Tesis sobre el tema "Yeast two-hybrid"
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1
Vafiadaki, Elizabeth. "An investigation of the role of dysferlin in skeletal muscle". Thesis, University of Newcastle Upon Tyne, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250105.
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Frei, Eva. "N-C Interaktionen des Ca2+-aktivierten Kaliumkanals, hSK3". [S.l. : s.n.], 2007. http://nbn-resolving.de/urn:nbn:de:bsz:289-vts-58883.
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INOUE, MASAHIKO, YOSHIHIRO WAKAYAMA, TAKAHIRO JIMI, SEIJI SHIBUYA, HAJIME HARA, AKIHIKO UNAKI y KIYOKAZU KENMOCHI. "SKELETAL MUSCLE SYNTROPHIN INTERACTORS REVEALED BY YEAST TWO-HYBRID ASSAY". Nagoya University School of Medicine, 2008. http://hdl.handle.net/2237/10550.
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Guy, Colin Paul. "RadB from archaea : bioinformatics, biochemistry and yeast two-hybrid analyses". Thesis, University of Nottingham, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.446393.
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Taylor, Danielle Nicola. "Yeast two-hybrid studies with tobacco mosaic virus replicase proteins". Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624336.
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Schmidt, Carsten. "Untersuchungen zu Angiogenin-Interakteuren mit Hilfe des Yeast-Two-Hybrid-Systems". [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=963014544.
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Hung, Kwok Wang. "Identification of the EphA4-interacting proteins by yeast two-hybrid screening /". View abstract or full-text, 2006. http://library.ust.hk/cgi/db/thesis.pl?BICH%202006%20HUNG.
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Kashuba, Elena. "Identification of EBNA binding cellular proteins, using yeast two-hybrid system /". Stockholm, 2002. http://diss.kib.ki.se/2003/91-7349-416-X/.
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Nguyen, Jacqueline Phuong Anh. "Finding Interactions of SCRAMBLED by Using the Yeast Two-Hybrid System". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p1453195.
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Thesis (M.S.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed July 1, 2008). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 40-42).
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Meddins, Anna Kathryn. "Isolating candidate cyclin-binding proteins using the Yeast Two-Hybrid assay". Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627268.
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McGillewie, L. "Identification of novel ligands of WDR47, using yeast two-hybrid analysis". Thesis, Stellenbosch : University of Stellenbosch, 2009. http://hdl.handle.net/10019.1/3002.
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Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics. Medical Biochemistry))--University of Stellenbosch, 2009.The mammalian neocortex contributes to the increasing functional complexity of the mammalian brain, partly because of its striking organisation into distinct neuronal layers. The development of the neocortex has been well studied because disrupted neurodevelopment results in several human diseases. The basic principles of neocortical development have been well established for some time; however the molecular mechanisms have only recently been identified. One major advance in our understanding of these molecular mechanisms was the discovery of Reelin, an extracellular matrix protein that directs the migration of neurons to their final positions in the developing neocortex. Reelin is a large multi-domain protein that exerts its functions by binding to its ligands on the cell surface and initiating a signal transduction cascade that ultimately results in cytoskeletal rearrangements. Several investigations have been undertaken to elucidate the functions of each of these domains to gain a better understanding reelin’s functions. We have previously identified the WR40 repeat protein 47 (WDR47), a protein of unknown function, as a novel putative ligand for the N-terminal reeler domain of reelin. To gain better understanding into the functional significance of this interaction, the present study sought to identify novel WDR47- interacting proteins. In order to achieve this, a cDNA encoding a polypeptide that contains the two N-terminal domains of WDR47, i.e. the Lis homology and the C-terminal Lis homology domain (CTLH) was used as bait in a Y2H screen of a foetal brain cDNA library. Putative WDR47 ligands were subsequently verified using 3D in vivo co-localisation. Results of these analyses showed that SCG10, a microtubule destabilizing protein belonging to the stathmin family of proteins, interacted with the N-terminal of WDR47. The identification of SCG10 as a novel WDR47 interacting protein not only sheds some light on the role and function of WDR47 but also aids in a better understanding of the reelin pathway and cortical lamination. Moreover, the data presented here, may also provide researchers with new avenues of research into molecular mechanisms involved in neuronal migration disorders.
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Durns, Tyler Adam. "Identifying Mucolipin Interactors Using the Split-Ubiquitin Yeast Two-Hybrid System". Thesis, The University of Arizona, 2011. http://hdl.handle.net/10150/144315.
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Tan, Elaine. "Interactions of p24 proteins characterized by yeast two-hybrid, mutagenesis, and overexpression". Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40758.
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The evolutionary conserved and abundant p24 proteins cycle through the early secretory pathway, but their cellular functions are still poorly understood. They are not essential in yeast, but in mice, p23 knockout is embryonic lethal. Moreover, p23 is involved in Alzheimer’s disease pathogenesis. The p24 proteins have the ability to form hetero-complexes. Here, the pairwise interactions among p24s, in yeast and human, were determined by yeast two-hybrid. The p24 interactions are specific and occur mostly through their GOLD domain with some contribution from their DOG sequence. Mutagenesis experiments suggested that two p24s interact differently with a common p24 partner. Finally, co-overexpression of p24s in yeast has a harmful effect on a subset of combinations. Some have a growth defect whereas others have a cell size increase phenotype or both. I propose that the p24 proteins might participate in transport of GPI-anchored proteins involved cell wall maintenance or cell cycle pathways.Les abondantes protéines de la famille p24 sont conservées dans l’évolution et circulent dans la voie sécrétoire précoce, mais leurs fonctions cellulaires sont encore mal définies. Ils sont dispensables chez la levure, mais un knock-out de p23 est létal embryonnaire chez la souris. De plus, p23 est impliqué dans la pathogénèse de la maladie d’Alzheimer. Les protéines p24 peuvent former des hétéro-complexes. Dans cette étude, les interactions des p24s sont déterminées par double hybride. Elles sont spécifiques et se font surtout par leur domaine GOLD avec une contribution de leur séquence DOG. Les expériences de mutagénèse révèlent que deux p24s interagissent différemment avec un p24 commun. La co-surexpression de certains p24s dans la levure cause la mort ou l’élargissement des cellules. Je propose que les protéines p24s participent dans le transport des protéines à ancrage GPI impliquées dans l’entretien de la paroi cellulaire et dans les voies du cycle cellulaire.
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Wilcox, Andrew. "Cloning of novel insulin-signalling proteins using the yeast two-hybrid system". Thesis, University of Nottingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272849.
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Millward, Laurie M. "Yeast Two-Hybrid Analysis of Cellular Proteins Interacting with HTLV-1 p30". The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1267727377.
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Aikebaierjiang, Abasi. "Protein-Protein Interaction Assay in Phytophthora sojae Using Yeast Two-Hybrid System". Bowling Green State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1585911019625665.
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Suliman, Muhtadi. "Interactome analysis of pancreatic cancer expressed proteins : a yeast two hybrid approach". Aix-Marseille 2, 2008. http://theses.univ-amu.fr.lama.univ-amu.fr/2008AIX22002.pdf.
