An extracellular bacteriocin-like substance produced by Clostridium acetobutylicum was detected during studies on an industrial fermentation process. The bacteriocin-like substance was not inducible by either ultraviolet light or mitomycin C, and its production was not associated with the induction of a protease. Studies on the mode of action of the bacteriocin-like substance indicated that it had no significant effect on DNA, RNA, or protein synthesis, and it did not cause the loss of intracellular ATP. However, the bacteriocin-like substance was able to lyse SDS-treated cells and cell walls of C. acetobutylicum and was identified as an autolysin. Some of the characteristics of this extracellular autolysin were determined, and after purification it was shown to be a glycoprotein with a molecular weight of 28 000.

The crystalline compound resulting from thermal cyclization of the Baylis-Hillman product, methyl 3-hydroxy-2-methylene-3-(2-pyridyl)propanoate, has been identified as the indolizine derivative, methyl indolizine-2-carboxylate, and this approach involving the reaction of pyridine-2-carboxaldehydes and acrylate analogues has been established as a general route to 2-substituted indolizines. The ease of cyclization the Baylis-Hillman products to indolizines has been shown to increase by converting the hydroxy group to an acetoxy group, and a range of acetylated Baylis-Hillman products were prepared and cyc1ized to the corresponding 2-substituted indolizines, generally in good overall yield. In the reaction of pyridine-2-carboxaldehyde and methyl vinyl ketone, the intermediate cyclized readily and directly to the corresponding indolizine. One- and two-dimensional ¹H and ¹³C NMR analysis of the 2-substituted indolizine products has permitted complete assignment of all ¹H and ¹³C NMR signals, as well as the measurement of all coupling constants for these compounds. A kinetic and mechanistic study has been conducted on the Baylis-Hillman reaction using ¹H NMR spectroscopy. A range of substrates has been examined and the reaction has been found to be third-order overall. A mechanism involving an addition - elimination sequence is proposed, which fits the kinetic data and accounts for observed substituent effects. Reaction of N,N-dimethylacrylamide with pyridine-2-carboxaldehyde in the presence of the tertiary amine catalyst, DABCO, in chloroform, yielded an unexpected product which has been identified by single crystal X-ray diffraction analysis as 1-(2,2,2-trichloro-1-hydroxyethyl)pyridine. Attempted extension of the general indolizine route to the preparation of chromene systems by reacting salicylaldehyde with methyl acrylate in the presence of DABCO, also led to an unexpected, crystalline material, identified by single crystal X-ray diffraction analysis as the coumarin derivative, 3-[(2-formylphenoxy)methyl]coumarin.A series of chloroquine analogues have been prepared from indolizine-2-carboxylic acid, pyrrolo[I,2-a]quinoline-2-carboxylic acid and imidazo[I,2-a]pyridine-2-carboxylic acid by reaction with suitable amines in the presence of the coupling reagent 1, I' -carbonyldiimidazole. This route has been shown to be a vast improvement on earlier procedures and has provided access to both secondary and tertiary indolizine-2-carboxamides. A range of N,N-dialkylindolizine-2-carboxamides have been prepared by this route, and the influence of substituents on their N-CO rotational energy barriers has been determined using variable temperature ¹H and ¹³C NMR techniques. Intercalation with natural DNA by both chloroquine and the synthesized chloroquine analogues has been examined using UV spectrophotometry, and ¹H and ³¹P NMR spectroscopy. The pyrrolo[I,2-a]quinolines have been shown to be DNA intercalators with binding affinities similar to that of the known antimalarial intercalator, chloroquine. In a preliminary study the synthesis of a short oligonucleotide has been undertaken and changes have been observed in the ¹H and ³¹P NMR spectra of the oligonucleotide on addition of the intercalator, chloroquine.

Covalent binding of dinuclear metal carboxylate compounds to purine DNA nucleobases has been shown to be a source of anticancer activity, and has resulted in intense research to understand the coordination of metal complexes to DNA. This investigation focuses on the formation of dirhenium metal:dinucleotide complexes of purine nucleobases. To our knowledge, complexes formed by the reaction of dinuclear rhenium metal compounds with dinucleotides have not been reported in the literature.

