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

Hillebrand, Sandro
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 09/09/2005 PT
Relevância na Pesquisa
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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...

Determination of nuclear transcription factor activity using a modified electrophoretic mobility shift assay

Wang,Yong; Huang,Wenhua; Peng,Daizhi; Hu,Yuanbin
Fonte: Instituto de Tecnologia do Paraná - Tecpar Publicador: Instituto de Tecnologia do Paraná - Tecpar
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/08/2008 EN
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1359.034%
In this work, several major procedures of the electrophoretic mobility shift assay (EMSA) were modified including swift extraction of the nucleic protein, labeling of the probe and radioautography. The modified assay required shorter time, simplified the nucleic protein extraction, increased the radioactivity of the labeling probe, skipped the tedious process of gel drying, and produced clear images. Its results were comparable, reproducible and stable. It thus has merited for wide application.

Gel Shift Assay of Nuclear Extracts from Histoplasma capsulatum Demonstrates the Presence of Several DNA Binding Proteins

Ignatov, Atanas; Keath, Elizabeth J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2002 EN
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A gel shift assay was optimized to detect several general DNA binding proteins from Histoplasma capsulatum strain G217B. The electrophoretic mobility shift assay (EMSA) technique also detected protein(s) recognizing a pyrimidine-rich motif found in several Histoplasma promoters. Establishment of EMSA conditions provides an important framework to evaluate regulation of homeostatic or phase-specific genes that may influence virulence in Histoplasma and other dimorphic fungal pathogens.

Rapid and Simple Detection of Hot Spot Point Mutations of Epidermal Growth Factor Receptor, BRAF, and NRAS in Cancers Using the Loop-Hybrid Mobility Shift Assay

Matsukuma, Shoichi; Yoshihara, Mitsuyo; Kasai, Fumio; Kato, Akinori; Yoshida, Akira; Akaike, Makoto; Kobayashi, Osamu; Nakayama, Haruhiko; Sakuma, Yuji; Yoshida, Tsutomu; Kameda, Yoichi; Tsuchiya, Eiju; Miyagi, Yohei
Fonte: American Society for Investigative Pathology Publicador: American Society for Investigative Pathology
Tipo: Artigo de Revista Científica
Publicado em /09/2006 EN
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A simple and rapid method to detect the epidermal growth factor receptor hot spot mutation L858R in lung adenocarcinoma was developed based on principles similar to the universal heteroduplex generator technology. A single-stranded oligonucleotide with an internal deletion was used to generate heteroduplexes (loop-hybrids) bearing a loop in the complementary strand derived from the polymerase chain reaction product of the normal or mutant allele. By placing deletion in the oligonucleotide adjacent to the mutational site, difference in electrophoretic mobility between loop-hybrids with normal and mutated DNA was distinguishable in a native polyacrylamide gel. The method was also modified to detect in-frame deletion mutations of epidermal growth factor receptor in lung adenocarcinomas. In addition, the method was adapted to detect hot spot mutations in the B-type Raf kinase (BRAF) at V600 and in a Ras-oncogene (NRAS) at Q61, the mutations commonly found in thyroid carcinomas. Our mutation detection system, designated the loop-hybrid mobility shift assay was sensitive enough to detect mutant DNA comprising 7.5% of the total DNA. As a simple and straightforward mutation detection technique, loop-hybrid mobility shift assay may be useful for the molecular diagnosis of certain types of clinical cancers. Other applications are also discussed.

Identification of a Calcitriol-Regulated Sp-1 Site in the Promoter of Human CD14 using a Combined Western Blotting Electrophoresis Mobility Shift Assay (WEMSA)

Moeenrezakhanlou, Alireza; Nandan, Devki; Reiner, Neil E.
Fonte: Biological Procedures Online Publicador: Biological Procedures Online
Tipo: Artigo de Revista Científica
Publicado em 17/02/2008 EN
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825.5243%
Calcitriol (1α, 25-dihydroxyvitamin D3) induces the expression of CD14 in mononuclear phagocytes. The mechanisms accounting for this have been unclear since the promoter of CD14 does not contain a canonical vitamin D response element (VDRE). Calcitriol has been shown to regulate the activity of the transcription factor Sp-1 and our analysis of the proximal promoter of CD14 indicated the presence of four Sp-1-like binding sequences. To identify which of these sites might be involved in the response to calcitriol, we used a system incorporating an electrophoretic mobility shift assay (EMSA) coupled to Western blot analysis (WEMSA). Using WEMSA, we found that only one of the Sp-1-like binding sequences, located at position -91 to -79 (relative to the transcription start site), bound the transcription factor Sp1. Sp-1 binding to this site was demonstrable using nuclear extracts from control cells. Notably, binding activity was attenuated in nuclear extracts prepared from cells that had been incubated with calcitriol, thus suggesting Sp-1 involvement in calcitriol induction of CD14 expression. Notably, these results show that like EMSA, WEMSA can be broadly applied to aid in the identification of transcription factors involved in regulating gene expression. WEMSA...