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L'adénocarcinome pancréatique (PDAC) est aujourd’hui un problème important de santé publique. Les traitements conventionnels contre le cancer ont peu d'impact sur le cours de cette maladie. Les changements génétiques impliqués dans la progression du PDAC concernent souvent des gènes qui codent pour des protéines liées à différentes voies de transduction de signal. Ce fait indique l'importance d’identifier le rôle et les relations entre les multiples voies de signalisation impliquées dans le PDAC. Dans ce travail nous utilisons une approche double-hybride chez la levure pour identifier des interactions impliquant des protéines exprimées dans des cellules cancéreuses pancréatiques. Dans la première partie de cette thèse, un groupe particulier de protéines, les protéines d’échafaudage, contenant de multiples domaines (SH3), a été étudié. Nous avons identifié plusieurs molécules interagissant avec ces protéines, modulant la prolifération cellulaire, la survie (CIZ1, BIRC6, RBBP6), la signalisation (LTBP4, Notch2, TOM1L1, STK24) et la dynamique des membranes (PLSCR1, DDEF2, VCP). Certaines protéines interagissant avec (Vav2, ITSN1, Stac) sont connues pour leurs différents rôles dans le vésicules de transport, l'endocytose ainsi que dans d’autres processus de régulation. Nous avons aussi dentifié des interactions protéiques assurées par d’autres protéines exprimées dans le pancréas, et ce pour identifier les événements qui sont spécifiques aux cellules cancéreuses pancréatiques. PTFIA est un activateur de transcription qui joue un rôle déterminant dans l’organogenèse pancréatique, il est aussi impliqué dans la maintenance du pancréas exocrine. Cette protéine contient un domaine HLH qui facilite la conversion des monomères inactifs en dimères s’activant pendant les étapes appropriées du développement. Nous avons identifié plusieurs molécules interagissant avec cette protéine, modulant la prolifération cellulaire (GCIP); différentiation de macrophage (PRKX) et la tumorigenesis d'intestinal. KLF6 est un facteur de transcription qui peut activer ou inhiber des gènes impliqués dans la régulation du cycle cellulaire. Quelques études présentent KLF6 comme étant un facteur de transcription impliqué dans la prolifération cellulaire. Il a été récemment démontré qu’il était déréglé dans des multiples cancers (prostate et ovaires), dérèglement traduit par une perte d’hétérozygotie. Nous avons identifié plusieurs molécules interagissant avec cette protéine, modulant la Ribosylation (PARP10) et l’adhésion (MSN). ArgBP2 a été récemment identifié comme étant un nouveau marqueur pour le cancer pancréatique et potentiellement une nouvelle cible thérapeutique. ArgBP2 est un substrat et interacteur des tyrosines kinases Arg et Abl, il a aussi été trouvé concentré sur les fibres d'actine. ArgBP2 appartiennent à une famille de protéines d'échafaudage qui incluent la vinexine et le CAP/ponsin; ils participent à la régulation de l’adhésion cellulaire, l’organisation du cytosquelette d'actine et la signalisation, en aval, des récepteurs de facteurs de croissance. La protéine ArgBP2 est caractérisée par un domaine (SoHo) dans la région N-terminal et trois domaines SH3 dans la région de C-terminal. Nous avons identifié la protéine CIP4 (Cdc42 interacting protein 4) comme étant un nouvel interacteur d’ArgBP2. Nous avons montré que, par cette interaction, ArgBP2 pouvait augmenter la capacité de c-Abl à phosphiryler CIP4 et que CIP4, après interaction, pouvait inhiber la phosphorylation d'ArgBP2. L’utilisation de petites molécules capables de perturber les interactions et l'analyse de l’effet de ces pertes d’interactions sur la croissance des cellules cancéreuses pancréatiques, in vivo et in vitro, pourraient permettre d’identifier des agents thérapeutiques potentiels pour le traitement du cancer pancréatique, une des maladies les plus meurtrières connues.
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Graham, Kevin Campbell. "Production of two S. cerevisiae strains designed to enhance utilization of the yeast two-hybrid system". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1996. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/MQ32118.pdf.
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Benzing, Jörg. "Identifikation intrazellulärer Interaktionspartner der Rezeptortyrosinkinasen UFO und MET im Two-Hybrid-System". Ulm : Universität Ulm, Medizinische Fakultät, 2001. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB9394024.
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Popat, Paiyal V. "Use of the yeast two-hybrid system to define the function of THAP5 protein". Honors in the Major Thesis, University of Central Florida, 2009. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/1310.
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This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.Bachelors
Medicine
Microbiology and Molecular Biology
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Mir, Kiran D. "The rotavirus nonstructural protein 4 (NSP4) interacts with both the N- and C- termini of caveolin-1". Texas A&M University, 2003. http://hdl.handle.net/1969.1/3976.
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Rotavirus (RV) is an etiologic agent of viral gastroenteritis in children and infants
worldwide, accounting for an estimated 500,000 deaths annually. NSP4, the first
described viral enterotoxin, contributes to RV pathogenesis by mobilizing intracellular
calcium through multiple mechanisms that promote abnormal ion transport and
subsequent secretory diarrhea. NSP4 and the enterotoxic peptide 114-135 preferentially
interact with model membranes mimicking caveolae in lipid composition and radius of
curvature. Our laboratory has recently reported the colocalization and
coimmunoprecipitation of NSP4 with caveolin-1, the structural protein of caveolae.
Moreover, the caveolin-1 binding domain of NSP4 has been localized to the enterotoxic
peptide. We now report that caveolin-1 binds NSP4 via the N- and C-termini and one
terminus is sufficient for binding. A panel of caveolin-1 deletion mutants was expressed
in a yeast two-hybrid assay against an NSP4 bait. Caveolin-1 mutants retaining at least
one terminus were capable of binding the NSP4 bait. An in vitro binding assay
confirmed the two-hybrid results and localized the NSP4 binding domains to caveolin-1
residues 2-22 and 161-178. These data support the hypothesis that caveolin-1 mediates
NSP4 signaling and/or intracellular trafficking.
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de, Bock Charles Edo St George Clinical School UNSW. "Novel protein interactors of urokinase-type plasminogen activator receptor". Awarded by:University of New South Wales. St George Clinical School, 2005. http://handle.unsw.edu.au/1959.4/23009.
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The plasminogen activator (PA) system plays an important role in cell adhesion, migration and invasion, and may require the coordinated expression of various proteins. The human urokinase-type plasminogen activator (uPA) receptor (uPAR) is a central protein component of the PA system. By binding its ligand uPA, uPAR can direct proteolysis of the extracellular matrix. Also, it is now apparent that uPAR can initiate proteolytic independent signal transduction to influence angiogenesis, inflammation, wound repair and tumour progression. To determine whether any novel proteins interacted with uPAR, a yeast two-hybrid screening analysis was undertaken using alternate uPAR domain constructs as baits. These included full-length three domain uPAR (uPAR-DIDIIDIII), two domain uPAR (uPAR-DIIDIII), and each individual uPAR domain (uPAR-DI, uPAR-DII and uPAR-DIII). A number of proteins were identified as putative candidate interactors for the alternate constructs, with two of special interest for uPAR-DIDIIDIII. These were the heat shock protein Mrj, and the extracellular matrix protein fibulin-2. The protein Mrj was shown to bind uPAR both in vitro and in vivo using GST-pull down and co-immunoprecipitation assays respectively. The GST-pull down assay identified the interaction between Mrj and uPAR dependent on the C-terminal domain of Mrj and DI of uPAR. Using in vivo co-immunoprecipitation analysis, Mrj also bound to uPAR. Preliminary data suggest the association between uPAR and Mrj may play a role in the regulation of apoptosis. In regard to the uPAR interactor of fibulin-2, a calcium dependent binding interaction with uPAR was identified using the GST-pull down assay. However due to the large molecular weight and stringent conditions needed to solubilise fibulin-2, it was not possible to co-immunoprecipitate both uPAR and fibulin-2. Together, the identification of both Mrj and fibulin-2 amongst other candidate interactors of uPAR presented here provides further insight into the intricate relationship between uPAR and other proteins which may influence a range of biological functions.
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Kirkpatrick, Robert Daniel. "Interactions of the DNA repair protein Rad23 in the yeast two-hybrid system". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ45071.pdf.