No description available.

Die Zellreplikation ein kontrollierter Prozess aus sequentieller und zeitlich koordinierter Aktivierung und Abbau von Zyklinen, die einen schnellen Übergang zwischen den Zyklusphasen ermöglichen. Dabei ist der Erfolg bei der Ermittlung der wichtigsten Komponenten und Aufgliederung der Schaltmechanismen im Wesentlichen auf die gleichzeitige Anwendung von Modellsystemen wie Hefe, Frosch und Fliege zurückzuführen. Das heutige Verständnis des Zellzyklus muss erweitert werden, um zu überprüfen ob die Erkenntnisse auch auf in-vivo Modelle von Säugetieren wie der Maus zutreffen. Es existieren solche Modelle, die sich auf spezifische Kontrollpunkte oder Übergänge konzentrieren, allerdings noch kein integriertes Modell, in dem der Zellzyklus durch eine Verletzung im Säugetier induziert wird. Das Modellsystem der Leberregeneration bei Nagern wurde gewählt, da es sich durch das am höchsten verbreitete Phänomen der Synchronisation der Zellproliferation auszeichnet. Mit dem Fokus auf die Frage, wie die Zellen durch pro-inflammatorische Signale nach Verletzungen ins Priming in der G1/S Phase eintreten, gingen wir in einen durch Zytokine und Wachstumsfaktoren induzierten Säugetier-Zellzyklus über. Weiterhin wurden mitotische Ereignisse modelliert, die zum Alles-oder-Nichts G2/M Übergang und dem mitotischen Ausgang führen. Wir konzentrieren uns auf die vielversprechende Funktion von Cdh1 in der Zellzykluskontrolle, welches bekanntlich eine Schlüsselrolle in der G1 Phase spielt. Weiterhin haben wir dessen Rolle bei der Verzögerung der G2 Phase untersucht. Wir vermuten eine zentrale Rolle von Cdh1 im Zellzyklus durch die Kontrolle der Dynamik der Zykline. Das Modell ist ein Versuch, die Kernmechanismen der Zellzykluskontrolle bei Säugetieren zu verstehen. Besseres Verständnis der Mechanismen in der Säugetierzelle würde das Studium der Zellphysiologie im Hinblick auf Störungen der humanen Zellzyklusmaschinerie, welche zu Krankheiten wie Krebs führen. / Cell replication is a controlled process with sequential and timely activation and degradation of cyclins leading to swift transitions between the phases of the cell cycle. The essential achievement in identifying the key components and in dissecting the mechanisms of the cell cycle circuitry has been attributed to the simultaneous use of model systems like yeast, frogs, and flies. Present understanding of the cell cycle needs to be extended to investigate whether those findings also apply to mammalian in-vivo models like mice. We chose liver regeneration in mammals as the model system because it is the most synchronised cell proliferation phenomenon, where 95\% of the cells simultaneously enter cell cycle. The G1-S phase transition was modelled, focusing on how injury induced pro-inflammatory signals \textit{prime} the cells in G1 phase and consequently both cytokine and growth factor induced pathways lead to further cell cycle progression. The model was further extended to mitotic events leading to the all-or-none G2-M transition and mitotic exit. I focussed on the emerging role of Cdh1 in the mammalian cell cycle. Cdh1 known for its role in G1 phase was further investigated for its role G2 delay. Cdh1 was suggested to be at the core of the cell cycle machinery controlling cyclin dynamics. This model is an attempt in understanding core machinery of the mammalian cell cycle. Better understanding of the cell cycle control system in mammalian cells would enable understanding perturbations of the human cell cycle machinery which lead to diseases like cancers.