Analysis of factor interactions with RNA polymerase II elongation complexes using a new electrophoretic mobility shift assay

Cheng, Bo; Price, David H.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
EN
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943.5034%
The elongation phase of transcription by RNA polymerase II (RNAP II) is controlled by a carefully orchestrated series of interactions with both negative and positive factors. However, due to the limitations of current methods and techniques, not much is known about whether and how these proteins physically associate with the engaged polymerases. To gain insight into the detailed mechanisms involved, we established an experimental system for analyzing direct factor interactions to RNAP II elongation complexes on native gels, namely elongation complex electrophoretic mobility shift assay (EC-EMSA). This new assay effectively allowed detection of interactions of TFIIF, TTF2, TFIIS, DSIF and P-TEFb with elongation complexes generated from a natural promoter using an immobilized template. As an application of this assay system, we characterized the association of transcription elongation factor DSIF with RNAP II elongation complexes and discovered that the nascent transcript facilitated recruitment of DSIF. Examples of how the system can be manipulated to address different questions are provided. EC-EMSA should be useful for further investigation of factor interactions with RNAP II elongation complexes.

Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions

Hellman, Lance M.; Fried, Michael G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2007 EN
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843.5034%
The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.

ELECTROPHORETIC MOBILITY SHIFT ASSAY OF ZINC FINGER PROTEINS: COMPETITION FOR ZN2+ BOUND TO SP1 IN PROTOCOLS INCLUDING EDTA

Kothinti, Rajendra; Tabatabai, Niloofar M.; Petering, David H.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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The electrophoretic mobility shift assay (EMSA) offers a principal method to detect specific DNA·protein interactions. As commonly conducted, the reaction and electrophoresis running buffers contain large concentrations of EDTA. EDTA has large affinity for Zn2+ and readily competes with zinc-finger peptides for Zn2+ resulting in protein unfolding. Nevertheless, EMSA is routinely used to detect zinc-finger protein·DNA adducts. This paper examines the chemistry that permits the detection of zinc-finger·DNA complexes in the presence of EDTA, using Zn3-Sp1 and a cognate DNA binding site, GC1. Twice as much adduct was detected when the reaction was conducted in the absence than in the presence of EDTA. The observation of Zn-Sp1·GC1 was shown to depend on three properties: the inertness of Zn-Sp1·GC1 to reaction with EDTA and the comparatively similar rates of reaction of EDTA and GC1 with Zn3-Sp1 under the conditions of the assay that permit some Zn3-Sp1·GC1 to form. Inquiring about the mechanism of stabilization of Zn3-Sp1 by GC1, EDTA readily reacted with Zn3-Sp1 bound to a non-specific DNA, poly(dI-dC). Two structurally similar but oppositely charged chelators, nitrilotriacetate (NTA) and tris-(2-ethylaminoethyl) amine (TREN), that react with free Zn3-Sp1 failed to compete for zinc bound in the Zn3-Sp1·GC-1 adduct. On the basis of these and other results indicated that the stability of Zn3-Sp1·GC-1 has a thermodynamic not a kinetic origin. It is concluded that the observation of zinc finger proteins in the EMSA rests on a fortuitous set of chemical properties that may vary depending on the structures involved.

Transcription Factor Proteomics: Identification by a Novel Gel Mobility Shift-Three-Dimensional Electrophoresis Method Coupled with Southwestern Blot and HPLC-Electrospray-Mass Spectrometry Analysis