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Mendez, Jamie Elizabeth. "Investigation of Hsf1 Interacting Partners via a Genome-wide Yeast Two-hybrid Screen". Scholar Commons, 2013. http://scholarcommons.usf.edu/etd/4543.
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Heat shock factor 1 (HSF1) is the master transcriptional regulator of the heat shock response (HSR), an evolutionarily conserved cellular stress response. HSF1 promotes the expression of a variety of molecular chaperones that aid in restoring protein homeostasis upon exposure to proteoxic stress. However, all of the proteins responsible for regulating the HSR together with HSF1 are unknown. A genome-wide yeast two hybrid screen was performed to identify new S. cerevisiae Hsf1 protein interacting partners. Two GAL4 DNA binding domain-Hsf1 fusion proteins (baits) were constructed with mutations in the Hsf1 C-terminal activation domain to dampen Hsf1 mediated auto-activation of the reporter gene. Each haploid bait strain was mated with a haploid prey strain containing one of ~6,000 S. cerevisiae open reading frames fused to the GAL4 activation domain (prey). Interaction between the bait and prey reconstituted the GAL4 protein enabling it to bind to a GAL4 DNA binding site and activate the HIS3 reporter gene. The identified proteins from 4 screens were pooled generating 240 putative Hsf1 interacting partners. This list was narrowed to 38 candidates by selecting the 15 strongest interactions identified based on colony size and 33 candidates conserved in C. elegans. Hsf1 interactions with the 14 candidates in which protein expression was confirmed were then re-tested by a manual yeast two-hybrid assay. Hsf1 interactions with Sti1, Rim2 and Prp46 were repeatable in this manual assay. A study of the impact of knockdown of each of their C. elegans homolog on the HSR was performed using RNAi in an hsp70-promoter::GFP reporter strain of C. elegans. Preliminary results suggest that knockdown of Sti1 may impact the HSR in the worm. Further study of Sti1 and other potential Hsf1 interacting partners identified in this screen is warranted.
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Zhang, Aijing. "Identification of TEF cofactor(s) in skeletal muscles utilizing yeast two hybrid system". Free to MU Campus, others may purchase, 2004. http://wwwlib.umi.com/cr/mo/fullcit?p1420951.
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Farzadfar, Rahin. "Identification of Artemis and PARP-1 interacting factors: A yeast two hybrid screening approach". Thesis, University of Ottawa (Canada), 2009. http://hdl.handle.net/10393/28481.
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The genomic integrity of the cell is under the constant threat of DNA damage from endogenous and exogenous sources. In response to DNA damage mammalian cells elicit a highly complex, yet coordinated series of cellular responses, which are triggered by the detection of DNA lesions and ultimately lead to cell survival (DNA repair) or apoptosis.
Artemis and PARP-1 are two proteins with significant implications in the various branches within the DNA damage response network. A series of yeast two hybrid screening (Y2H) experiments were conducted using both classical and interacting mating Y2H systems to find Artemis and PARP-1 interacting factors. The ATRX protein was identified as a putative interactor of Artemis, Caspase-7 and an "unkown" protein were identified as putative interactors of PARP-1 binding factors. In addition a rare case of false positives were identified and characterized. Furthermore, the strengths and limits of each Y2H system, as well as troubleshooting techniques developed to increase the efficiency of each system were thoroughly examined.
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Zhang, Linhua 1968. "Identification of Fyb as an interacting protein with CD45 in yeast two-hybrid screening". Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98530.
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CD45, also known as Leukocyte-common antigen (LCA), is a type-1 receptor-like protein tyrosine phosphatase (PTPase), which contains two PTPase domains, the membrane proximal domain (DI) and the membrane distal domain (D2). It is uniquely expressed on the surface of all leukocytes and their hematopoietic precursors and plays an important role in immunoreceptor signaling. In this study, I identified a novel CD45-interacting protein, Fyb, through screening human leukocyte cDNA library with a modified yeast two-hybrid system. Fyb is an adaptor protein mainly involved in the regulation of T cell receptor-induced integrin clustering and adhesion. It contains several regions with potential to mediate protein-protein interactions and has been shown to bind to the Src family kinase Fyn, the adapter protein SLP-76, Ena/VASP proteins, the adapter protein SKAP55, and SKAP55-HOM in T cell receptor signaling. I also demonstrated that the interaction of Fyb with CD45 was through involves the C-terminal region of Fyb, where several tyrosine residues, such as Tyr 594 and Tyr624, are critical for its interaction with other proteins in signaling. The interaction of Fyb with CD45 is phosphotyrosine-dependent since Src kinase is required for the interaction. Mutation analysis revealed that the tyrosine residue 624 of Fyb is the pivotal tyrosine residue involved in its association with CD45, whereas tyrosin 594 was not found to be required for binding. By mutation of CD45, it was demonstrated that the phosphatase catalytic D1 domain of CD45 was involved in the interaction with Fyb. More significantly, I found that only the substrate-trapping mutant (Asp to Val), but not the wild type CD45 interacted with Fyb, suggesting that Fyb is a potential CD45 substrate. Furthermore, I demonstrated the association of the C-terminal fragment of Fyb with CD45 in mammalian cells. However, I was not able to determine the interaction of the wild type Fyb with CD45 in 293 cells.
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Lanthrop, Jeremy R. "Identification of proteins that interact with CeABF-1 using A yeast two-hybrid system". Scholarly Commons, 2004. https://scholarlycommons.pacific.edu/uop_etds/3101.
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The helix-loop-helix (HLH) family of transcription regulatory proteins are fundamental regulators in the processes of cell proliferation and differentiation, cell lineage determination, myogenesis, neurogenesis, and sex determination in a wide range of multicellular organisms. A gene encoding a novel class II HLH protein has recently been identified from a human B-cell eDNA library using a yeast two-hybrid screen. The predicted human ABF -1 polypeptide sequence was used to search the Caenorhabditis elegans genome database for a C. elegans ABF-1 homolog. This bHLH protein, called C. elegans ABF -1 (CeABF -1 ), has a bHLH domain that shares 72% amino acid similarity with its human ABF-1 relative. The expression of the CeABF-1 mRNA has been detected in larval stages L2, L3, L4, and adult, however the mRNA is most highly expressed at the L3 and L4 stages. CeABF -1 protein is capable of heterodimerizing with the human E2A gene product, E4 7. Like human ABF -1, CeABF -1 expression in the presence of the E4 7 protein results in a reduction in E2A mediated gene activation. It has therefore been concluded that CeABF -1 , like human ABF -1 , also acts as a transcriptional
repressor. Because C. elegans shares many conserved genes with higher eukaryotic organisms it has become a model organism for in depth genetic studies. It has therefore become increasingly desirable to investigate the possibility of alternative protein-protein interactions that can potentially occur within C. elegans, so it was necessary to construct a C. elegans eDNA library along with the appropriate bait vector expressing the CeABF- 1 protein. The titer ofthe primary library was calculated to be 9.7 x I06 clones, 10-fold greater than minimum titer requirement of I x I 06 clones for a good representational library. Sequencing of the CeABF -I insert confirmed successful construction of a mutation-free bait construct suitable for use in yeast two-hybrid screening. Yeast-two hybrid analysis revealed two new interactors, one of which was identified as an aldose reductase homolog, while the other remains uncharacterized.
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Gundurao, Ramya Mavinkaihalli. "Systematic analysis of protein-protein interactions of oncogenic Human Papilloma Virus". Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/8829.
