Proteases are essential for regulating a wide range of physiological and pathological processes. The imbalance of protease activation and inhibition will result in a number of major diseases including cancers, atherosclerosis, and neurodegenerative diseases. Although fluorescence resonance energy transfer (FRET)-based protease probes, a small molecular dye and other methods are powerful, they still have drawbacks or limitations for providing significant information about the dynamics and pattern of endogenous protease activation and inhibition in a single living cell or in vivo. Currently protease sensors capable of quantitatively measuring specific protease activity in real time and monitoring activation and inhibition of enzymatic activity in various cellular compartments are highly desired. In this dissertation, we report a novel strategy to create protease sensors by grafting an enzymatic cleavage linker into a sensitive location for changing chromophore properties of enhanced green fluorescent protein (EGFP) following protease cleavage, which can be used to determine protease activity and track protease activation and inhibition with a ratiometric measurement mode in living cells. Our designed protease sensors exhibit large relative ratiometric optical signal change in both absorbance and fluorescence, and fast response to proteases. Meanwhile, these protease sensors exhibiting high enzymatic selectivity and kinetic responses are comparable or better than current small peptide probes and FRET-based protease probes. Additionally, our protease sensors can be utilized for real-time monitoring of cellular enzymogen activation and effects of inhibitors in living cells. This novel strategy opens a new avenue for developing specific protease sensors to investigate enzymatic activity in real time, to probe disease mechanisms corresponding to proteases in vitro and in vivo, and to screen protease inhibitors with therapeutic effects. Strong fluorescence was still retained in the cleaved EGFP-based protease sensors, which stimulated us to identify the EGFP fragment with fluorescence properties for further understanding chromophore formation mechanisms and investigating protein-protein interactions through fluorescence complementation of split EGFP fragments. Through fusing EF-hand motifs from calbindin D9k to split EGFP fragments, a novel molecular probe was developed to simultaneously track the calcium change or calcium signaling pathways and calcium-dependent protein-protein interaction in living cells in real time.

Zinc (Zn) is an essential micronutrient for the growth and development of all organisms. Zinc deficiency is a widespread micronutrient disorder worldwide, which reduces crop yields and the nutritive value of the grain. Understanding the process of zinc absorption and translocation in crop is essential for this purpose. Zinc is taken up by plants and translocated within plants through high-affinity zinc transporter proteins embedded in the plasma membrane. The Zn transporters are induced under Zn deficiency, and it is speculated that the expression levels of some of zinc transporters are critical for improved tolerance to low zinc. A number of Zn transporters have been cloned from higher plants including rice and Arabidopsis, but little has been done in barley and wheat. This project aims to investigate genes involveld in zinc efficiency mechanism by complementation analysis in yeast, which is double mutant of zinc-transporters, using cDNA expression library from a most zinc efficient barley cultivar, Tokak-157.

This thesis investigates fundamental questions surrounding the phenomenon of control, with an emphasis on control in Turkish, as well as the behaviour of control verbs in non-infinitival environments, which have received little attention previously. I focus solely on the cases of obligatory control (OC) which constitute the only kind of control that is conditioned by the matrix verb alone. This approach is couched in Combinatory Categorial Grammar (CCG) where the control verb projects the necessary syntactic and semantic information. In particular, I argue that the control behaviour is an entailment associated with the verb itself, and that variable, split and partial control are instances of OC. Hence, no special mechanism/structure is needed to account for their interpretation. As to the syntactic and semantic status of the complement, I maintain that the complement is a bare VP in syntax and denotes a property in semantics. Building upon the conclusions reached about OC, I attempt to account for additional complementation patterns of OC verbs. I argue that here too the matrix verb has a crucial role in ruling in and out possible complement types. Finally, I note that control involves much more than just figuring out the reference of the &ldquo / unexpressed&rdquo / subject of the complement, and I furthermore propose that the additional frames of an OC verb provide important clues as to its lexical meaning, which are argued to be relevant for the acquisition of control.

