit to illustrate the relationship between code-diversity and dynamics of self-assembly; and (ii) the use of DNA as an active unit that interacts and regulates a target protein. In the study of DNA as a coding unit in code-diversity and dynamics of self-assembly, we developed the DNA-Based Diversity Modelling and Analysis (DDMA) method. Using Polymerase Chain Reaction (PCR) and Real Time Polymerase Chain Reaction (RT-PCR), we studied the diversity and evolution of synthetic oligonucleotide populations. The manipulation of critical conditions, with monitoring and interpretation of their effects, lead to understanding how PCR amplification unfolding could reshape a population. This new take on an old technology has great value for the study of: (a) code-diversity, convenient in a DNA-based selection method, so semi-quantitation can evaluate a selection development and the population\'s behaviour can indicate the quality; (b) self-assembly dynamics, for the simulation of a real evolution, emulating a society where selective pressures direct the population's adaptation; and (c) development of high-entropy DNA structures, in order to understand how similar unspecific DNA structures are formed in certain pathologies, such as in auto-immune diseases. To explore DNA as an active unit in Tumour Necrosis Factor α (TNF-α) interaction and activity modulation, we investigate DNA's influence on its spatial conformation by physical environment regulation. Active TNF-α is a trimer and the protein-protein interactions between its monomers are a promising target for drug development. It has been hypothesised that TNF-α forms a very intricate network after its activation between its subunits and receptors, but the mechanism is still not completely clear. During our research, we estimate the non-specific DNA binding to TNF-α in the low micro-molar range. Cell toxicity assays confirm this interaction, where DNA consistently enhances TNF-α's cytotoxic effect. Further binding and structural studies lead to the same conclusion that DNA binds and interferes with TNF-α structure. From this protein-DNA interaction study, a new set of tools to regulate TNF-α's biological activity can be developed and its own biology can be unveiled.

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