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Human papilloma virus (HPV) is a ubiquitous virus implicated in a growing list of cancers, particularly cervical cancer‐ the second most common cancer among women worldwide. Although persistent infection with high‐risk oncogenic HPVs such as types ‐16 or ‐18 is necessary, additional factors like co‐infection with other viruses can play a role in cancer progression. Protein‐protein interactions play a central role in the infection, survival and proliferation of the virus in the host. Although some interactions of HPV proteins are well characterised, it is essential to discover other key viral interactions to further improve our understanding of the virus and to use this knowledge for the development of newer biomarkers and therapeutics. The aim of this study was to systematically analyse the interactions of HPV‐16 proteins using yeast two‐hybrid (Y2H). To achieve this, a clone collection of the viral proteome was generated by recombinatorial cloning and three independent Y2H screens were performed: (i) Intra‐viral screen to identify interactions among the HPV‐16 proteins; (ii) Inter‐viral screen to identify interactions with proteins of Herpes Simplex Virus (HSV) which is suggested to be a co‐factor; and (iii) Virus‐host screen to identify novel cellular binding partners. The intra‐viral Y2H screen confirmed some of the previously known interactions and also identified binding of the E1 and E7 proteins. Deletion mutagenesis was performed to map the interaction domains to the amino‐terminal 92 amino acids of E1 and carboxy‐terminal CxxC domain of E7. Replication assays suggest a possible repression of E1‐mediated episomal replication by direct binding of E7. The inter‐viral Y2H screen identified interactions of HPV proteins with seventeen HSV‐1 proteins including transcriptional regulator ICP4 and neurovirulance factor ICP34.5. The biological relevance of these interactions in the context of co‐infection is discussed. The virus‐host screen performed against a human cDNA library identified 54 interactions, a subset of which was validated by biochemical pull‐down assays. The functional relevance of an interaction between E7 and a proto‐oncogene spermatogenic leucine zipper protein (SPZ1) was further investigated suggesting a role of SPZ1 in E7‐mediated cell proliferation. The work presented in this thesis identifies several novel interactions of HPV proteins. Future work will involve the in‐depth elucidation of biological relevance of these interactions. In particular, the interactions of E7 with E1 and SPZ1 are of great interest to improve our understanding of the life cycle and pathogenesis of the virus which can be applied for improved strategies of prevention and treatment of malignancies caused by HPV.
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Huang, Xinhua. "DIII Domain of Calpain 10 and Cpl Towards an Understanding of Calpain 10 Function". University of Toledo Health Science Campus / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=mco1096641027.
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Petzold, Herman E. III. "Discovery of New Protein-DNA and Protein-Protein Interactions Associated With Wood Development in Populus trichocarpa". Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/89363.
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The negative effects from rising carbon levels have created the need to find alternative energy sources that are more carbon neutral. One such alternative energy source is to use the biomass derived from forest trees to fulfill the need for a renewable alternative fuel. Through increased understanding and optimization of regulatory mechanisms that control wood development the potential exists to increase biomass yield. Transcription factors (TFs) are DNA-binding regulatory proteins capable of either activation or repression by binding to a specific region of DNA, normally located in the 5-prime upstream promoter region of the gene. In the first section of this work, six DNA promoters from wood formation-related genes were screened by the Yeast One-Hybrid (Y1H) assay in efforts to identify novel interacting TFs involved in wood formation. The promoters tested belong to genes involved in lignin biosynthesis, programmed cell death, and cambial zone associated TFs. The promoters were screened against a mini-library composed of TFs expressed 4-fold or higher in differentiating xylem vs phloem-cambium. The Y1H results identified PtrRAD1 with interactions involving several of the promoters screened. Further testing of PtrRAD1 by Yeast Two-Hybrid (Y2H) assay identified a protein-protein interaction (PPI) with poplar DIVARACATA RADIALIS INTERACTING FACTOR (DRIF1). PtrDRIF1 was then used in the Y2H assay and formed PPIs with MYB/SANT domain proteins, homeodomain family (HD) TFs, and cytoskeletal-related proteins. In the second section of this work, PPIs involving PtrDRIF1s' interaction partners were further characterized. PtrDRIF1 is composed of two separate domains, an N-terminal MYB/SANT domain that interacted with the MYB/SANT domain containing PtrRAD1 and PtrDIVARICATA-like proteins, and a C-terminal region containing a Domain of Unknown Function 3755 (DUF3755). The DUF3755 domain interacted with HD family members belonging to the ancient WOX clade and Class II KNOX domain TFs. In addition, PtrDRIF1 was able to form a complex between PtrRAD1 and PtrWOX13c in a Y2H bridge assay. PtrDRIF1 may function as a regulatory module linking cambial cell proliferation, lignification, and cell expansion during growth. Combined, these findings support a role for PtrDRIF1 in regulating aspects of wood formation that may contribute to altering biomass yield.Ph. D.
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Rahm, Jonas. "Biologically plausible visual representation of modular decomposition". Thesis, University of Skövde, School of Humanities and Informatics, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-953.
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Modular decompositions of protein interaction networks can be used to identify modules of cooperating proteins. The biological plausibility off these modules might be questioned though. This report describes how a modular decomposition can be completed with semantic information in the visual representation. Possible methods for creating modules of functionally related proteins are also proposed in this work. The results show that such modules, with advantage can be combined with modules from a graph decomposition, to find proteins that are likely to cooperate to perform certain functions in organisms
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Bahte, Svenja-Katharina Paula. "Identifikation neuer Bindungspartner von PKC- d [PKC-delta] mit Hilfe des Yeast-two-hybrid-Screens". [S.l.] : [s.n.], 2004. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=013081490&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Bormann, Ulrich. "Yeast-two-hybrid-Systeme zur Identifikation zytoplasmatischer Interaktionspartner der neuronalen Zelladhäsionsmoleküle MAG, P0 und NCAM". [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=96360760X.
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Scott, Adam. "Modified yeast two-hybrid screening identifies SKAP-HOM as a novel substrate of PTP-PEST". Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=21986.