In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation and salt, probably by stabilizing protein structures and lipid membranes. Trehalose-6-phosphate synthase 1 (TPS1) is a subunit of trehalose synthase complex in fungi / it plays a key role in the biosynthesis of trehalose. In this study, a TPS1 gene fragment in R. oryzae was cloned successfully by PCR with primers designed according to eight hypothetical proteins found from BLAST search which was performed by using S. cerevisiae TPS1 gene template. The full length of R. oryzae TPS1 gene (designated RoTPS1) was attained by RTPCR with primers specific to the 3&amp / #8242 / and 5&amp / #8242 / end of the RoTPS1 cDNA. The RoTPS1cDNA was composed of 2505 bps encoding a protein of 834 amino acids with a molecular mass of 93.8 kDa. The amino acid sequence has relatively high homology with the TPS1s of several other organisms. RoTPS1 was further characterized by transformation into S. cerevisiae tps1 mutant. In galactose media, the growth curves of wild type, tps1 mutant and transformant S. cerevisiae cells had a comparable pattern in general, tps1 mutant reached to a higher maximum cell concentration compared to the others and wild type had a slightly lower specific growth rate compared to the tps1 mutant and transformed cells. Trehalose levels of transformant and wild type cells were increased up to 37 mg/gdw in the stationary phase.

The largest class of cell surface receptors in mammalian genomes is the superfamily of G protein-coupled receptors (GPCRs) which are activated by a wide range of extracellular responses such as hormones, pheromones, odorants, and neurotransmitters. Drugs which have therapeutic effects on a wide range of diseases are act on GPCRs. In contrast to traditional idea, it is recently getting accepted that G-protein coupled receptors can form homo- and hetero-dimers and this interaction could have important role on maturation, internalization, function or/and pharmacology. Bimolecular fluorescence complementation technique (BiFC) / is an innovative approach based on the reassembly of protein fragments which directly report interactions. In our study we implemented this technique for detecting and visualizing the GPCR interactions in yeast cells. The enhanced green fluorescent protein (EGFP) fractionated into two fragments at genetic level which does not possess fluorescent function. The target proteins which are going to be tested in terms of interaction are modified with the non-functional fragments, to produce the fusion proteins. The interaction between two target proteins, in this study Ste2p receptors which are alpha pheromone receptors from Saccharomyces cerevisiae, enable the fragments to come in a close proximity and reassemble. After reassembly, EGFP regains its fluorescent function which provides a direct read-out for the detection of interaction. Further studies are required to determine subcellular localization of the interaction. Moreover, by using the fusion protein partners constructed in this study, effects of agonist/antagonist binding and post-translational modifications such as glycosylation and phosphorylation can be examined. Apart from all, optimized conditions for BiFC technique will guide for revealing new protein-protein interactions.

DNA diagnostics are important technologies in molecular and cellular biology. By allowing identification of specific sequences, DNA-based diagnostics potentially provide more accurate and rapid results than protein- or antigen-based diagnostics, primarily because phenotypic changes come much later than changes in genotype. Despite this advantage, there are fewer diagnostic or imaging systems that target DNA than those targeting proteins, antibodies, or antigens.Each type of DNA-based diagnostic has its own, unique set of limitations; however, most can be attributed to issues related to sequence restriction, signal detection, specificity, or some combination thereof. For example, while PCR-based methods allow amplification and assessment of specific DNA sequences, they lack the ability to report information of specific cells, or cell types, within the heterogeneous pool of cells typically found in a tumor biopsy. In addition, none of the currently available DNA detection methods has the potential to be utilized in living cells, a disadvantage which limits the potential applications.The work presented here describes the design and development of a new methodology for the detection of specific double-stranded DNA sequences. This detection method is based on the concept that two inactive fragments of a reporter protein, each coupled to engineered zinc finger DNA-binding motifs, are able to reassemble and form an active complex in the presence of a predefined DNA sequence. This system, designated sequence-enabled reassembly (SEER), can achieve single base-pair specificity, and has the potential to be utilized in living cells.In this dissertation, we discuss the efforts from constructing to refining the system, as well as the future applications of SEER in diagnostics and therapeutics. Chapter I will provide an introduction to DNA detection methods, on which the principles of the SEER system are based. Chapter II describes the design and construction of an enzymatic SEER system, SEER-LAC, using beta-lactamase as the enzyme. In Chapter III, we outline the in vitro characterization of the SEER-LAC system, followed by its optimization in Chapter IV. Chapter V illustrates the efforts to develop SEER system for mammalian cell culture applications. In the final chapter, the future developments and applications of SEER are discussed.

The interactions between protein-protein, protein-nucleic acid, and protein-small molecules are central to biological processes and are key for the design of new therapeutics. Rapid and easy to implement methodologies are needed that enable the interrogation of these interactions in a complex cellular context. Towards this goal, I have utilized the concept of split-protein reassembly, also called protein complementation, for the creation of a variety of sensor architectures that enable the interrogation of protein-nucleic acid, protein-protein, and protein-small molecule interactions. Utilizing the enzymatic split-reporter β-lactamase and existing zinc finger design strategies we applied our recently developed technology termed SEquence-Enabled Reassembly (SEER) towards the creation of a sensor capable of the specific detection of the CryIA transgene. Additionally, the split β-lactamase reporter was utilized for the sitespecific determination of DNA methylation at cytosine residues that is involved in epigenetic regulation. This method, dubbed mCpG-SEER, enabled the direct detection of femtomole levels of dsDNA methylation in sequence specific manner. In a separate endeavor, we have developed and optimized the first cell-free split-reporter systems for GFP, split β-lactamase, and firefly luciferase for the successful dsDNA-dependent reassembly of the various reporters. Our cell free in vitro translation systems eliminates previous bottlenecks encountered in split-reporter technologies such as laborious transfection/cell culture or protein purification. Capitalizing on the ease of use and speed afforded by this new technology we describe the sensitive detection of protein-protein, protein-nucleic acid, and protein-small molecule interactions and inhibitors thereof. In a related area, we have applied this rapid cell-free split-firefly luciferase assay to the specific interrogation of a large class of helix-receptor protein-protein interactions. We have built a panel consisting of the clinically relevant Bcl-2 family of proteins, hDM2, hDM4, and p53 and interrogated the specificity of helix-receptor interactions as well as the specificity of peptide and small-molecule inhibitors of these interactions. Finally, we describe the further applications of our cell-free technology to the development of a large number of general split-firefly luciferase sensors for the detection of ssRNA sequences, the detection of native proteins, the evaluation of protease activity, and interrogation of enzyme-inhibitor interactions. The new methodologies provided in this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.

Streptococcus gordonii, a commensal oral bacterium, is considered a good candidate to function as a live oral vaccine vector. The introduction of vaccine antigen genes into S. gordonii relies on the use of antibiotic resistance genes as selectable markers, which is undesirable. In this study, we used auxotrophic complementation (deletion of an essential gene from the chromosome and insertion into a plasmid) as a means to create an antibiotic marker-free gene delivery system in S. gordonii. S. gordonii ?thyA was created and complemented by an antibiotic marker-free expression plasmid containing the intact thyA gene, pDL276/thyAdelkan. Transformation of pDL276/thyAdelkan into the mutant gave an unexpected 100-fold increase in transformation efficiency as compared to pDL276. The transformants arose from both single and double crossing over. The increase in transformation efficiency suggests that a highly efficient antibiotic marker-free system to deliver genes to the chromosome has been created using thyA complementation.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 15, 2008). Lys,3 is superscript in the title. Includes bibliographical references.

