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Estudos das interações da septina 4 humana; Study of Human Septin 4 interactions

Silva, Nayara Cavalcante
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 09/09/2009 PT
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Septinas são proteínas ligantes a GTP encontradas desde fungos até metazoários. A primeira função identificada para septinas foi o seu papel central na organização e dinâmica do septo de divisão de leveduras. Uma das características marcantes é que septinas se organizam em heterofilamentos de 7 a 9 nm de espessura que foram purificados de diversos organismos tais como Saccharomyces cerevisiae, Drosophila e cérebro de camundongos. Hoje se sabe que septinas não estão envolvidas apenas nos processos de divisão celular, mas em uma variedade de processos como tráfico de vesículas, exocitose, interação com proteínas do citoesqueleto e com a membrana plasmática, o que resulta em alterações da morfologia celular. Neste trabalho foram desenvolvidos estudos da septina 4 humana (SEPT4) nos quais foi realizado a expressão e purificação da SEPT4 pelo uso do sistema de expressão heteróloga em E. coli e em células de insetos (Sf-9) via baculovírus. A tentativa de expressão usando o vetor pETTEV em E.coli não obteve sucesso, pois a proteína não foi expressa na forma solúvel. A construção do baculovírus recombinante AcSept4 e expressão da SEPT4 nas células de insetos foi realizada com êxito, mas o processo de purificação não foi satisfatório. Com o intuito de obter informações sobre possíveis proteínas que interagem com a SEPT4 e conseqüentemente sobre as funções desempenhadas por ela na célula...

Análise de interações da subunidade catalítica da fosfatase do tipo 1 (PP1c) de Dictyostelium discoideum identificadas através do sistema de duplo-híbrido em leveduras; Analysis of yeast two-hybrid system interactions of Dictyostelium discoideum type-1 protein phosphatase catalytic subunit (PP1c)

Raposo, Renato Astolfi
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 04/11/2010 PT
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65.96%
A proteína fosfatase do tipo-1 (PP1) é uma das principais proteínas serina/treonina fosfatases (PSTPs) e desempenha papeis fisiológicos tão diversos quanto importantes, tais como a regulação do metabolismo de carboidratos e do ciclo celular. A holoenzima PP1 é constituída por uma subunidade catalítica conservada (PP1c) que está associada a subunidades não-catalíticas que modulam sua localização subcelular, especificidade de substrato e atividade enzimática. Mais de 100 proteínas que interagem com a PP1c já foram identificadas em distintos organismos eucarióticos. Proteínas que interagem com a PP1c são, portanto, a chave para compreender os diferentes papéis biológicos da PP1. A subunidade catalítica da PP1 da ameba social Dictyostelium discoideum (DdPP1c) é codificada por um gene em cópia única o qual é expresso ao longo de todo o ciclo de vida desse organismo. Algumas proteínas que interagem e possivelmente modulam a atividade da PP1 de D. discoideum já foram identificadas, utilizando-se tanto buscas por similaridades na sequência genômica deste microorganismo como ensaios utilizando o sistema de duplo-híbrido em leveduras, utilizando-se a PP1c como isca. Com esta última abordagem, foram selecionados mais de 25 clones distintos de cDNA que codificam proteínas que potencialmente interagem com a DdPP1c...

Estudo funcional e estrutural de Nip7p, uma proteina conservada envolvida na sintese de ribossomos; Functional and structural analysis of Nip7p, a conserved protein involved in ribosome biogenesis

Patricia Pereira Coltri
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 10/12/2007 PT
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66.03%
A síntese de ribossomos é um processo conservado em eucariotos e se inicia com a transcrição dos rRNAs no nucléolo. Mais de 170 fatores atuam de forma transitória no processamento dos precursores para gerar os rRNAs maduros que formarão as subunidades ribossomais no citoplasma. Entre as proteínas envolvidas na síntese de ribossomos está a Nip7p, uma proteína nucleolar de 21 kDa associada ao complexo pré-60S em Saccharomyces cerevisiae. Nip7p é conservada e possui ortólogas em eucariotos e em Archaea. A análise da seqüência primária revela a presença de um domínio conservado na região C-terminal, denominado PUA, encontrado em diversas proteínas associadas a modificações no RNA. Neste trabalho, foram realizadas análises estruturais e funcionais com o objetivo de investigar a função molecular da proteína Nip7 no processamento e modificação do rRNA. A estrutura tri-dimensional de PaNip7, ortóloga de Nip7p em Pyrococcus abyssi foi resolvida por difração de raios-X até 1,8Å de resolução, utilizando o método SIRAS. Comparação estrutural seguida por ensaios in vitro confirmaram o envolvimento do domínio PUA na interação com RNA. Além disso, tanto Nip7p como suas ortólogas PaNip7 e HsNip7 interagem com seqüências ricas em uridina...

Analysis of the interaction between human kidney anion exchanger 1 and kanadaptin using yeast two-hybrid systems

Wongthida,Phonphimon; Akkarapatumwong,Varaporn; Limjindaporn,Thawornchai; Kittanakom,Saranya; Keskanokwong,Thitima; Eurwilaichitr,Lily; Yenchitsomanus,Pa-thai
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2006 EN
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96.05%
Kidney anion exchanger adaptor protein (Kanadaptin) is a protein which interacts with the cytoplasmic N-terminal domain of kidney anion exchanger 1 (kAE1) and was first detected in mice using the yeast two-hybrid system and was also found to co-localize with kAE1 in rabbit a-intercalated cells. Impaired trafficking of human kAE1 can result in the kidney disease-distal renal tubular acidosis (dRTA), and defective interaction between human kAE1 and kanadaptin may cause this trafficking impairment and be the basis for dRTA pathogenesis. However, it is unknown whether kAE1 can really interact with kanadaptin in humans. We have thus investigated the interaction between human kAE1 and human kanadaptin by using both Gal4 and LexA yeast two-hybrid systems. It was found that co-expression of Gal4DBD fused to the cytoplasmic N-terminal domain of kAE1 and Gal4AD fused to kanadaptin could not activate the transcription of the ADE2, HIS3 and lacZ reporters in the Gal4 system. A similar result was obtained for the interaction between B42AD fused to the cytoplasmic N-terminal domain of kAE1 and LexA fused to kanadaptin in activation of lacZ transcription in the LexA system. The absence of interaction between the fusion proteins in both yeast two-hybrid systems raises the possibility that kAE1 may not interact with kanadaptin in human cells. Considerably different structures of both kAE1 and kanadaptin in mice and humans may lead to different binding properties of the proteins in these two species.

Neurospora crassa mat A-2 and mat A-3 proteins weakly interact in the yeast two-hybrid system and affect yeast growth

Silva,Carla C. da; Cruz,Rosana C.; Bucciarelli-Rodriguez,Mônica; Vilas-Boas,Adlane
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2009 EN
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95.99%
Mating-type genes control the entry into the sexual cycle, mating identity and sexual development in fungi. The mat A-2 and mat A-3 genes, present in the mat A idiomorph of the filamentous fungus Neurospora crassa, are required for post-fertilization functions but are not essential for mating identity. Their putative roles as transcription factors are based on the similarity of mat A-2 with the Podospora anserina SMR1 gene and an HMG motif present in the mat A-3 gene. In this work the yeast two-hybrid system was used to identify transcriptional activity and protein-protein interaction of N. crassa mat A-2 and mat A-3 genes. We observed that the mat A-3 protein alone is capable of weakly activating transcription of yeast reporter genes; it also binds with low specificity to the GAL1 promoter sequence, possibly due to its HMG domain. Our results also indicate that mat A-3 is capable to form homodimers, and interact with mat A-2. Interference on yeast growth was observed on some transformants suggesting a toxic action of the mat A-2 protein. Our data on pattern of interactions of mat proteins contributes towards understanding the control of vegetative and sexual cycles in filamentous fungi.

Interaction of Proteus mirabilis Urease Apoenzyme and Accessory Proteins Identified with Yeast Two-Hybrid Technology

Heimer, Susan R.; Mobley, Harry L. T.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/2001 EN
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Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)3. To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay...

Genetic Analysis of nif Regulatory Genes by Utilizing the Yeast Two-Hybrid System Detected Formation of a NifL-NifA Complex That Is Implicated in Regulated Expression of nif Genes

Lei, Shi; Pulakat, Lakshmidevi; Gavini, Narasaiah
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/1999 EN
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66.07%
In diazotrophic organisms, nitrogenase synthesis and activity are tightly regulated. Two genes, nifL and nifA, are implicated as playing a major role in this regulation. NifA is a transcriptional activator, and its activity is inhibited by NifL in response to availability of excess fixed nitrogen and high O2 tension. It was postulated that NifL binds to NifA to inhibit NifA-mediated transcriptional activation of nif genes. Mutational analysis combined with transcriptional activation studies clearly is in agreement with the proposal that NifL interacts with NifA. However, several attempts to identify NifA-NifL interactions by using methods such as coimmunoprecipitations and chemical cross-linking experiments failed to detect direct interactions between these proteins. Here we have taken a genetic approach, the use of a yeast two-hybrid protein-protein interaction assay system, to investigate NifL interaction with NifA. A DNA fragment corresponding to the kinase-like domain of nifL was PCR amplified and was used to generate translation fusions with the DNA binding domain and the DNA activation domain of the yeast transcriptional activator GAL4 in yeast two-hybrid vectors. Similarly, a DNA fragment corresponding to the catalytic domain of nifA was PCR amplified and used to generate translation fusions with the DNA-binding domain and the DNA-activation domain of GAL4 in yeast two-hybrid vectors. After introducing appropriate plasmid combinations in yeast cells...

Protein-peptide interactions analyzed with the yeast two-hybrid system.

Yang, M; Wu, Z; Fields, S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 11/04/1995 EN
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The yeast two-hybrid system was used to screen a library of random peptides fused to a transcriptional activation domain in order to identify peptides capable of binding to the retinoblastoma protein (Rb). Seven peptides were identified, all of which contain the Leu-X-Cys-X-Glu motif found in Rb-binding proteins, although their activity in the yeast assay varied over a 40-fold range. Mutagenesis of the DNA encoding two of these peptides followed by screening in the two-hybrid system allowed the delineation of residues apart from the invariant Leu, Cys and Glu that affect binding to Rb. Binding affinities of a peptide and one of its variants to Rb, determined by surface plasmon resonance, correlated with results from the two-hybrid assay. This method offers several advantageous features compared to existing technology for screening peptide libraries: in vivo detection of protein-peptide interactions, high sensitivity, the capacity for rapid genetic screening to identify stronger and weaker binding peptide variants, and the use of a simple assay (transcriptional activity) as a means to assess binding affinity.

A Strategy for Constructing Large Protein Interaction Maps Using the Yeast Two-Hybrid System:Regulated Expression Arrays and Two-Phase Mating

Zhong, Jinhui; Zhang, Huamei; Stanyon, Clement A.; Tromp, Gerard; Finley, Russell L.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /12/2003 EN
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66.03%
Maps representing the binary interactions among proteins have become valuable tools for understanding how proteins work together to mediate biological processes. One of the most effective methods for detecting biologically important protein interactions has been the yeast two-hybrid system. Here we present an efficient two-hybrid strategy to facilitate construction of protein interaction maps on a genome-wide scale. The strategy begins with two arrays of yeast expressing known proteins fused to either a DNA binding domain (BD), or a transcription activation domain (AD). The fusion proteins are conditionally expressed using regulated promoters that can be repressed during construction and amplification of the yeast arrays. Interaction assays are conducted in two phases. In the first phase, small pools of AD strains are mated with the array of BD strains. In the second phase, individual BD strains are mated with appropriate subsets of the AD array corresponding to positive pools in the first phase. This strategy has several advantages over previously described approaches, including the ability to detect interactions with proteins that inhibit yeast growth or that activate transcription as BD fusions. Moreover, by minimizing the number of mating operations and sequencing reactions needed to test large sets of binary interactions...

Yeast Two-Hybrid Systems and Protein Interaction Mapping Projects for Yeast and Worm

Walhout, Albertha J. M.; Boulton, Simon J.; Vidal, Marc
Fonte: Hindawi Publishing Corporation Publicador: Hindawi Publishing Corporation
Tipo: Artigo de Revista Científica
Publicado em //2000 EN
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The availability of complete genome sequences necessitates the development of standardized functional assays to analyse the tens of thousands of predicted gene products in high-throughput experimental settings. Such approaches are collectively referred to as ‘functional genomics’. One approach to investigate the properties of a proteome of interest is by systematic analysis of protein–protein interactions. So far, the yeast two-hybrid system is the most commonly used method for large-scale, high-throughput identification of potential protein–protein interactions. Here, we discuss several technical features of variants of the two-hybrid systems in light of data recently obtained from different protein interaction mapping projects for the budding yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans.

A densely overlapping gene fragmentation approach improves yeast two-hybrid screens for Plasmodium falciparum proteins

Brown, Hakeenah F.; Wang, Ling; Khadka, Sudip; Fields, Stanley; LaCount, Douglas J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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66.06%
Use of the yeast two-hybrid assay to study Plasmodium falciparum protein-protein interactions is limited by poor expression of P. falciparum genes in yeast and lack of easily implemented assays to confirm the results. We report here two methods to create gene fragments – random fragmentation by partial DNAse I digestion and generation of densely overlapping fragments by PCR – that enable most portions of P. falciparum genes to be expressed and screened in the yeast two-hybrid assay. The PCR-based method is less technically challenging and facilitates fine-scale mapping of protein interaction domains. Both approaches revealed a putative interaction between PfMyb2 (PF10_0327) and PFC0365w. We developed new plasmids to express the proteins in wheat germ extracts and confirmed the interaction in both the split-luciferase assay and in co-purification experiments with glutathione-S-transferase and HA-tagged proteins. The combination of improved yeast two-hybrid screening approaches and convenient systems to validate interactions enhances the utility of yeast two-hybrid assays for P. falciparum.

High throughput flow cytometry based yeast two-hybrid array approach for large-scale analysis of protein-protein interactions

Chen, Jun; Carter, Mark B.; Edwards, Bruce S.; Cai, Hong; Sklar, Larry A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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66.08%
The analysis of protein-protein-interactions is a key focus of proteomics efforts. The yeast two-hybrid system has been the most commonly used method in genome-wide searches for protein interaction partners. However, the throughput of the current yeast two-hybrid array approach is hampered by the involvement of the time-consuming LacZ assay and/or the incompatibility of liquid handling automation due to the requirement for selection of colonies/diploids on agar plates. To facilitate large-scale yeast two-hybrid assays, we report a novel array approach by coupling a GFP reporter based yeast two-hybrid system with high throughput flow cytometry that enables the processing of a 96 well plate in as little as 3 minutes. In this approach, the yEGFP reporter has been established in both AH109 (MATa) and Y187 (MATα) reporter cells. It not only allows the generation of two copies of GFP reporter genes in diploid cells, but also allows the convenient determination of self-activators generated from both bait and prey constructs by flow cytometry. We demonstrate a Y2H array assay procedure that is carried out completely in liquid media in 96-well plates by mating bait and prey cells in liquid YPD media, selecting the diploids containing positive interaction pairs in selective media and analyzing the GFP reporter directly by flow cytometry. We have evaluated this flow cytometry based array procedure by showing that the interaction of the positive control pair P53/T is able to be reproducibly detected at 72 hrs post-mating compared to the negative control pairs. We conclude that our flow cytometry based yeast two-hybrid approach is robust...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Tian, Qing-Yun; Zhao, Yun-Peng; Liu, Chuan-ju
Fonte: MyJove Corporation Publicador: MyJove Corporation
Tipo: Artigo de Revista Científica
Publicado em 17/01/2012 EN
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65.96%
Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

Li, Hao; Dou, Wei; Padikkala, Emil; Mani, Sridhar
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/11/2013 EN
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66.04%
As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g. hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole’s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or “gain-of-function” second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug...

Screening of hepatocyte proteins binding with C-terminally truncated surface antigen middle protein of hepatitis B virus (MHBst167) by a yeast two-hybrid system

LI, ZHI QUN; LINGHU, ENQIANG; JUN, WAN; CHENG, JUN
Fonte: D.A. Spandidos Publicador: D.A. Spandidos
Tipo: Artigo de Revista Científica
EN
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66.03%
The function of middle hepatitis B surface protein C-terminally truncated at amino acid position 167 (MHBst167) is not currently clear. This study aimed to screen and identify the proteins that interact with MHBst167 in hepatocytes using a yeast two-hybrid system, and to explore the effects of MHBst167 in the development of hepatocellular carcinoma and precancerous diseases of the liver. The MHBst167 gene was amplified by polymerase chain reaction (PCR) and cloned into a pGEM-T vector. The target region was sequenced and the constructed bait plasmid, pGBKT7-MHBst167, was transformed into AH109 yeast cells. The transformed AH109 cells were then mated with Y187 yeast cells containing the fetal liver cDNA library plasmid using a yeast two-hybrid system. The false positives were eliminated and the true positive clones were selected by PCR and sequencing analysis. The pGBKT7-MHBst167 bait plasmid was successfully constructed and 66 clones grew in the selective synthetic defined media lacking leucine, tryptophan, histidine and adenine. Fifty-two clones were identified following X-α-Gal selection and segregation analysis. Seven proteins were found to be expressed that could interact with MHBst167 in hepatocytes by the yeast two-hybrid system. These results have provided novel insights into the biological functions of MHBst167.

Selection of Intracellular Single-Domain Antibodies Targeting the HIV-1 Vpr Protein by Cytoplasmic Yeast Two-Hybrid System

Matz, Julie; Hérate, Cécile; Bouchet, Jérôme; Dusetti, Nelson; Gayet, Odile; Baty, Daniel; Benichou, Serge; Chames, Patrick
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 01/12/2014 EN
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The targeting of HIV-1 using antibodies is of high interest as molecular tools to better understand the biology of the virus or as a first step toward the design of new inhibitors targeting critical viral intracellular proteins. Small and highly stable llama-derived single-domain antibodies can often be functionally expressed as intracellular antibodies in the cytoplasm of eukaryotic cells. Using a selection method based on the Sos Recruitment System, a cytoplasmic yeast two-hybrid approach, we have isolated single-domain antibodies able to bind HIV-1 Vpr and Capside proteins in the yeast cytoplasm. One anti-Vpr single domain antibody was able to bind the HIV-1 regulatory Vpr protein in the cytoplasm of eukaryotic cells, leading to its delocalization from the nucleus to the cytoplasm. To our knowledge, this is the first description of a functional single-domain intrabody targeting HIV-1 Vpr, isolated using an in vivo cytoplasmic selection method that alleviates some limitations of the conventional yeast two-hybrid system.

Systematic identification of factors involved in post-transcriptional processes in wheat grain

Lopato, S.; Borisjuk, L.; Milligan, A.; Shirley, N.; Bazanova, N.; Langridge, P.
Fonte: Kluwer Academic Publ Publicador: Kluwer Academic Publ
Tipo: Artigo de Revista Científica
Publicado em //2006 EN
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Post-transcriptional processing of primary transcripts can significantly affect both the quantity and the structure of mature mRNAs and the corresponding protein products. It is an important mechanism of gene regulation in animals, yeast and plants. Here we have investigated the interactive networks of pre-mRNA processing factors in the developing grain of wheat (Triticum aestivum), one of the world’s major food staples. As a first step we isolated a homologue of the plant specific AtRSZ33 splicing factor, which has been shown to be involved in the early stages of embryo development in Arabidopsis. Real-time PCR showed that the wheat gene, designated TaRSZ38, is expressed mainly in young, developing organs (flowers, root, stem), and expression peaks in immature grain. In situ hybridization and immunodetection revealed preferential abundance of TaRSZ38 in mitotically active tissues of the major storage organ of the grain, the endosperm. The protein encoded by TaRSZ38 was subsequently used as a starting bait in a two-hybrid screen to identify additional factors in grain that are involved in pre-mRNA processing. Most of the identified proteins showed high homology to known splicing factors and splicing related proteins, supporting a role for TaRSZ38 in spliceosome formation and 5′ site selection. Several clones were selected as baits in further yeast two-hybrid screens. In total...

Identifizierung und Charakterisierung von neuen Genen für die Entwicklung des cerebralen Cortex; Identification and characterization of new genes for the development of the cerebral cortex

Glaubitz, Joachim
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
DE_DE
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In dieser Arbeit wurde eine Microarray-Expressionsanalyse mit dem Ziel durchgeführt, neue Gene mit regionalisierter Expression im cerebralen Cortex der Maus zu finden. Die auf diese Weise gefundenen Gene wurden annotiert und mittels in-situ-Hybridisierung verifiziert. Neben bekannten Genen wurden 26 neue ESTs gefunden, von denen 5 in dieser Arbeit ausführlicher dargestellt wurden. Diese konnten als neue Markergene für die Cortexentwicklung identifiziert werden. In dem Wettstreit der beiden Theorien um die Cortexspezifizierung, dem Protocortex-Modell auf der einen Seite, welches die "Fernprogrammierung" des Cortex durch einwachsende thalamische Afferenzen annimmt, und dem Protomap-Modell auf der anderen Seite, welches intrinsische Faktoren vorzieht, konnten in dieser Arbeit weitere 2 Gene gefunden werden, die bereits eine intrinsische Identifizierung zulassen. Die Gene ERM (Etv5) und TRIM46 zeigten Expression in ihren speziellen corticalen Arealen bereits vor dem Eintreffen thalamo-corticaler Projektionen zum Stadium E14 und behielten diese bis zum Abschluss der embryonalen Cortexentwicklung bei bzw. weiteten diese darüber hinaus aus. Das Gen TRIM46 wurde für eine detailliertere Analyse ausgewählt, weil es einer bekannten Proteinfamilie angehört...

Identification of Therapeutic and Diagnotic Targets Through Yeast Two Hybrid System: Molecular Biology in Medicine; Identificação de Alvos Terapêuticos e de Diagnóstico Através do Yeast Two Hybrid System: A Biologia Molecular na Medicina

Freitas, Maria João; Laboratório de Transdução de Sinais. Centro de Biologia Celular. Departamento de Biologia. Universidade de Aveiro. Aveiro. Portugal.; Korrodi-Gregório, Luís; Laboratório de Transdução de Sinais. Centro de Biologia Celular. De
Fonte: Ordem dos Médicos Publicador: Ordem dos Médicos
Tipo: info:eu-repo/semantics/article; review; info:eu-repo/semantics/publishedVersion Formato: application/x-pdf
Publicado em 28/01/2013 POR
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In the last decades, molecular biology development was driven medicine, mainly in identification of novel therapeutic and diagnosticstargets. In cells, proteins are the main responsible for the functioning of all cellular processes, from DNA synthesis to RNA and proteinproduction, transport of cellular components and structural composition of the cell. Proteins are also an important component of signalingpathways between cells. Studies show that proteins normally do not function as singular units but as protein complexes. Understandprotein interactions and discover compounds that interfere with such protein complexes are important to develop new pharmacologictreatments. There are already some drugs with such characteristics. Trichostatin A, a histone diacetilase, acts in Phosphatase protein1 – Histone diacetilase complex, being a good target for anti-cancer therapy.In 1989, in a revolutionary way, Fields and Songs developed the Yeast Two Hybrid system (YTH). This method is based in the geneticproperties of Saccharomyces cerevisiae and allows the detection of protein interactions in vivo. Since its development it suffered a fewmodifications that allowed its application in translational medicine. For example, this technique allows a high throughput screening toassess if a drug can interfere with a protein interaction. In the other hand...

Analysis of the interaction of viral RNA replication proteins by using the yeast two-hybrid assay.

O'Reilly, E K; Paul, J D; Kao, C C
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1997 EN
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The yeast two-hybrid system has been a useful tool in the genetic evaluation of protein-protein interactions. However, the biological relevance of these two-hybrid interactions to viral positive-strand RNA replication has not been demonstrated. The brome mosaic virus (BMV) system has been characterized extensively both genetically and biochemically, providing numerous mutations in the BMV 1a helicase-like and 2a polymerase-like proteins. We have tested wild-type 1a and 18 insertion mutations of 1a and found a perfect correlation between the in planta phenotypes and their ability to interact with 2a in the two-hybrid system. This finding allowed further characterization of the interaction between and among the BMV viral proteins. Using the two-hybrid assay, we have found that the interaction between the helicase-like region of 1a and the N terminus of 2a is stabilized by the presence of the centrally conserved polymerase-like domain of 2a. We have also identified a novel interaction between the 1a helicase-like protein and itself. Additionally, we have found this interaction in two related tripartite RNA viruses, cowpea chlorotic mottle virus and cucumber mosaic virus. We have demonstrated that this protein-protein interaction is specific to homologous pairings of the protein.