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PTP-PEST is a soluble intracellular phosphatase that is ubiquitously expressed in mammalian tissues. The biological importance of this enzyme is highlighted by the fact that it is essential for murine embryogenesis and that it has been implicated in actin dynamics as well as other processes such as immunoreceptor signalling and cell death. Having five proline-rich regions in its C-terminal tail, PTP-PEST has a large potential for protein:protein interactions, several of which are known. To better understand the physiological importance of PTP-PEST, we explored the possibility that other substrates or binding partners existed. To this end, we carried out a modified yeast two-hybrid assay using full-length PTP-PEST with the D199A substrate trapping mutation as bait and screened a library of cDNA clones from 17-day-old mouse embryos. Screening identified the cytosolic adaptor protein, SKAP-HOM, as a novel substrate of PTP-PEST and GST pull-down assays confirmed this interaction in HeLa299 cells. We showed that PTP-PEST's PTP-domain and proline-1 region are essential for this interaction, as are SKAP-HOM's Y260 residue and SH3 domain. Further mutagenesis studies revealed that PTP-PEST residues S39 and S434 play a role in inhibiting and promoting the interaction with SKAP-HOM, respectively. Preliminary evidence was also obtained suggesting that p59fyn kinase plays a role in promoting the PTP-PEST D199A:SKAP-HOM interaction through a mechanism other than SKAP-HOM phosphorylation. Understanding the biological significance of this interaction could lead to novel discoveries in the fields of cell metastasis and inflammation.PTP-PEST est une phosphatase soluble intracellulaire qui est exprimée de façon ubiquitaire dans les tissus de mammifères. L'importance biologique de cette enzyme relève du fait qu'elle est essentielle durant l'embryogénèse chez la souris, et qu'elle est de plus impliquée dans l'organisation dynamique du cytosquelette d'actine, ainsi que dans d'autres processus tels la signalisation par les immunorécepteurs et la mort cellulaire. Avec ses cinq régions riches en motifs proline dans la région C-terminale, PTP-PEST possède un large potentiel d'interactions protéine : protéine, parmi lesquelles plusieurs sont déjà identifiées. Afin de mieux comprendre l'importance physiologique de PTP-PEST, nous avons exploré la possibilité de l'existence d'autres substrats ou ligands. À cette fin, nous avons développé un test modifié de levures à deux-hybrides, utilisant une construction pleine longueur de PTP-PEST insérée d'une mutation de piégeage de ligands D199A comme appât. Suite à l'analyse d'une banque d'ADNc provenant d'embryons de souris de 17 jours, nous avons identifié une protéine adaptatrice cytosolique, SKAP-HOM, en tant que nouveau substrat de PTP-PEST. L'essai de capture de la protéine GST a de plus confirmé cette interaction dans les cellules HeLa299. Nos résultats démontrent que le domaine PTP de PTP-PEST, ainsi que la région proline-1, sont essentiels à cette interaction, tout comme le résidu Y260 et le domaine SH3 de SKAP-HOM. Une analyse de mutagénèse plus approfondie a identifié les résidus S39 et S434 de PTP-PEST comme jouant respectivement un rôle dans l'inhibition et dans la promotion de l'interaction avec SKAP-HOM. Nous avons aussi obtenus des résultats préliminaires suggérant que la protéine kinase p59fyn joue aussi un rôle dans la promotion de l'interaction PTP-PEST D199A:SKAP-HOM, par un mécanisme autre que la phosphorylation de SKAP-HOM. La compréhension de l'importance biologique de cette interaction pour
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Esteves, Sara Luísa de Castro. "Characterization of human brain protein phosphatase 1a interacting proteins using the yeast two-hybrid system". Master's thesis, Universidade de Aveiro, 2008. http://hdl.handle.net/10773/917.
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Maric, Martina. "Identification of cellular factors involved in herpes simplex virus type 1 nucelar egress". Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/3346.
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The herpesvirus life cycle involves a step where newly formed capsids leave the nucleus by translocating across the intact nuclear envelope (NE). Little is known about the role of cellular factors during nuclear egress. We sought to identify novel cellular proteins that interact with the conserved herpes simplex virus-1 (HSV-1) pUL34 by performing a yeast two-hybrid screen. pUL34 was chosen due to its crucial and multifunctional role during nuclear egress. From 42 cellular factors that interacted with pUL34 in yeast, twelve were further evaluated in mammalian cells by co-localization studies using immunofluorescence. No specific co-location between the tested cellular factors and pUL34 was observed in infected cells, thus the screen failed to convincingly identify novel pUL34 interactors. In the second part of the thesis we addressed the functional significance of the cellular protein torsinA (TA) in the HSV-1 life cycle. We became interested in TA due to its role in maintaining normal NE morphology. We showed that perturbing the normal function of TA through overexpression impaired HSV-1 replication and caused a defect in capsid nuclear egress. In mouse embryonic fibroblasts that failed to express TA (TA-/-MEFs), HSV-1 replication was also inhibited, but a defect in capsid nuclear egress was not apparent. Strikingly, infection in TA-null MEFs induced a NE breakdown, the extent of which was dependent on viral products involved in nuclear egress. The viral growth defect and NE envelope breakdown, however, seem to be TA-null cell line specific rather than a functional consequence of TA loss as indicated by TA-/-MEFs reconstituted with TA and 293T with reduced TA levels. In conclusion, overexpression and loss of TA have different effects on the HSV-1 life cycle.
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Friese, Anke. "Etablierung eines Zwei-Hybrid-Screening-Systems zur Suche und Charakterisierung von Ras-Raf-Effektoren". [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=964787466.
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Poon, Wing Yee Louisa. "Identification of sensory-neuron-specific sodium channel NAVI.8 interacting proteins by yeast-two hybrid screen". Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.404822.
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Todd, Carol. "Identification of novel sarcomeric modifiers of hypertrophy in hypertrophic cardiomyopathy using the yeast two-hybrid system". Thesis, Stellenbosch : Stellenbosch University, 2013. http://hdl.handle.net/10019.1/79819.
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Thesis (MScMedSc)--Stellenbosch University, 2013.ENGLISH ABSTRACT: Left ventricular hypertrophy (LVH) occurs when the cardiomyocytes in the left ventricle become enlarged by increasing in mass in response to haemodynamic pressure overload. This can either be attributed to a normal physiological response to exercise or can be the result of a maladaptive process or disease state, such as chronic hypertension. Hypertrophic cardiomyopathy (HCM) is the most common form of Mendelian-inherited cardiac disease. A defining characteristic thereof is primary LVH that occurs when there are no other hypertrophy-predisposing conditions present. Therefore, HCM provides a unique opportunity to study the molecular determinants of LVH in the context of a Mendelian disorder, instead of in more complex disorders such as hypertension. Over 1000 HCM-causing mutations in 19 genes have been identified thus far, most of them encoding sarcomeric proteins residing in the sarcomeric C-zone. However, for many HCM patients no disease-causing genes have been identified. Moreover, studies have shown phenotypic variation in presentation of disease in, as well as between, families in which the same HCM-causing mutation segregates. This has led many investigators to conclude that genetic modifiers of hypertrophy exist. The aim of the study was to identify novel plausible HCM-causing or modifier genes by searching for interactors of a known HCM-causing protein, namely titin. The hypothesis was that genes encoding proteins, which interact with proteins that are encoded by known HCM-causative genes, may also be considered HCM-causing or may modify the HCM phenotype. To this end, the aim was to identify novel interactors of the 11-domain super-repeat region of titin, which resides within the sarcomeric C-zone, using yeast two-hybrid analysis. Five putative interactors of the 11-domain super-repeat region of titin were identified in this study. These interactions were subsequently verified by colocalisation in H9C2 rat cardiomyocytes, providing further evidence for possible interactions between titin and these proteins. The putative interactor proteins of titin determined from the Y2H library screen were: filamin C (FLNC), phosphatidylethanolamine-binding protein 4 (PEBP4), heart-type fatty acid binding protein 3 (H-FABP3), myomesin 2 (MYOM2) and myomesin 1 (MYOM1). The FLNC gene could be a candidate for cardiac diseases, especially cardiomyopathies that are associated with hypertrophy or developmental defects. The putative interaction of titin and PEBP4 is speculated to be indicative of the formation of the interstitial fibrosis and myocyte disarray seen in HCM. Heart-type fatty acid-binding protein 3 has prognostic value to predict recurrent cardiac events. Its suggested interaction with titin is speculated to play a role in inhibiting its functional abilities. Myomesin 2 is jointly responsible, with MYOM1, for the formation of a head structure on one end of the titin string that connects the Z and M bands of the sarcomere. This is speculated to be linked to a developmental error with the result being a defect in sarcomeric structure formation, which could result in pathologies such as HCM. Therefore, these identified proteins could likely play a functional role in HCM due to their interactions with titin. This research could thus help with new insights into the further understanding of HCM patho-aetiology.
AFRIKAANSE OPSOMMING: Linker ventrikulêre hipertrofie (LVH) ontstaan wanneer die kardiomyosiete in die linkerventrikel vergroot as gevolg van 'n verhoging in massa in reaksie op hemodinamiese drukoorlading. Dit kan toegeskryf word aan 'n normale fisiologiese respons op oefening of kan die gevolg wees van 'n wanaangepaste of siektetoestand, soos chroniese hipertensie. Hipertrofiese kardiomiopatie (HKM) is die mees algemene vorm van Mendeliese oorerflike hartsiekte. 'n Bepalende eienskap daarvan is primêre LVH, wat plaasvind wanneer daar geen ander hipertrofie-predisponerende voorwaardes teenwoordig is nie. Gevolglik bied HKM 'n unieke geleentheid om die molekulêre derterminante van LVH te bestudeer, in die konteks van 'n Mendeliese oorerflike siekte, in plaas van om dit in die meer komplekse siektes soos hoë bloeddruk te bestudeer. Meer as 1000 HKM-veroorsakende mutasies is tot dusver in 19 gene geïdentifiseer. Die meeste van hulle kodeer vir sarkomeriese proteïene wat in die C-sone voorkom. Egter, vir baie HKM-pasiënte is geen siekte-veroorsakende gene al geïdentifiseer nie. Daarbenewens het studies getoon dat variasie in fenotipiese aanbieding van die siekte in, sowel as tussen, families voorkom wat dieselfde HKM-veroorsakende mutasie het. Dit het daartoe gelei dat baie navorsers tot die gevolgtrekking gekom het dat genetiese wysigers van hipertrofie wel bestaan. Die doel van die studie was om nuwe moontlike HKM-veroorsakende of wysiger-gene te identifiseer deur te soek vir interaktors van 'n bekende HKM-veroorsakende proteïen, naamlik titin. Die hipotese was dat gene wat vir proteïene kodeer, wat in wisselwerking is met proteïene wat geïnkripteer word deur bekende HKM-veroorsakende gene, ook oorweeg kan word om HKM te veroorsaak. Dit kan ook die HKM fenotipe verander. Dus was die doel om nuwe interaktors van die 11-domein super-herhaalstreek van titin, soos gevind binne die sarkomeriese C-sone, te identifiseer deur middel van gis-twee-hibried-analise. Vyf vermeende interaktors van die 11-domein super-herhaalstreek van titin is in hierdie studie geïdentifiseer. Hierdie interaksies is later geverifieer met behulp van ko-lokalisering in H9C2-rotkardiomyosiete, wat verdere bewyse vir moontlike interaksies tussen titin en hierdie proteïene verskaf. Die vermeende interaktor-proteïene van titin wat bepaal is vanaf die gis-twee-hibried-biblioteeksifting was as volg: filamin C (FLNC), phosphatidylethanolamine-bindingsproteïen 4 (PEBP4), hart-tipe-vetsuur bindingsproteïen 3 (H-FABP3), myomesin 2 (MYOM2) en myomesin 1 (MYOM1). Die FLNC-geen kan 'n kandidaat vir kardiale siektes, veral kardiomiopatieë, wees wat geassosieer word met hipertrofie of ontwikkelingsafwykings. Die vermeende interaksie van titin en PEBP4 dui daarop om 'n aanduiding te wees vir die vorming van die interstisiële fibrose en miokardiale wanorde, soos gesien in HKM. Hart-tipe-vetsuur bindingsproteïen 3 het prognostiese waarde om herhalende kardiale gebeure te voorspel. Verder dui sy voorgestelde interaksie met titin moontlik daarop dat dit 'n rol kan speel in die inhibering van sy funksionele vermoëns. Myomesin 2 tesame met MYOM1 is verantwoordelik vir die vorming van 'n kopstruktuur aan die een kant van die titinstring wat dan die Z- en M-bande van die sarkomeer verbind. Daar word vermoed dat dit gekoppel is aan 'n ontwikkelingsfout, met die gevolg dat daar 'n defek is in sarkomeriese struktuurvorming, wat weer kan lei tot patologieë soos HKM.
Mrs Wendy Ackerman
Prof Paul van Helden
National Research Foundation (NRF)
Stellenbosch University
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Neubert, Jonathan Paul. "Characterizing the Role of HspB2 in Cardiac Metabolism and Muscle Structure Using Yeast and Mammalian Systems". BYU ScholarsArchive, 2012. https://scholarsarchive.byu.edu/etd/3748.
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HspB2 is a small heat shock protein encoded on human chromosome 11. Less than 1000 base pairs away from HSPB2 and situated in a head-to-head orientation lies the gene encoding another small heat shock protein, CRYAB. Because they are uncommonly close to one another they share regulatory elements. In addition, they share protein homology as sHSPs, suggesting that they perhaps perform aimilar functions. SHSPs such as HspB2 and CryAB are traditionally thought to provide protective effects to cells in response to a variety of stress inducers. In response to stress they form complexes around misfolded proteins or proteins in danger of denaturation. HspB2 has been shown to exhibit protective effects during cellular stress and to localize to the Z-line of skeletal muscle. It has also been implicated in cardiac energetics, specifically in the production of ATP, however little is known about its molecular targets. Here I report the use of yeast two-hybrid screening to uncover the molecular targets of HspB2. I also detail the process by which the screens are performed as well as the verification steps, including co-precipitation experiments in mammalian cells. Through these studies we identify many novelbinding partners of HspB2, including CryAB as well as multiple muscle and mitochondrial proteins. Proteins discovered to bind to HspB2 include such proteins as actin and myosin, enzymes catalyzing various steps of glycolysis and the electron transport chain, as well as redox-, small heat shock protein-, kinase-, and electrolyte-related proteins, among others. Studies of the binding partners of HspB2 in cardiac tissue will provide important information clarifying the involvement of HspB2 in cardiac muscle maintenance and metabolism.
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Wang, Ying. "Identification of a protein that interacts with Caenorhadbitis elegans CLK-2 in a yeast two-hybrid assay". Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=19733.
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The gene clk-2 of C. elegans is required in both the germline and the soma, for subsequent embryonic viability, and for developmental and behavioural rates, respectively, clk-2 encodes a protein that is homologous to Tel2p in yeast, which is required for the telomere length regulation. It has been demonstrated that clk-2 affects telomere length also in worms and human cells. By now the exact biochemical function of CLK-2 is unknown. In order to shed light onto the function of the gene clk-2, a two-hybrid screen was carried out to identify the interactors of the protein CLK-2. A potential interactor of CLK-2, Y105C5B.19, was identified in the screen. Y105C5B.19 is a novel gene and does not have homologues in other species. Y105C5B.19 contains an MSP (major sperm protein) domain, therefore it is possible that it could be involved in the processes that regulate oocyte maturation, gonadal sheath contractions, or sperm mobility. Interestingly, given that clk-2 is required for subsequent embryogenesis at some point during a narrow time between the end of oocyte maturation and the 2-cell stage, it is tempting to speculate that the interaction between CLK-2 and Y105C5B.19 might be functionally relevant. The lethality of clk-2(qm37) mutants might result from delayed consequences of defects in ovulation and/or fertilization, and perhaps such defects could result from the disruption of the interaction between CLK-2 and Y105C5B.19. The amino acid substitution C772Y resulting from the clk-2(qm37) mutation was found to disrupt the interaction between CLK-2 and Y105C5B.19 in a two-hybrid assay, lending support to the idea that the interaction between CLK-2 and Y105C5B.19 takes place in vivo.
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Cotsiki, Marina. "Identification of novel protein interactors of the SV40 large T antigen using the yeast two hybrid system". Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.268375.
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Wilkinson, Helen Louise. "The identification of a novel protein interacting with GABAâ†A receptors using the yeast two-hybrid system". Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326083.
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CASACA, Ana Leonor Vidal Gomes. "Uma abordagem yeast two-hybrid para o estudo da replicação e patogénese do vírus da hepatite delta". Doctoral thesis, Instituto de Higiene e Medicina Tropical, 2011. http://hdl.handle.net/10362/6054.
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O vírus da hepatite delta (HDV) é o agente etiológico de uma das formas mais graves de hepatite viral e é ainda endémico em diversas regiões do globo, nomeadamente em África, na Amazónia e no Extremo Oriente. O HDV co-infecta ou super-infecta hepatócitos infectados com o vírus da hepatite B (HBV) aumentando em cerca de 10 vezes o risco de cirrose e hepatite fulminante. A associação clínica entre os dois vírus deve-se ao facto do invólucro do HDV ser constituído pelos antigénios de superfície do HBV (HBsAgs) que são necessários para a propagação da infecção.
O genoma do HDV é constituído por uma molécula de RNA de cadeia simples, circular, com cerca de 1.7 Kb, que possui cerca de 70% de emparelhamento interno. Foi identificada uma única grelha de leitura aberta (ORF) no RNA viral que codifica para o antigénio delta (HDAg). A ocorrência de um mecanismo de editing do RNA, resulta na expressão de duas formas do HDAg, a pequena (S-HDAg) e a grande (L-HDAg).
Várias funções essenciais para a replicação do HDV têm sido atribuídas a ambas as formas do HDAg, sendo a S-HDAg essencial para a acumulação de RNA viral e a L-HDAg responsável pela interacção com os HBsAgs para formar partículas virais. No entanto, dada a simplicidade dos seus componentes, admite-se que a replicação viral depende das interacções estabelecidas entre os HDAgs e factores celulares do hospedeiro. Apesar do número considerável de factores celulares descritos como interactores dos HDAgs ou RNA virais, a importância de muitas destas interacções não foi elucidada e muitas etapas do ciclo de replicação do HDV permanecem pouco claras. Para além disso, dado o número limitado de factores do hospedeiro que estão envolvidos na sua replicação, é muito provável que um número elevado de interactores do HDV permaneça por identificar.
Este trabalho teve como objectivo a identificação de proteínas de fígado humano capazes de interagir com os HDAgs, utilizando o sistema yeast Two-Hybrid (YTH). Identificaram-se trinta proteínas com capacidade de interagir com a S-HDAg no sistema YTH, sendo que estas proteínas se encontram envolvidas em diferentes processos celulares. Com base nas características funcionais, foram seleccionadas três destas proteínas e as suas interacções com a S-HDAg foram investigadas com maior detalhe. As três proteínas seleccionadas foram a ribonucleoproteína nuclear heterogénea C (hnRNPC), a embryonic lethal abnormal vision like1 (ELAVL1/HuR) e a proteína 2 de ligação a EBNA1 (EBP2). As duas primeiras são proteínas de ligação a RNA, previamente descritas como envolvidas em processos de replicações de outros vírus com genoma RNA, enquanto a EBP2, é uma proteína de localização preferencialmente nucleolar, tal como por vezes acontece com os HDAgs.
As interacções foram analisadas recorrendo a vários ensaios bioquímicos. No caso da hnRNPC e da HuR, após validação no sistema YTH, a capacidade de interacção com a S-HDAg foi confirmada quer in vitro por blot overlay quer in vivo por co-imunoprecipitação em células de hepatoma humano. Nas mesmas células, observou-se uma co-localização considerável entre os HDAgs e os RNAs virais. Finalmente, de modo a investigar a contribuição das proteínas hnRNPC e HuR na replicação do HDV, procedeu-se ao silenciamento destas proteínas pela utilização de short hairpin RNAs (shRNAs) específicos para os mRNAs correspondentes Observou-se que o silenciamento de ambas as proteínas hnRNPC e HuR endógenas, individualmente resultou numa diminuição acentuada nos níveis de expressão dos HDAgs.
No que respeita à EBP2, a interacção com a S-HDAg foi confirmada em condições in vitro com recurso a ensaios de blot overlay e de cromatografia de afinidade. A análise por imunofluorescência indirecta e microscopia confocal revelou co-localização elevada entre os HDAgs e a EBP2, principalmente nos nucléolos de células de hepatoma humano.
Finalmente, foi ainda utilizado o sistema YTH para estudar os mecanismos de importação dos HDAgs. Assim, este sistema foi utilizado com o propósito de identificar proteínas celulares capazes de interagir com um domínio específico dos HDAgs, o sinal de localização nuclear (NLS). Na pesquisa YTH realizada obtiveram-se 161 clones positivos, sendo que um deles mostrou codificar para a carioferina α4 (KPNA4). A interacção da KPNA4 com a S-HDAg foi reproduzida em condições in vitro através de um ensaio de cromatografia de afinidade tendo sido utilizadas formas recombinantes das duas proteínas.
Este trabalho permitiu identificar várias proteínas celulares que interagem com a S-HDAg. Obtiveram-se evidências sugestivas de que algumas das proteínas identificadas podem desempenhar funções importantes no ciclo de replicação do HDV e que abrem novas perspectivas para o estudo do ciclo de replicação do vírus.Hepatitis delta virus (HDV) is the causative agent of one of the most severe forms of viral hepatitis and is still endemic in populations from Africa, Amazon basin and Far East. HDV infects liver cells already infected with Hepatitis B virus (HBV) and increases the severity and risks of fulminant disease. HDV replication occurs independently of the replication of the helper virus but needs its surface antigens (HBsAgs) to assemble in viral particles. The HDV genome consists of a 1.7 Kb single-stranded, negative-polarity circular RNA molecule which bears about 70% of internal base-pairing. The genome contains a single ORF from which two forms of the same protein, the small and the large delta antigens (S-HDAg and L-HDAg, respectively) are derived as a consequence of an editing mechanism. Several functions have been assigned to both forms of the delta antigen and it is consensual that S-HDAg is necessary for RNA accumulation, and L-HDAg interacts with HBsAgs playing an important role during virus packaging. However, due to its simplicity it is likely that HDV replication is highly dependent on the interactions between HDAgs and host cellular factors. Despite the increasing number of cellular factors described as HDV RNA or antigens’ partners, the role of most of these interactions in HDV replication were not elucidated, and many steps of the virus life cycle remain unclear. Furthermore, given the limited number of host factors found to play a role in its replication, it is highly probable that still a considerable number remains to be discovered. The main purpose of this work was to identify human liver proteins that interact with HDAgs using the yeast two-hybrid (YTH) system. Thirty known proteins involved in different cellular processes were identified as S-HDAg interactors. Given their functional properties three proteins were selected for further analysis. These proteins include heterogeneous nuclear ribonucleoprotein C (hnRNPC), embryonic lethal abnormal vision like 1 (ELAVL1/HuR) and EBNA1 binding protein 2 (EBP2). hnRNPC and HuR are RNA binding proteins that were shown to play important roles during replication of different virus. EBP2 is a preferentially nucleolar protein, as sometimes also occurs for HDAgs. The selected interactions were further explored using different biochemical approaches. Having validated hnRNPC and HuR interactions with S-HDAg in the YTH system, we were able to confirm both interactions in vitro using a blot overlay assay and in vivo using co-immunoprecipitation assays in human hepatoma cells. Furthermore, the two proteins were found to co-localize with HDAgs and HDV RNAs in liver cells. Finally, reducing hnRNPC and HuR expression in cells undergoing HDV replication, using specific short hairpin RNAs (shRNAs), it was possible to observe a marked decrease in both S-HDAg and L-HDAg protein expression. S-HDAg/EBP2 interaction was also confirmed in vitro using blot overlay and pull-down assays. Indirect immunofluorescence and confocal microscopy analysis revealed a strong co-localization of EBP2 and HDAg mainly in the nucleoli of human hepatoma cells. Finally, in order to investigate the nuclear import mechanism of HDAg we performed a YTH screening to identify cellular proteins able to interact with the nuclear localization signal (NLS) of HDAgs. 161 positive clones were obtained allowing the identification of karyopherin-α4 (KPNA4). Having established the S-HDAg/KPNA4 interaction in the YTH system, it was further confirmed in vitro using a pull down assay with bacterially expressed recombinant proteins in a pull down assay. This study allowed the identification of several cellular proteins as S-HDAg partners. We found evidences suggesting that some of these proteins may play important roles in HDV replication cycle, opening new possibilities for the study of the viral replication.
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Morgese, Fabio. "Identification of putative interactors of Fanconi anaemia proteins by yeast to hybrid system: characterization of two novel genes highly expressed during spermatogenesis". Doctoral thesis, Università degli studi di Trieste, 2009. http://hdl.handle.net/10077/3134.
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2007/2008Fanconi Anaemia (FA) is a rare human genetic disease characterized by bone marrow failure, malformations, chromosomal instability and cancer susceptibility. Thirteen genes belonging to a common pathway have been identified, but their function is still unclear even if evidence indicates a role in DNA-repair. In the attempt to gain new insights on FA-BRCA pathway, this work aimed at finding and characterizing novel putative interactors of the FA proteins. Using the yeast two-hybrid system, we screened a cDNA library of human testis and rescued two clones. Clone 54, which encoded for a putative ubiquitin-conjugating enzyme E2 (UBE2U), was first found to interact with FANCD2 and then with FANCL (E3 ubiquitin-ligase of the FA pathway), FANCC, FANCE and FANCF by direct interaction mating in yeast. Different assays indicated that the expression of this gene is limited to mouse and human testis (specifically in spermatocytes and spermatides). Interestingly, even mouse Fancd2 showed a high expression level in these two cell types, supporting the hypothesis of an interaction between the two proteins and a role of the FA-BRCA pathway during spermatogenesis. In order to confirm the binding between UBE2U and FANCD2, we transiently transfected cell lines with a tagged UBE2U. However, since we failed to detect the protein at any level, we tried to validate the interaction using mouse testis extract. Using the specific antibody we generated, we were however not able to confirm the binding, but, before excluding definitively the interaction, we should further investigate using more suitable antibodies. Clone 4, encoding for a novel putative exonuclease (ISG20L2), was instead found to interact with the C-terminus of FANCG. Though it was ubiquitously present at low levels in all the cells tested, it showed a stronger expression in mouse testis. In transiently transfected cells, ISG20L2 was detected primarily in nucleoli by immunofluorescence, but it was revealed also in the cytoplasmic fraction by western blot. Both nuclear and cytoplasmic distributions of the protein were confirmed at endogenous levels, after production of a specific antibody. Coimmunoprecipitation studies between ISG20L2 and FANCG did not confirm their interaction, but this might be in agreement with a recent report for ISG20L2 as a nucleolar exoribonuclease, not directly involved with DNArepair.
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Davidora, Albena. "Validation and characterization of putative NHE6-interacting proteins identified by yeast two-hybrid screening and tandem affinity purification :". Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=100793.
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Na+/H+ exchangers (NHE) are integral membrane proteins that catalyze the electroneutral exchange of Na+ (or K+) for H+. The nine human isoforms identified to date share the same topology of twelve membrane-spanning domains and a cytoplasmic regulatory tail, and diverge in their tissue expressions and subcellular localizations. This thesis focused on the identification and characterization of proteins interacting with the ubiquitous NHE6 isoform, which resides predominantly in recycling endosomes. Recent yeast two-hybrid (Y2H) screening of a human brain cDNA library using the regulatory tail of NHE6 as a probe resulted in the tentative identification of about 250 partial or full-length NHE6-interacting proteins. Thirty other potential NHE6-interacting partners were identified by isolating NHE6 complexes from embryonic kidney 293 cells by tandem affinity purification (TAP) and subjecting them to mass spectrometry analysis. The interaction between NHE6 and eight of these proteins (four from each Y2H and TAP analyses) was tested by co-immunoprecipitation, co-localization by immunofluorescence microscopy and in vitro GST-fusion protein pull-down assays. We show that apolipoprotein D interacts weakly with NHE6, while myosin light chain kinase and the mitochondrial ATP synthase subunit-alpha are true NHE6-binding partners. The remaining five proteins exhibited poor and/or non-reproducible association with NHE6.
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Yildirim, Figen. "Elucidation Of R Gene Mediated Yellow Rust Disease Resistance Mechanism In Wheat By Dual Bait Yeast Two-hybrid Analysis". Phd thesis, METU, 2005. http://etd.lib.metu.edu.tr/upload/12606473/index.pdf.
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Yellow rust, caused by Puccinia striiformis Westend. f. sp. tritici Eriksson is one of the most severe leaf diseases of wheat. Aim of this study is to illuminate the downstream signaling pathways upon incompetible infection of rust pathogen in wheat, thus to understand the genes involved in resistance mechanism. The strategy used is the dual bait yeast two-hybrid analysis which is the most powerful method for in vivo detection of protein-protein interactions. The bait proteins used arethe domains of Yr10 yellow rust resistance gene, Rad6 gene which is considered to have a critical role in R gene mediated signaling pathway, and WR5 gene fragment which is an unknown protein having homology to the WD40 repeat containing protein with apoptosis related activity. Screening of a yeast prey library with these baits revealed proteins having mostly apoptosis related functions (SRP72, POR1, CSE1), translation initiation control in response to stress conditions (Gcn2p, Eap1p), phosphorylation (SKY1) and dephosphorylation activities (GAC1), cell cycle control (FAR1), oxidative stress control (OXR1), protein degradation control (TOM1), protein folding control (CPR7) and ion homeostasis in the cell (POR1, GAC1). The significance of the study can be summarized as i) being the first yeast two hybrid analysis of a wheat R gene, ii) being able to detect interacting partners with anticipated functions, iii) most importantly, initiating further detailed analysis of the key interactors.
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Chia-Wei, Ho. "The yeast two-hybrid screen for Ndt80-interacting proteins". 2006. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0001-3006200616160100.
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Ho, Chia-Wei y 何家瑋. "The yeast two-hybrid screen for Ndt80-interacting proteins". Thesis, 2006. http://ndltd.ncl.edu.tw/handle/60397916041998730639.
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碩士國立臺灣大學
分子與細胞生物學研究所
94
In budding yeast Saccharomyces cerevisiae, cells defective in meiotic recombination and chromosome synapsis undergo checkpoint-mediated arrest at the pachytene stage. Previous study suggests that this checkpoint-mediated arrest is through Ndt80. Ndt80 is a meiosis-specific transcription factor that activates the expression of middle and late sporulation genes, including genes required for nuclear division and spore formation. Ndt80 is regulated by pachytene checkpoint at transcriptional and post-translational level. However, the detail mechanism is not clear. In our previous study, we found a dominant allele of Ndt80, Ndt80-bc. The deletion region of Ndt80-bc is in 346~402 a.a. We hypothesized that this region is the binding target of pachytene checkpoint to Ndt80. We proposed that the pachytene checkpoint regulates the activity of Ndt80 through protein-protein interaction. In this study, we perform yeast two-hybrid screen to identify potential Ndt80-interacting proteins. We find that Akr2 has the potential to interact with Ndt80. Moreover, overproduction of Ndt80 and Akr2 in zip1 mutant decreases the suppression effect of Ndt80 overproduction in zip1 mutant. We suppose that Akr2 may negatively regulate Ndt80 activity. However, zip1akr2 mutant is still arrest at pachytene stage, suggesting that Akr2 may not be the most critical factor that negatively regulates Ndt80 activity. Furthermore, Akr2 also interacts with Ndt80-bc, suggesting that the different activity of Ndt80 and Ndt80-bc is not caused by Akr2, and there should be other reason that leads to the difference. We suggest that there would be other factors involved in the control mechanism.