Jiang, Daifeng; Jia, Yinshan; Jarrett, Harry W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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725.5243%
Transcription factor (TF) purification and identification is an important step in elucidating gene regulatory mechanisms. In this study, we present two new electrophoretic mobility shift assay (EMSA)-based multi-dimensional electrophoresis approaches to isolate and characterize TFs, using detection with either southwestern or western blotting and HPLC-nanoESI-MS/MS analysis for identification. These new techniques involve several major steps. First, EMSA is performed with agents that diminish non-specific DNA-binding and the DNA-protein complex is separated by native PAGE gel. The gel is then electrotransferred to PVDF membrane and visualized by autoradiography. Next, the DNA-protein complex, which has been transferred onto the blot, is extracted using a detergent-containing elution buffer. Following detergent removal, concentrated extract is separated by SDS-PAGE (EMSA-2DE), followed by in-gel trypsin digestion and HPLC-nanoESI-MS/MS analysis, or the concentrated extract is separated by two-dimensional gel electrophoresis EMSA-3DE), followed by southwestern or western blot analysis to localize DNA binding proteins on blot which are further identified by on-blot trypsin digestion and HPLC-nanoESI-MS/MS analysis. Finally, the identified DNA binding proteins are further validated by EMSA-immunoblotting or EMSA antibody supershift assay. This approach is used to purify and identify GFP-C/EBP fusion protein from bacterial crude extract...

Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein

Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S; Prasad, Manoj
Fonte: Landes Bioscience Publicador: Landes Bioscience
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
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The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncation of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T] [T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein.

Mapping DNA Quantity into Electrophoretic Mobility through Quantum Dot Nanotethers for High Resolution Genetic and Epigenetic Analysis

Zhang, Yi; Liu, Kelvin J.; Wang, Tian-Li; Shih, Ie-Ming; Wang, Tza-Huei
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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Newly discovered nanoparticle properties have driven the development of novel applications and uses. We report a new observation where the electrophoretic mobility of a quantum dot-DNA nanoassembly can be precisely modulated by the degree of surface DNA conjugation. By using streptavidin-coated quantum dots (QD) as nanotethers to gather biotin-labeled DNA into electrophoretic nanoassemblies, the QD surface charge is modulated and transformed into electrophoretic mobility shifts using standard agarose gel electrophoresis. Typical fluorescent assays quantify based on relative intensity. However, this phenomenon uses a novel approach that accurately maps DNA quantity into shifts in relative band position. This property was applied in a quantum dot enabled nanoassay called Quantum Dot Electrophoretic Mobility Shift Assay (QEMSA) that enables accurate quantification of DNA targets down to 1.1-fold (9%) changes in quantity, beyond what is achievable in qPCR. In addition to these experimental findings, an analytical model is presented to explain this behavior. Finally, QEMSA was applied to both genetic and epigenetic analysis of cancer. First, it was used to analyze copy number variation (CNV) of the RSF1/HBXAP gene where conventional approaches for CNV analysis based on comparative genomic hybridization (CGH)...

Direct visualization of electrophoretic mobility shift assays using nanoparticle-aptamer conjugates

Wang, Min S.; Reed, Scott M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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839.4435%
Here we demonstrate that aptamers tethered to gold nanoparticles enable direct visualization of protein-oligonucleotide interactions during gel electrophoresis. This technique is used to confirm that an aptamer previously identified as binding to C-reactive protein (CRP) only binds to the monomeric form of CRP. While native, pentameric CRP (pCRP) is used in clinical assays to predict cardiovascular disease (CVD) risk, it is the monomeric isoform that is more strongly associated with pro-inflammatory and pro-atherogenic effects. To visualize this selectivity, the CRP-aptamer was conjugated to streptavidin-coated gold nanoparticles and the mobility of the free oligonucleotide-nanoparticle conjugate (ON-NP) and the protein/ON-NP complex bands were visualized and recorded during electrophoresis using a simple digital camera. At a concentration of 6 mg/mL, monomeric CRP showed a significant decrease in the observed ON-NP mobility, whereas no change in mobility was observed with pCRP up to 18 mg/mL. Advantages of this nanoparticle-based electrophoretic mobility shift assay (NP-EMSA) over traditional EMSA include real-time detection of protein-oligonucleotide interactions, the avoidance of harmful radioisotopes, and elimination of the need for expensive gel imagers. The availability of both the NP-EMSA technique and an mCRP specific probe will allow for improved clinical diagnostic to more accurately predict future CVD risk.

Microfluidic Screening of Electrophoretic Mobility Shifts Elucidates Riboswitch Binding Function

Karns, Kelly; Vogan, Jacob M.; Qin, Qian; Hickey, Scott F.; Wilson, Stephen C.; Hammond, Ming C.; Herr, Amy E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
644.2587%
Riboswitches are RNA sensors that change conformation upon binding small molecule metabolites, in turn modulating gene expression. Our understanding of riboswitch regulatory function would be accelerated by a high throughput, quantitative screening tool capable of measuring riboswitch-ligand binding. We introduce a microfluidic mobility shift assay that enables precise and rapid quantitation of ligand binding and subsequent riboswitch conformational change. In 0.3% of the time required for bench top assays (3.2 min vs. 1020 min), we screen and validate five candidate SAM-I riboswitches isolated from thermophilic and cryophilic bacteria. The format offers enhanced resolution of conformational change compared to slab gel formats, quantitation and repeatability for statistical assessment of small mobility shifts, low reagent consumption, and riboswitch characterization without modification of the aptamer structure. Appreciable analytical sensitivity coupled with high resolution separation performance allows quantitation of equilibrium dissociation constants (Kd) for both rapidly and slowly interconverting riboswitch-ligand pairs as validated through experiments and modeling. Conformational change, triplicate mobility shift measurements...