In the context of the somatic mutation theory of chemical carcinogenesis, mutations are thought to arise during the replication of DNA past carcinogen-DNA adducts. The work described in this thesis deals with the testing of a hypothetical mechanism whereby mammalian cells are able to replicate their DNA past polycyclic aromatic hydrocarbon DNA adducts. The second objective of this thesis work was to develop both in vivo and in vitro models to study the induction of mutations in a target human gene by chemical carcinogens from two different classes, polycyclic aromatic hydrocarbons and nitrosamines. To approach the hypothetical mechanism of bypass replication in mammalian cells, synchronized Chinese hamster ovary cells were treated with the ultimate carcinogenic form of benzo (a) pyrene, 7β, 8α-dihydroxy-9α, 10α-epoxy-7,8,9,10-tetrahydrobenzo (a) pyrene (BPDE I). Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. It was determined using agarose gel electrophoresis that the ligation of Okazaki size replication intermediates was blocked in BPDE I-treated, synchronized CHO cells. The data obtained were, therefore, supportive of the 'block-gap' model of DNA bypass replication in carcinogen damaged mammalian cells. To study mutagenesis of a specific sequence induced by chemical carcinogens, the human c-Ha-ras proto-oncogene was transfected into the mouse fibroblast cell line, NIH 3T3. Transfected NIH 3T3 cell lines (HHRN 1-4) were isolated that had a low copy number of the human c-Ha-ras proto-oncogene and a non-transformed phenotype. It was determined that the integrated human c-Ha-ras gene was hypomethylated, and expressed at the messenger level. The human c-Ha-ras protein, p21, was also detected in these transfected cell lines. Treatment of the HHRN cell lines with the nitrosamine, N-methyl-N-nitroso-N'-nitroguanidine (MNNG) resulted in transformed NIH 3T3 foci. In vitro MNNG treatment of the plasmid, z-6, and transfection into NIH 3T3 cells led to the isolation of transformed cell lines. Screening of the in vitro and in vivo treated, transformed cell lines by RNA:RNA duplex mismatch analysis led to the detection of no mutations within the first exon of the human c-Ha-ras oncogene.

An in vitro initiation of a DNA replication system was developed to study the function of Ø29 terminal protein. Cell free extracts prepared from Bacillus phage Ø29-infected cells catalyzed the formation of a complex between a 30,000 dalton protein and dAMP in the presence of MgCl₂, Ø29 DNA-protein template and α-³²P dATP. Uninfected cell extracts did not support this reaction. The molecular weight of this complex, the nature of linkage between dAMP and 30,000 dalton protein as well as nucleotide specificity for this reaction suggest that the protein moiety of this complex is the terminal protein of Ø29. Similar results were obtained with phages GA-1 and M2Y infected cell extracts. The similar requirements for complex formation and DNA replication in vitro implies that the complex formation is an initiation step in DNA replication. This notion was supported by characterizing the elongation product which formed in the presence of ddCTP. Two distinct antibodies were prepared for analyse the function of the terminal protein in Ø29 DNA replication. These antibodies react with native terminal protein as assayed by immunoprecipitation and ELISA methods. The inhibition of complex formation by these antibodies provides strong evidence that the terminal protein is involved in complex formation. The notion that complex formation is an initial step of DNA replication was demonstrated conclusively by inhibition of anti-TP on DNA replication in vitro. The results presented in this dissertation provide evidence supporting the protein-priming mode of linear Ø29 DNA replication.

La protéine PCNA est un facteur d'échafaudage polyvalent pour plus de cinquante protéines impliquées dans le métabolisme d'ADN, notamment dans la réplication et la réparation. Comment les échanges entre les partenaires de PCNA sont régulés est actuellement mal compris. Parmi ses partenaires, CDT1, p21 et PR-Set7/Set8 possèdent un motif d'interaction avec PCNA particulier, nommé « PIP degron », qui favorise leur protéolyse d'une manière dépendante de l'E3 ubiquitine ligase CRL4Cdt2. Après irradiation aux UV-C, le facteur d'initiation de la réplication CDT1 est rapidement détruit d'une manière dépendante de son PIP degron, mais le rôle de cette dégradation est inconnu. Dans cette étude, j'ai analysé la fonction du PIP degron de CDT1 et fourni des évidences expérimentales qui montrent que l'inhibition de la dégradation de Cdt1 par CRL4Cdt2 dans les cellules de mammifères compromet la relocalisation de l'ADN polymérase translesionnelle eta en foyers nucléaires induits par les irradiations UV-C. En élargissant cette étude à d'autres partenaires de PCNA, nous avons constaté que seuls les protéines qui contiennent un PIP degron, et pas un PIP box canonique comme celui de FEN1 et p15 (PAF), interfèrent avec la formation de foyers de pol eta. La mutagenèse du PIP degron de CDT1 a révélé qu'un résidu de thréonine conservé parmi les PIP degrons est essentiel pour l'inhibition de la formation des foyers de pol eta. Les résultats obtenus suggèrent que l'élimination de protéines contenant des PIP degrons par la voie CRL4Cdt2 régule le recrutement de pol eta au niveau des sites de dommages induits par les UV-C. / The sliding clamp PCNA is a versatile scaffold for more than fifty proteins involved in DNA metabolism such as replication and repair. How the switch between PCNA partners is regulated is currently not fully understood. Among its partners, Cdt1, p21 and PR-Set7/Set8 contain a specialized PCNA-binding motif named « PIP degron » that promotes their proteolysis in a fashion dependent on the E3 ubiquitin ligase CRL4Cdt2. Upon UV-irradiation, the replication initiation factor Cdt1 is rapidly destroyed in a PIP degron-dependent manner but the role of this degradation is unknown. Here we have analyzed the function of Cdt1 PIP degron and we provide evidence that interference with CRL4Cdt2-mediated destruction of Cdt1 in mammalian cells compromises PCNA-dependent relocalisation of the DNA translesion polymerase eta into UV-induced nuclear foci. By extending this analysis to other PCNA partners, we found that only PIP degrons, as compared to canonical PCNA-binding motifs of Fen1 and p15(PAF), interfere with pol eta focus formation. Mutagenesis of Cdt1 PIP degron revealed that a threonine residue conserved in PIP degrons is critical for inhibition of pol eta focus formation. Our results suggest that removal of high-affinity PIP degron-containing proteins from PCNA by CRL4Cdt2 pathway regulates pol eta recruitment to sites of UV-damage.

<p>The use of DNA as a polymeric building material transcends its function in biology and is exciting in bionanotechnology for applications ranging from biosensing, to diagnostics, and to targeted drug delivery. These applications are enabled by DNA’s unique structural and chemical properties, embodied as a directional polyanion that exhibits molecular recognition capabilities. Hence, the efficient and precise synthesis of high molecular weight DNA materials has become key to advance DNA bionanotechnology. Current synthesis methods largely rely on either solid phase chemical synthesis or template-dependent polymerase amplification. The inherent step-by-step fashion of solid phase synthesis limits the length of the resulting DNA to typically less than 150 nucleotides. In contrast, polymerase based enzymatic synthesis methods (e.g., polymerase chain reaction) are not limited by product length, but require a DNA template to guide the synthesis. Furthermore, advanced DNA bionanotechnology requires tailorable structural and self-assembly properties. Current synthesis methods, however, often involve multiple conjugating reactions and extensive purification steps.</p><p>The research described in this dissertation aims to develop a facile method to synthesize high molecular weight, single stranded DNA (or polynucleotide) with versatile functionalities. We exploit the ability of a template-independent DNA polymerase−terminal deoxynucleotidyl transferase (TdT) to catalyze the polymerization of 2’-deoxyribonucleoside 5’-triphosphates (dNTP, monomer) from the 3’-hydroxyl group of an oligodeoxyribonucleotide (initiator). We termed this enzymatic synthesis method: TdT catalyzed enzymatic polymerization, or TcEP.</p><p>Specifically, this dissertation is structured to address three specific research aims. With the objective to generate high molecular weight polynucleotides, Specific Aim 1 studies the reaction kinetics of TcEP by investigating the polymerization of 2’-deoxythymidine 5’-triphosphates (monomer) from the 3’-hydroxyl group of oligodeoxyribothymidine (initiator) using in situ 1H NMR and fluorescent gel electrophoresis. We found that TcEP kinetics follows the “living” chain-growth polycondensation mechanism, and like in “living” polymerizations, the molecular weight of the final product is determined by the starting molar ratio of monomer to initiator. The distribution of the molecular weight is crucially influenced by the molar ratio of initiator to TdT. We developed a reaction kinetics model that allows us to quantitatively describe the reaction and predict the molecular weight of the reaction products.</p><p>Specific Aim 2 further explores TcEP’s ability to transcend homo-polynucleotide synthesis by varying the choices of initiators and monomers. We investigated the effects of initiator length and sequence on TcEP, and found that the minimum length of an effective initiator should be 10 nucleotides and that the formation of secondary structures close to the 3’-hydroxyl group can impede the polymerization reaction. We also demonstrated TcEP’s capacity to incorporate a wide range of unnatural dNTPs into the growing chain, such as, hydrophobic fluorescent dNTP and fluoro modified dNTP. By harnessing the encoded nucleotide sequence of an initiator and the chemical diversity of monomers, TcEP enables us to introduce molecular recognition capabilities and chemical functionalities on the 5’-terminus and 3’-terminus, respectively.</p><p>Building on TcEP’s synthesis capacities, in Specific Aim 3 we invented a two-step strategy to synthesize diblock amphiphilic polynucleotides, in which the first, hydrophilic block serves as a macro-initiator for the growth of the second block, comprised of natural and/or unnatural nucleotides. By tuning the hydrophilic length, we synthesized the amphiphilic diblock polynucleotides that can self-assemble into micellar structures ranging from star-like to crew-cut morphologies. The observed self-assembly behaviors agree with predictions from dissipative particle dynamics simulations as well as scaling law for polyelectrolyte block copolymers.</p><p>In summary, we developed an enzymatic synthesis method (i.e., TcEP) that enables the facile synthesis of high molecular weight polynucleotides with low polydispersity. Although we can control the nucleotide sequence only to a limited extent, TcEP offers a method to integrate an oligodeoxyribonucleotide with specific sequence at the 5’-terminus and to incorporate functional groups along the growing chains simultaneously. Additionally, we used TcEP to synthesize amphiphilic polynucleotides that display self-assemble ability. We anticipate that our facile synthesis method will not only advance molecular biology, but also invigorate materials science and bionanotechnology.</p> / Dissertation

We can now manipulate the genetic material of living organism routinely and cheaply. This has inspired a burgeoning field of synthesis based on DNA as a building block. The development of this new synthetic field has mirrored the trajectory of synthetic organic chemistry from small molecular systems to complex macromolecular assemblies. At first, this field of synthetic biology delivered recombinant proteins that enhanced our understanding of the structure-function relationship of biological macromolecules. Now, as the synthetic tools and analysis methods have come of age, synthetic whole-cell and multicellular systems have come within reach. In Chapter 1 we review the significant advances in DNA synthesis and analysis that have brought us to this point. In this work, we first ask what practical applications will benefit most from the unique qualities of synthetic whole-cell system, such as their ability to replicate, sense and respond with molecular specificity. In Chapter 2, we implement a pathogen detection platform based solely on genetically modified yeast. This approach holds the potential to deliver ultra low-cost sensors that can be used and produced at the point-of-care. In Chapter 3, we develop methods to target these yeast-based sensors for the detection of any peptide biomarker of choice. We next look forward to the potential of synthetic multicellular systems. While natural multicellular systems can be directly manipulated, our ability to rationally build multicellular systems from the bottom-up is still in its infancy. There still remain gaps in the available tools to make and analyze such synthetic systems. In Chapter 4, we leverage the explosion of available genomic databases to uncover a highly extensible set of cell-cell signaling modules. In Chapter 5, we implement ratiometric fluorescent tags to track mixed cell populations in multiplex. Together these components will be useful in implementing and analyzing synthetic communication networks that will be key components of advanced synthetic multicellular systems.