Made available in DSpace on 2014-08-20T13:32:52Z (GMT). No. of bitstreams: 1 tese_sibele_borsuk.pdf: 16306468 bytes, checksum: 4743a54f442368d81ff802d8290c7cfc (MD5) Previous issue date: 2008-02-28 / Mycobacterium bovis BCG has the potential to be an effective live vector for multivalent vaccines. However, there are two problems regarding the utilization of recombinant BCG as vaccine. The first one is that most mycobacterial cloning vectors rely on antibiotic resistance gene as selectable marker, which is used for genetic transformation. The second one is the limited use of BCG in animals because it interferes in the tuberculosis diagnosis by tuberculin skin test, which elicits delayed type hypersensitivity to the purified protein derivative (PPD). In this work we developed and evaluated the use of auxotrophic complementation as a new selectable marker, characterized the proteins that are present in the bovine and avium PPD and developed a knockout BCG strain by homologous recombination. To test the auxotrophic complementation as selectable marker, an auxotrophic BCG strain for the amino acid leucine was constructed by knocking out the leuD gene by homologous recombination. Expression of leuD on a plasmid acted as a selectable marker in the auxotrophic M. bovis BCG leuD and M. smegmatis mc2144. The auxotrophic complementation selection was similar to selection by antibiotic resistance, but with the advantage of promoting stability of the plasmid. The new system was highly stable even during in vivo BCG growth. The identification of proteins from PPD was archived by LC-MS/MS (Liquid Chromatography/Mass Spectrometry/Mass Spectrometry). A total of 147 proteins among five PPD samples (2 bovine PPD and 3 avium PPD) were identified. The bovine PPD had a considerable higher number of proteins comparing to the avium PPD. We identifying a group of 28 proteins present only in bovine PPD and a group of five proteins deleted in M. bovis BCG vaccinal strain. These two groups are of special interest as they can be used in tests with improved specificity, and potentially able to differentiate vaccinated and infected individuals. A mutant BCG strain with the DPPD antigen deleted was constructed. The Mb0092 coding sequence was knocked out by homologous recombination. The 11 sequences flanking the target gene were cloned into a suicide vector. Double crossovers were selected using sacB. The knockout genotype was determined by PCR and by Southern blot. This mutant BCG strain can be useful in animal vaccination as it will not interfere in the tuberculosis diagnostic test, when performed using recombinant DPPD. The results show alternatives for the problems related to the use of M. bovis BCG as a recombinant vaccine. The auxotrophic complementation system was highly stable, efficient and it is suitable for expressing heterologous antigens in BCG. The identification of proteins present in PPD preparations and the mutant BCG obtained provide the possibility for the development of differential diagnostic test, thus allowing the use of BCG as vaccine also in animals. / Mycobacterium bovis BCG tem o potencial para ser um vetor efetivo para vacinas recombinantes multivalentes. No entanto, existem dois problemas quanto a sua utilização como vetor vacinal. O primeiro é a presença de genes que conferem resistência a antibióticos nos vetores utilizados para transformação genética. O segundo é a limitação de uso de BCG em animais, principalmente por comprometer o teste de tuberculina, utilizado como diagnóstico de tuberculose, o qual se baseia em reação de hipersensibilidade ao PPD (Derivado Protéico Purificado). Neste trabalho desenvolvemos e avaliamos a complementação auxotrófica como novo marcador de seleção, fizemos a caracterização das proteínas componentes de amostras de PPD aviário e bovino e desenvolvemos um mutante de BCG por recombinação homóloga. Para o uso de complementação auxotrófica como marcador de seleção, uma cepa de BCG auxotrófica para o aminoácido leucina foi construída por knockout do gene leuD por recombinação homóloga. A expressão do gene leuD em um plasmídio atuou como marcador de seleção nas cepas auxotróficas de M. bovis BCG leuD e M. smegmatis mc2144. A seleção por complementação de BCG auxotrófica se mostrou equivalente à seleção por resistência a antibiótico, com a vantagem adicional de proporcionar maior estabilidade do vetor plasmidial, já que a pressão seletiva é mantida mesmo durante multiplicação da bactéria in vivo. A identificação das proteínas que compõem o PPD foi feita por espectrometria de massa utilizando-se LCMS/ MS (cromatografia líquida associada à espectrometria de massa em tandem). Foram identificadas 147 proteínas entre 5 amostras de PPD (2 PPD bovino e 3 PPD aviário). O PPD bovino teve um número maior de proteínas comparado ao PPD aviário. Foi identificado um grupo de 28 proteínas presentes em PPD bovino, mas ausentes em PPD aviário. Além disso, 5 proteínas encontradas no PPD estão ausentes em M. bovis BCG. Estes são de 9 especial interesse, pois poderão vir a contribuir para o desenvolvimento de um teste de diagnóstico mais específico, e possivelmente capaz de diferenciar indivíduo vacinado com BCG e infectado com o bacilo da tuberculose. Um mutante de M. bovis BCG Pasteur foi construído. O gene Mb0092 (dppd) foi alvo de inativação gênica por recombinação homóloga. Seqüências que flanqueiam o gene alvo foram clonadas em um vetor suicida. Duplo crossover foi selecionado utilizando sacB. O genótipo mutante foi determinado por PCR e por Southern blot. Esta cepa poderá ser utilizada como vacina em animais, quando o diagnóstico for feito com DPPD recombinante. Os resultados obtidos apresentam alternativas para os problemas envolvidos quanto à utilização de M. bovis BCG como vacina recombinante. O sistema de seleção por complementação auxotrófica foi estável, e pode ser empregado na expressão de antígenos heterólogos em BCG. A identificação dos principais componentes protéicos do PPD e o desenvolvimento da cepa mutante de BCG possibilitam o desenvolvimento de testes diagnósticos diferencias, permitindo a utilização de BCG como vacina também em animas.