An Electrophoretic Mobility Shift Assay Identifies a Mechanistically Unique Inhibitor of Protein Sumoylation

Kim, Yeong Sang; Nagy, Katelyn; Keyser, Samantha; Schneekloth, John S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 18/04/2013 EN
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The dynamic, posttranslational modification of proteins with a SUMO tag has been recognized as an important cellular regulatory mechanism relevant to a number of cancers as well as normal embryonic development. As part of a program aimed towards the identification of inhibitors of SUMO conjugating enzymes, we have developed a microfluidic electrophoretic mobility shift assay to monitor sumoylation events in real time. We disclose herein the use of this assay to discover the first cell permeable compound capable of blocking the transfer of SUMO-1 from the E2 enzyme UBC9 to the substrate. We screened a small collection of compounds and identified an oxygenated flavonoid derivative that inhibits sumoylation in vitro. Next, we carried out an in-depth mechanistic analysis that ruled out many common false positive mechanisms such as aggregation or alkylation. Furthermore, we report that this flavonoid inhibits a single step in the sumoylation cascade: the transfer of SUMO from the E2 enzyme (UBC9) thioester conjugate to the substrate. In addition to being the first example of a compound with this unique mechanism of action, this inhibitor has a discreet structure-activity relationship uncharacteristic of a promiscuous inhibitor. Cell-based studies showed that the flavonoid inhibits the sumoylation of topoisomerase-I in response to camptothecin treatment in two different breast cancer cell lines...

Electrophoretic Mobility Shift Assays for Protein–DNA Complexes Involved in DNA Repair*

Tsai, Chun; Smider, Vaughn; Hwang, Byung Joon; Chu, Gilbert
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2012 EN
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836.98055%
The electrophoretic mobility shift assay (EMSA) can be used to study proteins that bind to DNA structures created by DNA-damaging agents. UV-damaged DNA-binding protein (UV-DDB), which is involved in nucleotide excision repair, binds to DNA damaged by ultraviolet radiation or the anticancer drug cisplatin. Ku, XRCC4/Ligase IV, and DNA–PKcs, which are involved in the repair of DNA double-strand breaks by nonhomologous end joining, assemble in complexes at DNA ends. This chapter will describe several EMSA protocols for detecting different DNA repair protein–DNA complexes. To obtain additional information, one can apply variations of the EMSA, which include the reverse EMSA to detect binding of 35S-labeled protein to damaged DNA, and the antibody supershift assay to detect the presence of a specific protein in the protein–DNA complex.

Analyzing the nuclear complexes of Notch signaling by Electrophoretic Mobility Shift Assay

Arnett, Kelly L.; Blacklow, Stephen C.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2014 EN
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An Electrophoretic Mobility Shift Assay (EMSA) is a sensitive technique for detecting protein-DNA and protein-protein interactions in which complexes are separated by native (non-denaturing) gel electrophoresis. EMSAs can provide evidence for specific binding between components prepared from a wide range of sources, including not only highly purified proteins but also components of crude cellular extracts. EMSA experiments were critical in identifying the minimal protein requirements for assembly of transcriptionally active nuclear Notch complexes as well as the DNA sequence specificity of Notch transcription complexes. Here, we describe a radioactive EMSA protocol for detection of Notch transcription complexes.

The two-component signal transduction system RR06/HK06 regulates expression of cbpA in Streptococcus pneumonia

Standish, A.; Stroeher, U.; Paton, J.
Fonte: Natl Acad Sciences Publicador: Natl Acad Sciences
Tipo: Artigo de Revista Científica
Publicado em //2005 EN
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829.27164%
Streptococcus pneumoniae encounters a number of environmental niches in the body, including the nasopharynx, lungs, blood, middle ear, and brain. Recent studies have identified 13 putative two-component signal-transduction systems in S. pneumoniae, which are likely to be important for gene regulation in response to external stimuli. Here, we present conclusive evidence for the regulation of choline binding protein A (CbpA), a major pneumococcal virulence factor and protective antigen, by one of these two-component signal-transduction systems. We have demonstrated divergent expression of cbpA in unmarked hk06 and rr06 deletion mutants relative to wild-type S. pneumoniae D39 by using Western immunoblotting and real-time RT-PCR. Electrophoretic mobility-shift and solid-phase binding assays have demonstrated the binding of RR06 to the promoter region of cbpA, suggesting that RR06/HK06 directly regulates cbpA transcription. We have also shown that this system is important for the ability of the pneumococcus to adhere to epithelial cells in vitro and to survive and proliferate in an in vivo mouse model. Thus, the RR06/HK06 system has a significant role in pathogenesis, both in colonization and invasive disease.; Alistair J. Standish, Uwe H. Stroeher...

Application of Medicinal Chemistry Methods on Different Classes of Drugs

BALLANTE, FLAVIO
Fonte: La Sapienza Universidade de Roma Publicador: La Sapienza Universidade de Roma
Tipo: Tese de Doutorado
EN
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839.092%
The present doctoral thesis is the result of the work carried out during the three years of my PhD scholarship at the Rome Center for Molecular Design laboratory (RCMD, Department of Chemistry and Drug Technologies, Sapienza University of Rome), under the supervision of Prof. Rino Ragno. The research activity was focused mainly on the design, optimization and application of computational strategies to derive quantitative structure-activity relationships (QSAR, 3-D QSAR, and COMBINE) on different molecular classes of current interest, such as: opioid receptor antagonists (OPAs), Hepatitis C Virus NS5B-Polymerase Inhibitors (NS5B-NNIs), Hystone Deacetylase Inhibitors (HDACIs), Anti- tubercular agents, vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors, HSP90 inhibitors, HIV-1 reverse transcriptase inhibitors (NNRTIs), Bovine Serum Amine Oxidase (BSAO) substrates, etc... Moreover two research periods abroad were performed: the first framed in a LLP Erasmus program collaboration, was conducted for six months at the Laboratoire d'Ingénierie et Moléculaire Pharmacologique Biochimie (LIMBP) of the Université de Lorraine Metz (France), directed by Prof. Gilbert Kirsch, and characterized by the application of organic synthesis to obtain new thienopyrimidinone derivatives as potential inhibitors of vascular endothelial growth factor receptor-2 (VEGFR-2); the second took place...

Gel mobility shift scanning of pectin-inducible promoter from Penicillium griseoroseum reveals the involvement of a CCAAT element in the expression of a polygalacturonase gene

Ribon, Andréa de O. B.; Ribeiro, João Batista; Gonçalves, Daniel B.; de Queiroz, Marisa V.; de Araújo, Elza F.
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
722.8206%
Previous reports have described pgg2, a polygalacturonase-encoding gene of Penicillium griseoroseum, as an attractive model for transcriptional regulation studies, due to its high expression throughout several in vitro growth conditions, even in the presence of non-inducing sugars such as sucrose. A search for regulatory motifs in the 5' upstream regulatory sequence of pgg2 identified a putative CCAAT box that could justify this expression profile. This element, located 270 bp upstream of the translational start codon, was tested as binding target for regulatory proteins. Analysis of a 170 bp promoter fragment by electrophoretic mobility shift assay (EMSA) with nuclear extracts prepared from mycelia grown in pectin-containing culture medium revealed a high mobility complex that was subsequently confirmed by analyzing it with a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished the formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied.

Gel mobility shift scanning of pectin-inducible promoter from Penicillium griseoroseum reveals the involvement of a CCAAT element in the expression of a polygalacturonase gene

Ribon,Andréa de O.B.; Ribeiro,João Batista; Gonçalves,Daniel B.; Queiroz,Marisa V. de; Araújo,Elza F. de
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2009 EN
Relevância na Pesquisa
1149.5904%
Previous reports have described pgg2, a polygalacturonase-encoding gene of Penicillium griseoroseum, as an attractive model for transcriptional regulation studies, due to its high expression throughout several in vitro growth conditions, even in the presence of non-inducing sugars such as sucrose. A search for regulatory motifs in the 5' upstream regulatory sequence of pgg2 identified a putative CCAAT box that could justify this expression profile. This element, located 270 bp upstream of the translational start codon, was tested as binding target for regulatory proteins. Analysis of a 170 bp promoter fragment by electrophoretic mobility shift assay (EMSA) with nuclear extracts prepared from mycelia grown in pectin-containing culture medium revealed a high mobility complex that was subsequently confirmed by analyzing it with a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished the formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied.