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1
Fauconnier, Aurélien. "Étude des modalités de transposition des séquences d'insertion bactériennes des familles IS91-ISCR." Electronic Thesis or Diss., Limoges, 2023. http://www.theses.fr/2023LIMO0101.
Анотація:
Les éléments de la famille IS91/ISCR constituent une famille hautement associée à des gènes de virulence et de résistance aux antibiotiques. Cette famille d'IS atypique code une transposase HUH médiant les évènements de transposition en utilisant un mécanisme de transposition en cercle roulant. Contrairement aux autres membres de la famille, IS91 contient un petit ORF codant potentiellement pour un polypeptide de 121 acides aminés, Orf121, en amont du gène de la transposase, tnpA. La première partie de ce travail a consisté à une étude in silico de deux éléments de la famille IS91, IS91 et IS1294b présentent des séquences conservées au niveau de la transposase et des régions terIS et oriIS. La deuxième partie du travail a été expérimentale concernant la régulation et l’efficacité de la transposition. Nous avons montré que i) la transcription du gène tnpA provient principalement du promoteur Porf121 et que la transcription des deux gènes est couplée, ii) l'expression de la protéine Orf121 en cis ou en trans a un effet inhibiteur sur la transposition in vivo de IS91, iii) Orf121 est nécessaire à la reconnaissance et au clivage précis de l'extrémité terIS91 pour limiter la mobilisation de l’ADN adjacent. En ce qui concerne la transposition de IS91, nous avons montré que seuls les intermédiaires circulaires ADN simple brin peuvent être insérés dans une nouvelle séquence cible et avons identifié deux doigts de zinc essentiels pour l’activité de la transposition, appelés ZF1 impliquant les cystéines 41, 68, 73, 76 et ZF2 impliquant les cystéines, 53, 58, 360 et 363. Enfin, nous avons démontré que les transposases des éléments de la famille IS91 (IS91, ISKnp22) et de la famille ISCR (ISCR1) sont capables de se mobiliser et de reconnaitre et cliver l’extrémité oriIS de IS91, ISCR1 et ISCR2 et l’extrémité terIS de IS91 et ISCR2The IS91/ISCR family is highly associated with virulence and antibiotic resistance genes. This atypical IS family encodes a HUH transposase mediating transposition events using a rolling circle transposition mechanism. Unlike the other members of the family, IS91 contains a small ORF potentially encoding a 121 amino acid polypeptide, Orf121, upstream of the transposase gene, tnpA. The first part of this work consisted of an in silico study of two members of the IS91 family, IS91 and IS1294b, that have conserved sequences of the transposase and terIS and oriIS regions. The second part of this work was experimental and focused on the transposition efficiency and regulation. We have shown that i) transcription of the tnpA gene originates mainly from the Porf121 promoter and that transcription of the two genes is coupled, ii) expression of the Orf121 protein in cis or in trans has an inhibitory effect on in vivo transposition of IS91, iii) Orf121 is required for recognition and precise cleavage of the terIS91 end to limit mobilization of the adjacent DNA. With regard to IS91 transposition, we showed that only single-stranded circular DNA intermediates can be inserted into a new target sequence and identified two zinc fingers essential for the transposase activity, called ZF1 involving cysteines 41, 68, 73 and 76 and ZF2 involving cysteines 53, 58, 360 and 363. Finally, we demonstrated that transposases from the IS91 family (IS91, ISKnp22) and the ISCR family (ISCR1) are able to mobilize and recognize and cleave the oriIS end of IS91, ISCR1 and ISCR2 and the terIS end of IS91 and ISCR2
2
Vieira, Débora Fernanda. "Produção e caracterização de enzimas de Streptomyces clavuligerus relacionadas com a síntese do ácido clavulânico." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-20122012-154241/.
Анотація:
Ácido clavulânico (AC) é um potente inibidor de β-lactamases, produzido por Streptomyces clavuligerus, usado clinicamente em combinação com antibióticos β-lactâmicos para tratar infecções bacterianas resistentes. Apesar da produção industrial de AC já ser bem estabelecida muitos aspectos importantes relacionados com sua biossíntese permanecem carentes de estudo. Sabe-se que a via de síntese do AC envolve no mínimo 8 passos enzimáticos sendo os primeiros passos mais abordados. Por exemplo, as enzimas N2-(2-carboxietil) arginina sintase (CEAS), β-lactama sintase (BLS) e proclavaminato amidino hidrolase (PAH) são as responsáveis pela primeira, segunda e quarta reações enzimáticas respectivamente. Estudos mutagênicos recentes em S.clavuligerus relacionaram cópias extras dos genes ceas, bls e pah (ceas1, bls1 e pah1) com essa via porém nenhum ensaio enzimático foi relatado. Embora os passos finais da via ainda não estejam completamente estabelecidos, a ação de algumas enzimas putativas, como a codificada por orf12, mostraram ser essenciais a produção do AC. Assim, com o objetivo de aumentar a informação disponível sobre a biossíntese do AC estudamos quatro de seus membros: CEAS1, BLS1, PAH1 e a proteína putativa codificada pela orf12. Os genes foram isolados a partir do DNA genômico de S. clavuligerus por PCR e clonados em vetores para produção das proteínas recombinantes em E.coli. Os protocolos de expressão foram estabelecidos para CEAS1, PAH1 e ORF12 e as proteínas recombinantes foram purificadas por cromatografia de afinidade por metal. BLS foi obtida de forma isolúvel. As proteínas solúveis foram caracterizadas por meio de técnicas bioquímicas e estruturais. As análises de CEAS1 e PAH1 foram comparadas com informações já obtidas para as isozimas CEAS2 e PAH2, respectivamente. Assim, as análises de oligomerização das proteínas resultaram em uma mistura de oligômeros (monômero, dímero e tetrâmero) para CEAS1, na forma hexamérica para PAH1 e na forma dimérica para ORF12, estando de acordo com as formas solúvel e cristalográfica de CEAS2 (dímero e tetrâmero) e PAH2 (hexâmero). Espectros de dicroísmo circular mostraram que CEAS1 e PAH1 possuem um enovelamento do tipo α/β sendo estáveis até 35ºC e numa ampla faixa de pH. Os parâmetros termodinâmicos da interação entre CEAS1 e o cofator Mg+2 foram determinados mostrando que é entropicamente dirigida, com uma estequiometria de ligação de 4 : 1, com uma constante de afinidade na ordem de micromolar (KD = 1,76 ± 0.23 µM). Análises realizadas com as técnicas de reação acoplada, de Cromatografia Líquida de Alta Pressão acoplada a Espectrometria de Massas (LC-MS) e de Calorimetria de Titulação Isotérmica mostraram que CEAS1, assim como CEAS2, apresenta atividade sob o substrato gliceraldeído-3-fosfato, porém sem a formação do produto final N2-(2-carboxietil)arginina. Por outro lado, a proteína recombinante PAH1 mostrou ser inativa sobre o substrato análogo, N-α-acetil-L-arginina. Assim, apesar das isozimas manterem um padrão estrutural, podem ter mecanismos de ação distintos. Em relação a ORF12 esta proteína foi classificada com uma β-lactamase com atividade esterase de acordo com nossos estudos realizados com os substratos cefalosporina C e p-nitrofenil acetato.Clavulanic acid (CA) is a potent inhibitor of β-lactamases, produced by Streptomyces clavuligerus, clinically used in combination with β-lactam antibiotics to treat resistant bacterial infections. Although CA industrial production is well-established, many important aspects related to its biosynthesis remains under study. It is known that CA pathway involves at least 8 enzymatic steps, being the earliest stages more addressed. For instance, N2-(2-carboxyethyl) arginine synthase (CEAS), β-lactam synthase (BLS) and proclavaminate amidinohydrolase (PAH) are responsible for the first, second and fourth enzymatic reaction, respectively. Recent mutagenic studies in S.clavuligerus have related extra copies of ceas, bls and pah genes ((ceas1, bls1 e pah1) to this pathway but none enzymatic assay was further reported. Although later stages the pathway remain unclear, the action of some putative enzymes like the codified by orf12 showed essential to CA production. Thus, aiming to increase the information available about CA biosynthesis we studied four of its members: CEAS1, BLS1, PAH1, and the putative protein codified by orf12. The genes were isolated from S.clavuligerus genomic DNA by PCR and further cloned into expression vectors in order to produce recombinant proteins in E.coli. Protocols of protein expression were established to CEAS1, PAH1 and ORF12 and recombinant proteins were purified by metal affinity chromatography. BLS was obtained as an insoluble form. Soluble proteins were characterized by means of biochemical and structural approaches. Analyses of CEAS1 and PAH1 were compared with information ever conducted to the isozymes CEAS2 and PAH2, respectively. Thus, oligomerization analysis of proteins resulted respectively in a mix of oligomers forms (monomer, dimer, tetramer) to CEAS1, hexameric form to PAH1 and dimeric form to ORF12, according to the soluble and crystallographic form of CEAS2 (dimer and tetramer) and PAH2 (hexamer). Circular Dichroism spectra showed that CEAS1 and PAH1 have an α-β conformation and were stable up to 35ºC over a wide pH range. Thermodynamic parameters of CEAS1 cofactor (Mg+2) binding were determined showing that is entropic driven, with a 4:1 binding stoichiometry, with a micro-molar affinity (KD = 1.76 ± 0.23 µM). Analyses by coupled assay, High Pressure Liquid Chromatography coupled to Mass Spectroscopy (LC-MS) and Isothermal Titration Calorimetry showed that CEAS1, as well as the CEAS2, presents activity at the substrate glyceraldehydes-3-phosphate, however without formation of final product, N2-(2-carboxyethyl)arginine. Meanwhile recombinant PAH1 showed none activity at analogous substrate, N-α-acetil-L-arginine. Thus despite isozymes maintain a structural pattern, they may have distinct action mechanism. Regards to ORF12, this protein was classified as a β-lactamase with an esterase activity according to our studies performed with the substrates cephalosporin C and p-nitrophenyl acetate.
3
Fang, Minggang. "The analysis of Autographa Californica multiple Nucleopolyhedrovirus EXONO (ORF141) function and its role in virus budding." Thesis, University of British Columbia, 2007. http://hdl.handle.net/2429/191.
Анотація:
Baculoviruses have a biphasic replication cycle producing two types of virions, budded virus (BV) and occlusion derived virus (ODV) which are required for the systemic spread or oral infection with the insect host respectively. Little is known about the events of the BV pathway and the mechanism by which nucleocapsids are selected and directed from the nucleus to plasma membrane to form BV. The Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) gene exon0 (orf141) is known to be required for the efficient production of BV and in this study the function and mechanism by which EXON0 affects BV production was investigated.
Confocal microscopic analysis showed that EXON0 localized in the nucleus in the ring zone of virogenic stroma where nucleocapsids are assembled. In addition EXON0 also concentrated in the cytoplasm at the plasma membrane. Analysis of virions revealed that EXON0 copurified with nucleocapsid fractions of both BV and ODV. In support of this yeast 2-hybrid screening, co-immunoprecipitation, and confocal microscopy revealed that EXON0 interacted with the known nucleocapsid proteins FP25 and BV/ODV-C42. Transmission electron microscopy showed that deletion of exon0 results in nucleocapsids being unable to efficiently egress from the nucleus to the cytoplasm.
Cellular protein interaction analyzed by tandem affinity purification and co-immunoprecipitation showed that beta-tubulin co-purified with EXON0. Immunofluorescence also showed that EXON0 and microtubules co-localized during virus infection. The microtubule inhibitors colchicine and nocodazole affected the localization of EXON0 and significantly reduced BV production. These data support the conclusion that egress of AcMNPV nucleocapsids is facilitated by interaction of EXON0 with beta-tubulin and microtubules.
Deletion and point mutation analysis mapped domains of EXON0 required for efficient budding, dimer formation and association with FP25, BV/ODV-C42 and beta-tubulin. The Leucine zipper domain was required for dimer formation, beta-tubulin and BV/ODV-C42 interaction and also reduced interaction with FP25. Multiple domains were also shown to affect BV production.
This study provides a detailed analysis of EXON0 which is one of the first baculovirus genes shown to be specific for the BV pathway. The results extend our understanding of the BV pathway which is a major determinant of baculovirus pathogenesis.
4
Lehiy, Christopher J. "A study of two highly conserved baculovirus genes." Diss., Kansas State University, 2010. http://hdl.handle.net/2097/13892.
Анотація:
Doctor of PhilosophyDepartment of Biology
A. Lorena Passarelli
Baculoviruses are enveloped, rod shaped viruses with circular, double-stranded DNA genomes. These viruses infect arthropods, primarily in the order Lepidoptera, although members of this virus family also infect species of Diptera, Hymenoptera, and Crustacea. The majority of these viruses undergo a bi-phasic cycle with one phase defined by the production of a budded virus (BV) form, responsible for cell to cell transmission, and the other defined by the production of an occlusion-derived virus (ODV) form, responsible for host to host transmission. The proto-typical member of the Baculoviridae family is considered to be Autographa californicaM Nucleopolyhedrovirus (AcMNPV). Its 133,894 base pair genome is predicted to encode for 156 proteins, a large number of which are essential for virus replication.. In this current work, we have further characterized two viral proteins that are highly conserved among baculoviruses. The first of these is an ortholog of the fibroblast growth factor family of proteins with sequence homology to the Drosophila Branchless protein as well as the mammalian FGF- 9, -16 and -20 subfamily. Despite its high degree of conservation among baculoviruses, the viral fibroblast growth factor (vFGF) is considered a non-essential protein, although its deletion from the genome does affect the lethality of the virus when ingested per os. In our study, we were able to localize vFGF to the membrane of BV. Its presence on the envelope affected the ability of the virus particle to bind to both heparin in vitro and to the cell surface in vivo, and may play a role in the attachment phase prior to virus entry. We also characterized AcMNPV’s open reading frame 109 (Ac-orf109). Unlike vFGF, Ac-orf109 is essential for virus replication since its deletion results in a complete lack of BV production. Transmission electron microscopy of cells transfected with an Ac-orf109 deletion virus shows the full range of virus-associated structures including mature capsid formation but there appears to be a deficiency in capsid egress out of the nucleus. Furthermore, the ODV retained in the nucleus appear to lack microvesicular membranes, an essential component for host to host transmission of infection.
5
Szczepańska, Agnieszka Katarzyna. "Characterisation of recombination functions of the orf12-15 DNA region from the bIL67 bacteriophage active against Lactococcus lactis subsp. Lactis." Paris 11, 2006. http://www.theses.fr/2006PA112281.
Анотація:
La recombinaison homologue est essentielle au maintien de la stabilité du génome et à son évolution. Les phages possèdent des mécanismes de recombinaison particuliers qui participent efficacement à leur multiplication et à leur évolution en favorisant la circularisation et la réplication de leur ADN ainsi que les échanges horizontaux de gènes. Les études menées sur le phage bIL67 virulent pour Lactococcus lactis subsp. Lactis avaient montré que la protéine codée par l’orf13 est homologue à la recombinase Erf du phage P22. Sur la base de données de génomique comparative des phages, nous avons identifié un module putatif de recombinaison qui comprend les orfs 12 à 15. Notre travail a ensuite montré que le phage bIL67 est capable d’effectuer sa propre recombinaison homologue en l’absence de la protéine bacterienne RecA. Nous avons aussi montré que ce phage inhibe l’activité exonucléase et celle de recombinaison du complexe RexAB chez L. Lactis. La caractérisation biochimique de la protéine codée par l’orf14 a montré que c’est une protéine de type SSB. Par des expériences de retard de migration en gel il a été démontré que Orf14 est capable de se lier d’une manière séquence non spécifique aux acides nucléiques simple brin. La modélisation de la structure tridimensionnelle de la protéine, combinée à une analyse in vitro de protéines mutantes purifiées, indique que le domaine de liaison à l’ADN de la protéine se situe dans un domaine OB putatif (oligosaccharide/oligonucleotide binding), une caractéristique partagée par toutes les protéines SSB. De plus, Orf14 est capable de complémenter partiellement in vivo le défaut de croissance d’un mutant ssb thermosensible d’E. ColiRecombination is involved in key stages of phage multiplication. Due to its importance for phage development, it is postulated that phages do not rely only on host-encoded functions but posses their own recombination proteins. Despite general indications that lactococcal phages can recombine, existence of recombination systems lacked direct proof. Results of this thesis supply evidence of phage bIL67-mediated recombination and studies that led to characterising individual recombination genes in the phage genome. This work presents in a general way that phage bIL67 encodes proteins proficient in promoting recombination. Phage bIL67 was shown to develop and recombine independently of host RecA protein, which suggested the presence of phage-encoded proteins promoting the same events as bacterial recombination proteins. The putative recombination region was defined to orfs12-15 of the early transcribed part of the phage genome. Finding orf13 gene product to be homologous to phage P22 Erf recombinase and an overall organisation similarity with P22 recombination module were the initial evidences suggesting localisation of the putative recombination gene cluster in this region. Identification of orf14-encoded SSB function just upstream of orf13 supplied further proof. Putative recombination region was shown to encode anti-Exo activity acting against main lactococcal Exo protein (RexAB). Molecular characterisation of Orf14 activity in vitro confirmed its affinity for single-stranded nucleic acids. Amino acid substitution of Orf14 led to identification of the functional domains involved in DNA binding and protein-protein interactions. In vivo complementation studies proved Orf14 to partially substitute SSB of E. Coli
6
Stockmeyer, Kerstin [Verfasser]. "Heterologe Expression und Funktionsanalyse des CMS-assoziierten offenen Leserahmens orf107 aus Sorghum bicolor in Nicotiana tabacum und Arabidopsis thaliana / Kerstin Stockmeyer." Kiel : Universitätsbibliothek Kiel, 2008. http://d-nb.info/1019543426/34.
7
Okada, Sachiko. "Characterization of the repeat-rich region of the Y chromosome harboring a male-specific gene, ORF162, in the liverwort, Marchantia polymorpha." Kyoto University, 2002. http://hdl.handle.net/2433/149933.
Анотація:
Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第9648号
農博第1276号
新制||農||847(附属図書館)
学位論文||H14||N3680(農学部図書室)
UT51-2002-G406
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 大山 莞爾, 教授 佐藤 文彦, 教授 關谷 次郎
学位規則第4条第1項該当
8
Diekmann, Ulrike Verfasser], and Lothar [Akademischer Betreuer] [Jänsch. "Characterisation of the KSHV protein ORF20 and its role in innate immunity / Ulrike Diekmann ; Betreuer: Lothar Jänsch." Braunschweig : Technische Universität Braunschweig, 2019. http://d-nb.info/1179909984/34.
9
Diekmann, Ulrike [Verfasser], and Lothar [Akademischer Betreuer] Jänsch. "Characterisation of the KSHV protein ORF20 and its role in innate immunity / Ulrike Diekmann ; Betreuer: Lothar Jänsch." Braunschweig : Technische Universität Braunschweig, 2019. http://d-nb.info/1179909984/34.
10
C, G. GUALBERTO JOSE MANUEL. "Etude des genes nad3, rps12, orf156 et coxiii du genome mitochondrial du ble (triticum aestivum) : structure, expression et edition de leurs transcrits." Strasbourg 1, 1990. http://www.theses.fr/1990STR13091.
Анотація:
Deux regions transcrites du genome mitochondrial du ble ont ete sequencees et analysees. Une de ces regions correspond a une unite de transcription contenant les genes nad3 et rps12, codant respectivement pour la sous-unite 3 de la nadh deshydrogenase et pour la proteine ribosomale s12. Ces deux genes sont precedes par un gene de trna (trns-gcu) et par un pseudo-gene de trna. Trois phases ouvertes de lecture, orf299, orf86 et orf156, sont presentes en aval de rps12. Des anticorps specifiques detectent in vivo les produits de rps12 et de l'orf156. L'autre region etudiee contient les genes coxiii et trne-uuc, codant respectivement pour la sous-unite 3 de la cytochrome oxydase et pour un trna#g#l#u. La transcription de ces sequences a ete etudiee. L'analyse de l'utilisation des codons cgg dans nad3, rps12 et coxiii a permis de demontrer qu'une activite d'edition du rna, qui remplace specifiquement des cytidines en uridines, existe dans les mitochondries vegetales. Les sites d'edition dans les genes nad3, rps12, orf299, orf156 et coxiii ont ete determines. Des similarites de sequence autour des sites d'edition suggerent que des rna anti-sens sont impliques dans la specificite de l'edition. Des clones partiellement edites de nad3 et rps12 ont ete identifies. L'ensemble des resultats montre que l'edition est un processus post-transcriptionnel qui permet la conservation des proteines mitochondriales chez les plantes superieures
11
Khazina, Elena [Verfasser], and Thilo [Akademischer Betreuer] Stehle. "Structural and functional analysis of the ORF1p protein of the human LINE-1 retrotransposon / Elena Khazina ; Betreuer: Thilo Stehle." Tübingen : Universitätsbibliothek Tübingen, 2011. http://d-nb.info/1162698985/34.
12
Serfiotis-Mitsa, Dimitra. "Biophysical and structural studies of the antirestriction proteins ArdA and KlcA." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4358.
Анотація:
Gene orf18, which is situated in the conjugative transposon Tn916 from the bacterial pathogen Enterococcus faecalis, encodes a putative ArdA (alleviation of restriction of DNA) protein. ArdA from Tn916 may be responsible for the apparent immunity of the transposon to DNA restriction and modification (R/M) systems and for ensuring that the transposon has a broad host range. The orf18 gene was engineered for overexpression in Escherichia coli and the recombinant ArdA protein was purified to homogeneity. Biophysical characterisation of ArdA demonstrated tight association between ArdA and the M.EcoKI. Also, ArdA was shown to efficiently inhibit restriction and modification by all four major classes of Type I R/M enzymes in vivo. Thus, ArdA can overcome the restriction barrier following conjugation and so helps to increase the spread of antibiotic resistance genes by horizontal gene transfer. The amino acid sequence of KlcA, from the incompatibility plasmid pBP136 from Bordetella pertussis, showed a high degree of similarity with the antirestriction protein ArdB from the IncN plasmid pKM101. In this study the solution structure of KlcA was solved with high-resolution NMR and its antirestriction function demonstrated. The structure of KlcA showed a rigid globular molecule with a novel fold. No antimodification function was observed for KlcA in vivo and the antirestriction function of KlcA has been successfully shown in vivo but not in vitro. Because no direct binding of KlcA to EcoKI was observed in vitro, the mechanism of the endonuclease blocking was assumed to be different from that of ArdA. Preliminary experiments including coimmunoprecipitation assays were conducted in order to elucidate the antirestriction mechanism of KlcA.
13
BELLAOUI, MOHAMMED. "Analyse de l'expression du gene orf138 associe a la sterilite male-cytoplasmique ogura chez le colza et des recombinaisons dans la region qui le contient." Paris 11, 1997. http://www.theses.fr/1997PA112191.
Анотація:
La fusion de protoplastes realisee par georges pelletier et al, (1983) entre le colza male-fertile et le colza male-sterile portant le cytoplasme de radis ogura a fourni un materiel ideal pour etudier l'expression du gene mitochondrial orf138 associe a la sterilite male-cytoplasmique ogura et des recombinaisons dans cette region. Ces cybrides ont ete utilises au cours de cette these pour etudier trois aspects de l'expression et de la dynamique du genome mitochondrial. Le sujet principal de cette these concerne le role des regions 3' non codantes dans le controle de l'expression du gene orf138 rencontre dans trois configurations differentes dans des cybrides de colza. Nous avons etabli le role de ces regions dans la difference d'accumulation des arnm observee pour les trois configurations, et montre qu'elles controlent la maturation et la stabilite post-transcriptionnelle de ces arnm. Dans un deuxieme temps, nous avons montre qu'il existe dans le genome mitochondrial des plantes etudiees differentes configurations des genes orf138 et orfb a l'etat substchiometrique. Des evenements de recombinaison homologue sont sans doute impliques dans l'interconversion de ces configurations. Nous avons montre que ces interconversions sont frequentes et qu'un mecanisme actif est sans doute implique dans le maintien sous forme de molecules substchiometriques des produits d'un evenement de recombinaison donne. Un effet du genome nucleaire sur la presence d'une configuration substchiometrique de l'orfb a ete egalement mise en evidence. Dans une troisieme partie, l'etude de l'effet du gene de restauration (le gene nucleaire rfo) sur l'expression du gene orf138 chez les cybrides de colza a ete realisee. Nous avons montre que le gene rfo agit de facon organe- et stade-specifique sur l'accumulation de la proteine orf138 sans affecter l'accumulation de l'arnm correspondant ni l'efficacite de sa traduction. Un controle post-traductionnel (direct ou indirect) de la stabilite de la proteine orf138 par le produit du gene rfo constitue donc probablement le mecanisme de restauration de la fertilite male dans ces plantes.
14
Henzel, Andréia. "Experimental pathogenesis of acute and latent genital infection by bovine herpesvirus type 1.2 in heifers." Universidade Federal de Santa Maria, 2012. http://repositorio.ufsm.br/handle/1/4062.
Анотація:
Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorThe feline calicivirus (FCV) and the felid herpesvirus type 1 (FeHV-1) are the main agents of the respiratory tract diseases of felines. Both viruses are distributed worldwide, however its distribution and prevalence in Brazil are not well known. In the present thesis we describe the isolation and one serosurvey of FCV and FeHV-1 in some counties of the Rio Grande do Sul State, Brazil; and the molecular characterization of the capsid protein gene of FCV isolates is also described. In Chapter 1, we describe the epidemiologic survey of FCV and FeHV-1 through investigation of the conjunctival, nasal, oral and oropharyngeal swabs from 302 domestic cats. The viral isolation was performed in Crandell-Reese feline kidney cells and the isolates were submitted to PCR and RT-PCR for confirmation of the presence of the FeHV-1 and FCV, respectively. Fifty five (18.2%) of the 302 cats analyzed were positive for the viral isolation; when tested by PCR, 29 cats (52.7%) were shedding the FCV, 21 (38.2%) shedding FeHV-1, and 5 cats (9.1%) shedding both viruses. In addition, isolates of FCV and FeHV-1 were standardized to be used as control in the PCR and virus neutralization (VN) assays (serological technical used in chapter 3 of the thesis). The SV65/90 (FCV) and the SV534/00 (FeHV-1) isolates were defined as standard viruses for the assays. In Chapter 2, the molecular characterization of 13 FCV isolates is described; ten isolates came from the survey performed in chapter 1, two were deposited in the virology laboratory of the UFSM and one was obtained from a cat with gingivitis-stomatitis syndrome. The ORF2 (open reading frame) region that codifies the major protein of the viral capsid was sequenced, which includes an immunodominant region of the virus. The ORF 2 was analyzed at the nucleotide and amino acid levels and these data was used for phylogenetic studies of the 13 Brazilian isolates of FCV. These isolates were compared to ten reference strains of FCV and to the San Miguel sea lion virus type 1 (SMSV-1) as an outgroup in phylogenetic study. The primers were designed using the complete sequence of FCV-F9. The comparison of the 13 Brazilian isolates with the F9 strain revealed a large molecular diversity among them, concentrated mainly along the linear epitopes of the region. The Chapter 3 describes a serosurvey against FCV and FeHV-1 in serum samples from domestic feline. From the 630 samples tested by the VN assay, 53.6% (338/630) were positive to one or both viruses with a prevalence of 39.2% for neutralizing antibodies against FCV and 30.6% for FeHV-1. The results from the present study demonstrated that the FCV and FeHV-1 are circulating among the feline population examined, and also, the presence of carriers of the viruses among these cats. Besides, the molecular characterization of the FCV evidenced the great genetic variability of the Brazilian isolates when compared to vaccine and reference strains.
O calicivírus felino (feline calicivirus FCV) e o herpesvírus felino tipo 1 (felid herpesvirus type 1 FeHV-1) são os principais agentes das doenças do trato respiratório dos felinos. Ambos os vírus são mundialmente distribuídos, mas no Brasil sua distribuição e prevalência são ainda pouco conhecidas. Na presente tese descrevemos o isolamento e sorologia do FCV e do FeHV-1 em alguns municípios do estado do Rio Grande do Sul (RS), Brasil; e a caracterização molecular do gene da proteína do capsídeo de isolados de FCV. No Capítulo 1, descreve-se o isolamento do FCV e do FeHV-1 por meio da coleta de suabes (conjuntival, nasal, oral e orofaringeano) de 302 gatos domésticos. O material coletado foi inoculado em cultivo celular de origem felina (CRFK) e submetidos a PCR e RT-PCR para confirmação em FeHV-1 e FCV, respectivamente. Dos 302 gatos analisados, 55 (18,2%) foram positivos no isolamento; e quando testados por PCR, 29 dos 55 gatos (52,7%) excretavam o FCV, 21 (38,2%) excretavam o FeHV-1 e 5 gatos (9,1%) apresentavam ambos os vírus. Além dessa descrição, realizou-se a padronização de um isolado de FCV e de FeHV-1, para serem utilizados como controle nas técnicas de PCR e na vírus neutralização [VN] (técnica sorológica utilizada no capítulo 3 da tese). O SV65/90 (FCV) e o SV534/00 (FeHV-1) foram os isolados definidos como vírus-padrão. No Capítulo 2, incluímos o estudo sobre a análise molecular de 13 isolados de FCV; dez isolados foram obtidos a partir do material descrito no capítulo 1, dois estavam armazenados no laboratório de virologia da UFSM e um foi obtido de um gato acometido pela síndrome gengivo-estomatite. A região analisada foi o gene da proteína do capsídeo, codificado pela ORF2 (open reading frame), na qual se localizam as regiões imunodominantes do FCV. A ORF2 foi analisada em nível de nucleotídeos e aminoácidos; e uma análise filogenética dos 13 isolados brasileiros de FCV, incluindo dez cepas de referência e o vírus dos leões marinhos de San Miguel tipo 1 (San Miguel sea lion virus type 1 SMSV-1) como um outgroup, foi também realizada. Os primers foram desenhados com base na sequência completa do genoma da cepa de referência a FCV-F9. Quando os 13 isolados brasileiros de FCV foram comparados a cepa F9, detectou-se grande diversidade molecular nas regiões variáveis do gene e nos epitopos lineares mapeados. O Capítulo 3 contem um estudo sorológico contra FCV e FeHV-1 em amostras de soro de felinos domésticos. Das 630 amostras testadas pela VN, 53,6% (338/630) foram positivas para um ou ambos os vírus; sendo a prevalência de anticorpos neutralizantes contra o FCV de 39,2% e do FeHV-1 de 30,6%. Os resultados aqui apresentados demonstram a circulação do FCV e do FeHV-1 na população de gatos domésticos analisados, assim como, a presença de gatos portadores para ambos os vírus. Além disso, a caracterização molecular do FCV demonstrou a grande variabilidade genética dos isolados brasileiros em comparação às cepas vacinais e às de referência.
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Heidari, Farshad. "In vitro modulation of host immune response by the Varicella zoster virus ORF1 gene product." Thesis, Kingston University, 2017. http://eprints.kingston.ac.uk/41971/.
Анотація:
Varicella-zoster virus causes chicken pox (Varicella) primary infection, which becomes latent in the dorsal root ganglia and trigeminal ganglia, and may reactivate to cause shingles, the most serious complication of which is post-herpetic neuralgia occurring in 50% of individuals over 60 years. Severity of lesions depends on host's immune response. Like many viruses, varicella-zoster virus appears to have evolved escape mechanisms from host immune surveillance by downregulating cell surface major histocompatibility complex class 1 expression and may delay of resolution of infection. Major histocompatibility complex class 1 is processed and transported to the cell surface through the Golgi apparatus. Varicella-zoster virus genome encodes a membrane gene, open reading frame type 1, which is localised in the enoplasmic reticulum and Golgi apparatus. Given the cellular localisation of open reading frame type 1 in the Golgi apparatus, this study investigated the expression of major histocompatability complex class 1 in human immortalised keratinocytes transfected with only empty vector. As a control, the expression of major histocompatability complex class 1 human immortalised keratinocytes parental cells were also examined using current molecular biology techniques. The results of this thesis demonstrate that the expression of varicella-zoster virus-open reading frame type 1 prevents the transport of major histocompatibility complex class 1 complexes to the cell surface and causes its retention in the Golgi apparatus, which is compensated by treatment with IFN-[alpha]. Varicella-zoster virus (VZV)-open reading frame type 1 does not affect the synthesis of human leukocyte antigen class 1 heavy chains or the expression of the transporter associated with antigen processing. Additionally, we determined that varicella-zoster virus-open reading frame type 1 impedes the surface expression of human leukocyte antigen class-A and human leukocyte antigen class-B, which present viral peptides to major histocompatibility complex class I-restricted cytotoxic T lymphocytes, but not the natural killer cell inhibitory ligands human leukocyte antigen class-C and non-classical human leukocyte antigen class-E. This selective downregulation of cell surface human leukocyte antigen class 1 molecules may allow the virus to establish infection by avoiding immune clearance of virus-infected cells by both cytotoxic T lymphocytes and natural killer cells. However, it remains to be seen if open reading frame type 1 expressing cells evade cytotoxic T lymphocytes killing, through downregulation of classical major histocompatibility complex class I, and natural killer killing, through lack of downregulation of non-classical major histocompatibility complex class I. The study outcome will be a valuable attempt to elucidate factors involved in varicella-zoster virus-related lesion progression, contribute to existing knowledge, and importantly allude to further investigations on the pathogenesis of this virus on human disease. The data obtained may also offer novel means of therapeutic intervention.
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Basso, Virginia. "Identification and characterization of transcription factors involved in morphogenesis in the human fungal pathogen Candida albicans." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC131/document.
Анотація:
Candida albicans est un champignon commensal de l’homme, mais aussi l'un des agents pathogènes fongiques les plus répandus. C. albicans alterne entre formes levure et filamenteuses (pseudohyphes ou hyphes), une transition morphologique déclenchée par divers signaux environnementaux et jouant un rôle important dans la virulence. Un réseau de régulation complexe, faisant intervenir de nombreux facteurs de transcription, gouverne la transition levure-hyphe. L’objectif de ce travail de thèse était d’étudier la fonction et l’interaction avec ce réseau de régulation de deux facteurs de transcription, Skn7 et Orf19 .217. L’identification des circuits de transcription gouvernés par Skn7, par des approches globales d’étude de la transcription et de la fixation à la chromatine, indique un double rôle dual dans la régulation de la réponse au stress osmotique/oxydatif et de la croissance filamenteuse. L’analyse des interactions génétiques a révélé des relations fonctionnelles entre Skn7 et les principaux régulateurs de la morphogenèse. En outre, Skn7 est indispensable pour limiter l'accumulation d'espèces réactives à l'oxygène (ROS) pendant la croissance filamenteuse sur milieu solide. La caractérisation de mutants spécifiques de Skn7 a mis en évidence un découplage de cette dernière fonction et de la fonction de Skn7 dans la morphogénèse.Orf19.217 est impliqué dans la virulence de C. albicans, mais sa fonction biologique reste incertaine. Notre étude montre que Orf19.217 régule divers processus, tels que la morphogenèse, la modification de la paroi cellulaire et la réponse au stress. D'autres expériences sont en cours pour élucider son rôle dans la virulenceCandida albicans is a diploid fungus, commensal of most healthy individuals, but also one of the most prevalent human fungal pathogens. C. albicans has the ability to switch between the unicellular yeast form and filamentous forms (pseudohyphae or hyphae). This transition is triggered by various environmental cues and plays important roles in C. albicans virulence. An intricate regulatory network involving many transcription factors controls the yeast-to-hypha transition. The aim of this PhD was to explore the function of two transcription factors, Skn7 and Orf19.217, whose overexpression triggers filamentation independently of hypha-inducing cues, and their interplay with the morphogenetic regulatory network. Mapping of the Skn7 transcriptional circuitry, through combination of genome-wide expression and location technologies, pointed to a dual regulatory role encompassing osmotic/oxidative stress response and filamentous growth. Genetic interaction analyses revealed close functional interactions between Skn7 and master regulators of morphogenesis. Furthermore, Skn7 was crucial for limiting the accumulation of reactive oxygen species (ROS) during filamentous growth on solid medium. Interestingly, functional domain mapping using site-directed mutagenesis allowed decoupling of Skn7 function in morphogenesis from protection against intracellular ROS. Orf19.217 was previously implicated in C. albicans virulence, but its biological function remains unclear. Preliminary data showed that Orf19.217 regulates several processes, including morphogenesis, cell wall modifications and stress response. Further experiments are ongoing to elucidate the role of this gene in virulence
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März, Alexander [Verfasser]. "Generierung von Antiseren gegen Domänen von ORF1 der TT-Viren J01B6 und P/1C1 / Alexander März." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2010. http://d-nb.info/1024364976/34.
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Bagdassarian, Eugénie. "Interaction du virus de l'hépatite E avec la réponse intérféron de l’hôte." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS266/document.
Анотація:
L’infection par le virus de l’hépatite E (VHE) peut entraîner une hépatite aiguë chez l’homme évoluant en hépatite fulminante dans 1-4% des cas, voire 20% chez les femmes enceintes dans les régions endémiques. Le VHE, transmis par voie entérique, est responsable de grandes épidémies d’origine hydrique dans les pays en voie de développement et de nombreux cas sporadiques d’origine zoonotique dans des pays industrialisés. La description récente de cas d’hépatites E chroniques ou d’atteintes neurologiques graves souligne l’importance de caractériser les interactions du VHE avec son hôte. L’objectif de ce projet de thèse était de caractériser les interactions entre le VHE et la réponse immune innée de l’hôte et en particulier avec le système interféron de type I (IFN-I).La première partie de ce projet a consisté en l’étude de la modulation des voies de signalisation de l’IFN-I par la polyprotéine non-structurale ORF1 du VHE. Celle-ci est constituée de plusieurs domaines fonctionnels putatifs tels qu’une methyltransférase (Met) ou une protéase à cystéine de type papaïne (PCP) dont les fonctions sur la signalisation IFN-I restent encore peu connues. Les résultats obtenus ont montré que le domaine MetPCP de l’ORF1 est capable d’inhiber l’activation du promoteur de l’IFN-β et de celui des gènes sous le contrôle de l’IFN contenant des éléments de réponse à l’IFN (ISRE), ainsi que l’expression de certains des gènes induits par l’IFN (ISGs). En recherchant le mécanisme impliqué dans l’inhibition du promoteur ISRE, nous avons montré que le domaine MetPCP inhibe la phosphorylation de STAT1 et sa relocalisation nucléaire. Nous avons également montré que le domaine MetPCP n’inhibe pas la phosphorylation de STAT2. Le mécanisme d’action du domaine MetPCP reste encore à préciser. La deuxième partie de ce projet a été de déterminer si l’infection par le VHE entraîne la production d’IFN-I par les cellules dendritiques plasmacytoïdes (pDCs). En effet, les pDCs sont la principale source d’IFN-I et jouent un rôle crucial dans la réponse immunitaire innée et adaptative. Les résultats obtenus suggèrent que les pDCs ne produisent que modérément de l’IFN-I lorsqu’elles sont co-cultivées avec des cellules infectées par le VHE. L’ensemble des résultats obtenus pendant ce travail de thèse suggère que le VHE utilise plusieurs mécanismes pour moduler la signalisation IFN-I de l’hôteHepatitis E virus (HEV) is the causative agent of acute hepatitis in humans that can lead to fulminant hepatitis in 1-4% of cases, and in 20% of pregnant women in endemic regions. HEV is an enterically-transmitted virus responsible for large waterborne epidemics in developing countries and numerous cases of zoonotic hepatitis E in industrialized countries. The recent description of cases of chronic hepatitis E and severe neurological disorders highlight the importance to characterize the interactions between HEV and the host. The objective of this PhD project was to characterize the interactions between HEV and the host innate immune response and particularly with the type I interferon system (IFN-I).The first part of this project aimed to study the ability of the ORF1 non-structural polyprotein of HEV to modulate the IFN-I signalling pathways. HEV ORF1 contains several putative functional domains including a methyltransferase (Met) and a papain-like cysteine protease (PCP) whose functions on the IFN-I signalling remain poorly understood. The results obtained showed that the MetPCP domain of ORF1 inhibits IFN-β and IFN-stimulated response element (ISRE) promoter activation and the expression of some IFN-stimulated genes (ISGs). We then investigated the mechanism involved in this inhibition of ISRE promoter activation. We showed that the MetPCP domain inhibits STAT1 phosphorylation and nuclear translocation. In contrast, we found that the MetPCP domain does not inhibit STAT2 phosphorylation. However, the mode of action of MetPCP remains to be fully characterised. The second part of this project aimed to determine the ability of plasmacytoid dendritic cells (pDCs) to produce IFN-I in response to HEV infection. Indeed, pDCs are the main IFN-I source and play a crucial role in innate and adaptive responses. The results obtained suggest that pDCs produce IFN-I moderately when co-cultured with HEV-infected cells. Taken together, the results obtained during this PhD project suggest that HEV has evolved different mechanisms to modulate the IFN-I host response
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Ankavay, Maliki. "Étude des modifications post-traductionnelles de la protéine de capside ORF2 du virus de l'hépatite E (HEV)." Thesis, Lille 2, 2019. http://www.theses.fr/2019LIL2S013.
Анотація:
L’infection par le virus de l’hépatite E (HEV) est un problème majeur de santé publiquequi touche plus de 20 millions de personnes et tue environ 70 000 chaque année à travers lemonde. Le HEV est la cause majeure d’hépatite virale aiguë dans le monde. En France, laséroprévalence du HEV est de 22,4% dans la population générale. Ce virus se transmet parvoie féco-orale ou par consommation de viande contaminée mal cuite. Très récemment, nousavons décrit un système de culture cellulaire permettant d’amplifier efficacement le HEV. Cesystème représente un outil unique pour caractériser les différentes étapes du cycle infectieuxdu HEV qui sont très mal connues à ce jour. Dans le cadre de ma thèse, je me suis intéresséplus particulièrement à la protéine de capside ORF2 qui est l’unité structurale des particulesvirales et est donc un acteur central du cycle infectieux du HEV. La protéine ORF2 est uneprotéine de 660 acides aminés (aa) qui possède un peptide signal (PS) et trois sites potentielsde N-glycosylation (N1, N2 et N3). J’ai donc étudié le rôle de la N-glycosylation de cetteprotéine ORF2 dans le cycle infectieux du HEV. Pour atteindre cet objectif, la soucheinfectieuse HEV-p6 génotype 3 a été utilisée pour infecter les cellules de la lignéehépatocytaire PLC3, sous clone de la lignée PLC/PRF/5. Les résultats obtenus ont montré quela protéine ORF2 est N-glycosylée uniquement au niveau des sites N1 et N3 ; le site N2 estdéfavorable à la N-glycosylation. Nos résultats ont montré que la N-glycosylation de laprotéine ORF2 n’est pas importante pour sa stabilité, son oligomérisation, sa reconnaissancepar des anticorps, l’assemblage et l’infectiosité des particules virales. Nous avons aussimontré que la protéine ORF2 est importée dans le noyau des cellules hôtes au cours du cycleinfectieux du HEV de manière indépendante de la N-glycosylation. Nous avons identifié pourla première fois un signal de localisation nucléaire (NLS) fonctionnel en position N-terminalede la protéine ORF2 lui permettant d'interagir probablement avec l’importine alpha1. Demanière surprenante, ce NLS régule non seulement l’import nucléaire de cette protéine virale,mais aussi sa translocation réticulaire. Nous avons également démontré que la protéine ORF2est exportée du noyau des cellules hôtes en interagissant avec l’exportine1 et possède 3signaux d’export nucléaire (NES) en position C-terminale. Ces résultats améliorent laconnaissance du trafic intracellulaire de la protéine ORF2 et pourraient orienter les stratégiesantivirales dirigées contre le HEVHepatitis E virus (HEV) infection is a major public health problem that affects more than20 million people and kills approximately 70,000 worldwide each year. HEV is the leadingcause of acute viral hepatitis in the world. In France, the seroprevalance of HEV is 22.4% inthe general population. This virus is transmitted by fecal-oral route or by consumption ofundercooked contaminated meat. Very recently, we have described an efficient cell culturesystem to amplify HEV. This system represents a unique tool for characterizing the variousstages of the infectious HEV lifecycle that are very poorly understood to date. As part of mythesis, I focused on the ORF2 capsid protein which is the structural unit of viral particles andis therefore a central player in the HEV lifecycle. ORF2 is a 660 amino acids protein thatcontains a signal peptide and three potential N-glycosylation sites (N1, N2 and N3). My workaimed to identify the role of the ORF2 N-glycosylation in the HEV lifecycle. Our resultsrevealed that the ORF2 protein is N-glycosylated on the N1 and N3 sites, whereas the N2 siteis not N-glycosylated. Also, the N-glycosylation has no significant relevance neither for thestability, oligomerization, antibody recognition of the ORF2 protein nor for viral assemblyand viral infectivity. We also revealed that, the ORF2 protein is translocated into the nucleusof infected cells, independently of N-glycosylation. We identified for the first time afunctional nuclear localization signal (NLS) located at the N-terminus of the ORF2 proteinthat interacts probably with the importin alpha1. Surprisingly, this NLS regulates both nuclearimport and endoplasmic reticulum translocation of the ORF2 protein. Finally, wedemonstrated that, the ORF2 protein possesses three nuclear export signals (NES) located atits C-terminus. The ORF2 protein interacts with the exportin1 and is exported from the cellnuclei. This interaction drives the ORF2 protein from the nucleus to the cytoplasm of theinfected cells. Hence, this study led to new insights into the molecular mechanisms of theORF2 protein and could help to the design of novel antiviral strategies against HEV
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Guo, Hailong. "Antigenic epitope composition and protectivity of avian hepatitis E virus (avian HEV) ORF2 protein and vertical transmission of avian HEV." [Ames, Iowa : Iowa State University], 2006.
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Ferrié, Martin. "Étude de la maturation protéolytique et du trafic intracellulaire de la protéine de capside ORF2 du virus de l'hépatite E (HEV)." Electronic Thesis or Diss., Université de Lille (2022-....), 2023. http://www.theses.fr/2023ULILS067.
Анотація:
L'infection par le virus de l'Hépatite E (HEV) est un problème majeur de santé publique qui toucherait 100 millions de personnes et tuerait 100 000 personnes chaque année dans le monde. Le HEV est la première cause d'hépatite aigüe dans le monde. En France, la séroprévalence s'élève à 22,4%. Ce virus se transmet par voie féco-orale ou par la consommation de viande contaminée mal cuite. Au cours de ma thèse, je me suis intéressé plus particulièrement à la protéine de capside ORF2, qui est l'unité structurale des particules virales et un acteur central du cycle infectieux du HEV. La protéine ORF2 est une protéine de 660 acides aminés qui possède un peptide signal N-terminal et trois sites potentiels de N-glycosylation. Au cours de son cycle infectieux, le HEV produit au moins trois formes de sa protéine de capside : (i) la forme ORF2g (pour glycosylée) et (ii) la forme ORF2c (pour clivée), abréviées ORF2g/c, qui sont des formes glycosylées et massivement sécrétées dans les surnageants de culture ou le sérum des patients infectés, et (iii) la forme ORF2i (pour infectieuse), qui n'est pas glycosylée et qui est associée aux particules virales.Dans le cadre de mes travaux de thèse, j'ai étudié les mécanismes de maturation protéolytique et de trafic intracellulaire de la protéine ORF2. Plus précisément, j'ai d'abord montré que la protéine ORF2 était importée dans le noyau des cellules infectées par le biais d'un mécanisme dépendant de l'Importine-alpha1, ceci grâce à un motif riche en résidus arginine situé à l'extrémité N-terminale de l'ORF2 et nommé ARM. La protéine ORF2 est ensuite exportée vers le cytoplasme par un mécanisme dépendant de l'exportine CRM1, ceci grâce à trois sites d'export nucléaire (NES9, NES10 et NES12) que nous avons identifiés dans la séquence de l'ORF2. J'ai également montré que le trafic nucléo-cytoplasmique de l'ORF2 régule probablement l'expression de certains gènes de l'immunité antivirale aux temps précoces de l'infection.Dans un second temps, j'ai participé à l'identification et à la caractérisation des usines virales du HEV. En ce sens, nous avons montré que les protéine virales ORF1, ORF2 et ORF3, ainsi que l'ARN viral sont enrichis dans des structures vésiculaires et tubulaires localisées dans les régions périnucléaires des cellules infectées. Nous avons montré que ces structures sont également enrichies en marqueurs du compartiment endosomal de recyclage (ERC), comme Rab11 et CD71, indiquant que les usines virales du HEV dérivent probablement de l'ERC. En réalisant des expériences d'extinction de Rab11 avec des ARN interférents, nous avons confirmé l'importance de l'ERC dans la production des particules virales du HEV.Dans un troisième temps, j'ai démontré que la protéine ORF2i, qui est associée aux membranes de la voie de sécrétion, est adressée aux usines virales par un mécanisme faisant intervenir le complexe adapteur AP-1.Dans un quatrième temps, j'ai caractérisé les mécanismes de maturation protéolytique des formes ORF2g/c et ORF2i. J'ai montré que la furine, une proproprotéine convertase de la voie de sécrétion, est impliquée dans la maturation protéolytique des glycoprotéines ORF2g/c. J'ai également montré que la préséniline, qui est la sous-unité catalytique du macro-complexe Gamma-sécrétase, est impliquée dans la maturation protéolytique de la forme infectieuse ORF2i. De manière intéressante, j'ai montré que l'inhibition pharmacologique de la préséniline réduit drastiquement l'infectiosité virale dans des lignées d'hépatocarcinome humain et dans des hépatocytes primaires humains. Ces données suggèrent que l'inhibition pharmacologique de la préséniline représente une stratégie antivirale prometteuse.En conclusion, les résultats obtenus dans le cadre de ma thèse permettent de mieux comprendre les mécanismes d'adressage subcellulaire et de maturation protéolytique de la protéine de capside ORF2, et ouvrent la voie au développement de nouvelles thérapies pour lutter contre le HEVHepatitis E virus (HEV) infection is a major public health problem, affecting an estimated 100 million people and killing 100,000 every year worldwide. HEV is the leading cause of acute hepatitis worldwide. In France, seroprevalence is 22.4%. This virus is transmitted via the feco-oral route, or by eating contaminated undercooked meat. During my thesis, I focused on the ORF2 capsid protein, which is the structural unit of viral particles and a central player in the HEV lifecycle. ORF2 is a 660 amino acid protein with an N-terminal signal peptide and three potential N-glycosylation sites. During its lifecycle, HEV produces at least three forms of its capsid protein: (i) the ORF2g (for glycosylated) form and (ii) the ORF2c (for cleaved) form, abbreviated ORF2g/c, which are glycosylated forms and massively secreted in culture supernatants or serum from infected patients, and (iii) the ORF2i (for infectious) form, which is not glycosylated and is associated with viral particles.As part of my thesis work, I studied the mechanisms of proteolytic maturation and intracellular trafficking of the ORF2 protein. More specifically, I first showed that ORF2 is imported into the nucleus of infected cells via an Importin-alpha1-dependent mechanism, thanks to an arginine-rich motif located at the N-terminus of ORF2 and named ARM. The ORF2 protein is then exported to the cytoplasm via a CRM1 exportin-dependent mechanism, thanks to three nuclear export sites (NES9, NES10 and NES12) that we have identified in the ORF2 sequence. I have also shown that nucleocytoplasmic trafficking of ORF2 probably regulates the expression of certain antiviral immunity genes in the early stages of infection.Secondly, I participated in the identification and characterization of HEV viral factories. We have shown that the viral proteins ORF1, ORF2 and ORF3, as well as viral RNA, are enriched in vesicular and tubular structures located in the perinuclear regions of infected cells. We have shown that these structures are also enriched in markers of the endosomal recycling compartment (ERC), such as Rab11 and CD71, indicating that HEV viral factories probably derive from the ERC. By performing Rab11 silencing experiments with siRNAs, we confirmed the importance of the ERC in the production of HEV viral particles. Thirdly, I demonstrated that the ORF2i protein, which is associated with the membranes of the secretion pathway, is addressed to viral factories by a mechanism involving the AP-1 adaptor complex.In a fourth step, I characterized the proteolytic maturation mechanisms of the ORF2g/c and ORF2i forms. I showed that furin, a proproprotein convertase of the secretory pathway, is involved in the proteolytic maturation of ORF2g/c glycoproteins. I have also shown that presenilin, which is the catalytic subunit of the macro-complex Gamma-secretase, is involved in the proteolytic maturation of the ORF2i form. Interestingly, I have shown that pharmacological inhibition of presenilin drastically reduces viral infectivity in human hepatocarcinoma lines and in primary human hepatocytes. These data suggest that pharmacological inhibition of presenilin represents a promising antiviral strategy. In conclusion, the results obtained during my thesis provide a better understanding of the mechanisms of subcellular addressing and proteolytic maturation of the ORF2 capsid protein, and pave the way for the development of new therapies to combat HEV
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White, Ian Alexander. "Hematopoietic stem cell expansion : under serum free and cytokine-limited conditions using primary endothelial cells transfected with the adenoviral E4-ORF1 gene /." Access full-text from WCMC, 2009. http://proquest.umi.com/pqdweb?did=1692100331&sid=5&Fmt=2&clientId=8424&RQT=309&VName=PQD.
23
Basso, Virginia. "Identification and characterization of transcription factors involved in morphogenesis in the human fungal pathogen Candida albicans." Electronic Thesis or Diss., Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC131.
Анотація:
Candida albicans est un champignon commensal de l’homme, mais aussi l'un des agents pathogènes fongiques les plus répandus. C. albicans alterne entre formes levure et filamenteuses (pseudohyphes ou hyphes), une transition morphologique déclenchée par divers signaux environnementaux et jouant un rôle important dans la virulence. Un réseau de régulation complexe, faisant intervenir de nombreux facteurs de transcription, gouverne la transition levure-hyphe. L’objectif de ce travail de thèse était d’étudier la fonction et l’interaction avec ce réseau de régulation de deux facteurs de transcription, Skn7 et Orf19 .217. L’identification des circuits de transcription gouvernés par Skn7, par des approches globales d’étude de la transcription et de la fixation à la chromatine, indique un double rôle dual dans la régulation de la réponse au stress osmotique/oxydatif et de la croissance filamenteuse. L’analyse des interactions génétiques a révélé des relations fonctionnelles entre Skn7 et les principaux régulateurs de la morphogenèse. En outre, Skn7 est indispensable pour limiter l'accumulation d'espèces réactives à l'oxygène (ROS) pendant la croissance filamenteuse sur milieu solide. La caractérisation de mutants spécifiques de Skn7 a mis en évidence un découplage de cette dernière fonction et de la fonction de Skn7 dans la morphogénèse.Orf19.217 est impliqué dans la virulence de C. albicans, mais sa fonction biologique reste incertaine. Notre étude montre que Orf19.217 régule divers processus, tels que la morphogenèse, la modification de la paroi cellulaire et la réponse au stress. D'autres expériences sont en cours pour élucider son rôle dans la virulenceCandida albicans is a diploid fungus, commensal of most healthy individuals, but also one of the most prevalent human fungal pathogens. C. albicans has the ability to switch between the unicellular yeast form and filamentous forms (pseudohyphae or hyphae). This transition is triggered by various environmental cues and plays important roles in C. albicans virulence. An intricate regulatory network involving many transcription factors controls the yeast-to-hypha transition. The aim of this PhD was to explore the function of two transcription factors, Skn7 and Orf19.217, whose overexpression triggers filamentation independently of hypha-inducing cues, and their interplay with the morphogenetic regulatory network. Mapping of the Skn7 transcriptional circuitry, through combination of genome-wide expression and location technologies, pointed to a dual regulatory role encompassing osmotic/oxidative stress response and filamentous growth. Genetic interaction analyses revealed close functional interactions between Skn7 and master regulators of morphogenesis. Furthermore, Skn7 was crucial for limiting the accumulation of reactive oxygen species (ROS) during filamentous growth on solid medium. Interestingly, functional domain mapping using site-directed mutagenesis allowed decoupling of Skn7 function in morphogenesis from protection against intracellular ROS. Orf19.217 was previously implicated in C. albicans virulence, but its biological function remains unclear. Preliminary data showed that Orf19.217 regulates several processes, including morphogenesis, cell wall modifications and stress response. Further experiments are ongoing to elucidate the role of this gene in virulence
24
津下, 誠治. "カリフラワーモザイクウイルスの増殖機構に関する研究 : ORF1とORF3の遺伝子産物の役割". 京都大学, 2000. http://hdl.handle.net/2433/151627.
25
Peng, Yi Hewi, та 彭懿慧. "線狀噬菌體phiLf反股基因orf155、orf137及orf102之研究". Thesis, 2002. http://ndltd.ncl.edu.tw/handle/46605824916611718878.
Анотація:
碩士國立中興大學
分子生物學研究所
90
Abstract Lf is a filamentous phage specifically infecting Xanthomonas campestris pv. campestris, the pathogenic bacterium causing black rot in crucifers. The genome of Lf is similar to that of the Escherichia coli filamentous phage Ff (f1, fd, and M13), except that Lf lacks the gene homologous to Ff gene IV (gIV ) which is required for phage export. In addition, the Lf origin of replication (ori) is situated within gII, a location different from the case of Ff phage in which the oris for the replication of both complementary and viral strands as well as the DNA packaging signal are in the 508-bp intergenic region (IR) that is rich in secondary structures and encodes no proteins bigger than 16 amino acids. In Lf, the major IR is between gI and gII containing four open reading frames: one (orf98, nt 5720-8) in viral strand and three in complementary strand (orf155, nt 5721-5254; orf137, nt 5338-4925 and orf102, nt 4858-4550). Due to the property that Ff has no DNA length limit during phage packaging and that an insertion of DNA fragment into the IR region does not interfere with phage viability, the phage has wildly been used as cloning vectors. The purpose of this study was to search for the site(s) in the Lf IR region, 1,480-bp long locating between gI and gII, into which an insertion of or a replacement with an antibiotic-resistance cartridge does not affect its viability and stability. For this purpose, we constructed three recombinant phages: LfCD, which had the integration core region (nt 5339-5353) being replaced by a Gmr cartridge, disrupting the 3’ end of orf155; LfHD, which had the IHF binding site (nt5290-5301) being replaced by a Gmr cartridge, disrupting the 5’ end of orf137 and 3’ end of orf155; Lf155G which had the 114-bp EcoRI fragment (nt 5635-5521) being replaced by a Gmr, disrupting orf155. Comparing to those of the wild-type Lf, the plaques manifested by these recombinant phages were turbid, exhibiting degrees of plaque turbidity in the order Lf155G>LfHD>LfCD. Among the four orfs in the Lf IR, ORF155 shares 62% similarity with the Cf1c ORFII which plays a role in the determination of plaque turbidity. The results of transduction showed that the Lf155G has the highest transduction efficiency. The viability of the transductants of Lf155G, LfCD and LfHD were 60, 52, and 48.6%, respectively. These data suggest that orf155 may be harmful to P20H, resulting in death of part of the host cells after transfection. orf137 was cloned and over-expressed and the protein was purified for the preparation of antiserum. Using the antibody for Western hybridization, a protein of the same size as ORF137 was detected in Lf-infected and the non-infected P20H and Xc17. It is still uncertain whether Lf expresses ORF137 protein or not.
26
Hou, Hsuan-Wu, and 侯萱吾. "Expression and Functional Characterization of orf151 and orf106 of Xanthomonas fragariae Bacteriocin." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/87214634391203392156.
Анотація:
碩士國立中興大學
分子生物學研究所
102
A high-molecular weight bacteriocin produced by plant pathogen Xanthomonas fragarae (Xf) was identified in this laboratory and classify to be a phage tail-like bacteriocin. The complete nucleotide sequences of the cloned Xf bacteriocin gene cluster were determined to include 22,921 bp and predicted to have 32 putative orfs. The lysis cassette, orf74 (orf79), orf169, and the downstream orf151 and orf106 of Xf bacteriocin has been suggested to play the same role as holin (H), lysozyme (LSer), Rz and Rz1 as λ phage. Biochemical and functional characterization of the putative Rz (ORF151) and the putative Rz1 (ORF106) were investigated in this study. Polyclonal antibodies raised against ORF151 and ORF106 were prepared from immunized mice with purified recombinant proteins overexpressed in E. coli. Sucrose density gradient fractionation and Western blot analyses were applied to determine the subcellular localizations of the ORF151 and ORF106. Result showed that the overexpressed ORF151 and ORF106 could be identified in the inner and outer membrane fractions, respectively. The truncated ORF151 lacking the trans-membrane domain and fused with the CBD-Ssp intein sequence (designated as intein-ORF151ΔTMD), and the truncated ORF106 lacking the lipobox (designated as ORF106ΔLpp), were used for soluble proteins production in E. coli. Co-immunoprecipitation and gel-filtration chromatography results indicated that these two proteins forming a complex through direct interaction with each other. To elucidate the effect of lysis cassette genes on the growth of their host cells, each of the lysis cassette genes (orf74, orf79, orf169 , orf151 and orf106) and genes encoded by two (orf74 and orf169, orf151 and orf106) or four components of the cassette, were separately cloned into pBBad22k vector and individually introduced into E. coli DH5α. In addition, the 15 residues of (Gly4 Ser)3 was used as linker to fuse H and LSer protein, the constructs designated as HlL74 (or HlL79), were also used in this study. Changes of the culture turbidity were monitored after induction the expression of the recombinant lysis cassette proteins with arabinose. Results demonstrated that expression of the all four lysis genes (HLRR) or orf74 and orf169 (HL and HlL79) displayed significant lysis of E. coli hosts than the other constructs. The viable cell counts and the release of the cytoplasmic enzyme β-galactosidase correlated well with the rate turbidity reduction. The same lysis cassette genes were then introduced into M13 phage. However, after infection with the modified M13 phage, the E. coli JM103 hosts showed no significant difference in viable cell count.
27
Wang-Wei-Ching and 王薇菁. "Expression of ORF112 of Filamentous Bacteriophage." Thesis, 1993. http://ndltd.ncl.edu.tw/handle/71486491822441242755.
Анотація:
碩士國立中興大學
植物學系
81
Xanthomonas campestris 是許多種作物的病原細菌, 在 campestris種之 下包括一百三十餘個病原小種 (pathovars 簡稱 pv.)。phiLf 是一種感 染十字花科蔬菜黑腐病菌 Xanthomonas campestris pv. campestris (簡 稱 Xc) 的線狀噬菌體。此噬菌體的最有趣之處在於其能嵌入寄主的染色 體內。整個 phiLf 的基因組之核酸已由本實驗室定序完成 (一共由6, 008 個核酸所組成)。 ORF 分析結果與大腸桿菌之 Ff 噬菌體 (含 M13, f1, fd) 的前九個基因排列順序分別相互對應, 亦即為 II-X-V- VII-IX-VI II-III- VI-I。Ff 與 phiLf 在轉錄時也是依照此方向進行。 但在 phiLf基因組上, 位置與 Ff 的基因 IV 相對應之區段卻仍無法斷定 其上有何基因存在。Ff 的基因 IV 主導一個分子量為 45 kD 的蛋白質 。 此蛋白為一種 membrane-bound transport protein, 其功能為負責將 裝配完成的噬菌體釋出寄主細胞。 電腦分析的結果,發現在 phiLf 基因 組上, 相對應於 Ff 的基因 IV 之區段有一個轉錄方向相反, 而長度有 339 個核酸的 open reading frame, 簡稱 ORF112。此 ORF 距離其上 游的基因 II (ORF3 46) 及下游的基因 I (ORF440) 分別有 746 及 394 個核酸。換言之,在 phiLf 的基因 II 與基因 I 之間的 DNA, 除了 ORF112 之外, 即與 Ff的 intergenic region (IR) 區域類似。此一區段 若以 EcoRI 加 PstI 切割, 可切下一個 863 bp 的片段。而在此 863 bp 的片段上含有 ORF112 整主導區、啟動子 (promoter) 及核糖體附著 位 (ribosome binding site)。實驗利用 maxicell 及 fusion protein 的表現方式, 在E.coli證明 ORF112 是一個有表現的基因, 其可製造出一 個 13 kD 的蛋白質。其次, 利用一個具有啟動子選殖 (promoter proving vector) 功能的泛寄主 (broad host range) 載體證實, 此 ORF112 上游 242bp 的 DNA 片段內含有啟動子 (promoter) 的功能。 ORF112 所製出 13 kD 的蛋白產物有何功能尚不確定。此外, 也以 maxicell 表現, 觀察到 phiLf 基因 V 所主導出的 11 kD 的單股 DNA 附著蛋白 (single-stranded DNA binding protein)。在 maxicell 表現 的同時, 也發現有一個 12 kD 蛋白質產物,懷疑其係由 phiLf 基因 X 所 主導之蛋白產物。
28
Huang, Yu-Liang, and 黃有良. "Immuno-protection of porcine circovirus type 2 ORF1 and ORF2 recombinant protein." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/12062489232254298987.
Анотація:
碩士國立屏東科技大學
獸醫學系
92
Porcine circovirus type 2 (PCV2) has been reported to be associated with the development of porcine multisystemic wasting syndrome (PMWS), which often results in the death of pigs due to secondary infection. The genome of PCV2 might contain eleven open reading frames (ORFs). ORF 1 and ORF 2 encode the replication-associated proteins and capsid protein, respectively. In this study, the immunogenicity of E. coli-expressed PCV2 ORF 1 and PCV2 ORF 2 and baculovirus-expressed PCV2 ORF 2 were compared in mice and pigs. In addition, ORF 2 gene with Pseudomonas aeruginosa exotoxin A (expressed as PE407 and PE532) gene added at 5’ terminal, and/or the KDEL amino acids gene (expressed as K) added at 3’ terminal were constructed and expressed in E. coli. A total of seven recombinant proteins including PCV2 ORF1 (21 KDa), PCV2 ORF 2 (54 KDa), PCV2 ORF2K (56 KDa), PE407-ORF2 (81 KDa), PE407-ORF2K (100 KDa), PE532-ORF2 (95 KDa) and PE532-ORF2K (97 KDa) were successfully expressed in E. coli. A recombinant protein of 34 KDa (Bac-PCV2ORF2) was also expressed in baculovirus. Antibodies against PCV2 could be detected when pigs or mice were immunized with PCV2ORF1, PCV2ORF2, PE407-ORF2K and Bac-PCV2ORF2. Among the recombinant proteins tested, Bac-PCV2ORF2 induces the best antibody titer in animals. The aforementioned recombinant proteins can be used as the components for the development of PCV2 subunit vaccine in the future.
29
Wu, Shu-En, and 吳樹恩. "The characterization of SARS-CoV encoded nonstructural protein ORF11." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/80445396193091622031.
Анотація:
碩士國立陽明大學
微生物及免疫學研究所
92
SARS-CoV is identified as the agent that causes human severe acute respiratory syndrome (SARS). It is a new member of coronaviridae family and shares the similar genome organization as that of other members. The basic order of genes for the family of coronaviridae is replicase, spike, envelope, membrane protein and nucleocapsid, from 5’ to 3’ end. All except replicase are structure genes that are essential for the maturation of virion. The replicase is considered as a nonstructural (NS) protein, which is essential for the replication of viral genome. There are at least nine additional NS proteins potentially derived from open reading frames (ORFs) that scatter among and within the aforementioned viral gene loci. They are, as predicted by bioinformatics, ORFs 3, 4, 7, 8, 9, 10, 11, 13, and 14. Of the nine ORFs, ORF3 protein is indirectly implicated to have been actually expressed in vivo by the detection of anti-ORF3 antibody in the sera of SARS patients. The status of in vivo expression and functions of the remaining 8 ORFs remain to be explored. The purpose of this thesis is to study the expression and function of ORF11. The ORF11 gene is unique in that it doesn’t have a counterpart in the Civet SARS coronavirus. Thus, it might have unique contributions to the severity of SARS-CoV infection in human. Both viral subgenomic RNAs 2.0 and 2.5 are potential templates for the translation of ORF11. The in vitro transcription-translation coupled assay using both templates have failed to ascertain the presence of ORF11 due to the obscurity caused by the presence of one background band. However, the in vivo expression of ORF11 is indirectly substantiated by the presence of anti-ORF11 antibody in one SARS patient, using GST-ORF11 recombinant protein as the antigen in a Western blot assay. The intracellular localization of ORF11 has been determined to be in the cytoplasm of Vero E6 cell transfected with an ORF11-flag expression vector. The functionality of ORF11 is evaluated through the use of a yeast two-hybrid assay with human leukocyte cDNA library. A RYBP (Ring1 and YY1 binding protein) has been identified as the binding partner of ORF11 protein. This interaction has also been verified in vitro by the binding of both proteins produced in an in vitro transcription-translation coupled system, in the presence of canine microsomal membranes. RYBP is known to be possibly involved in apoptosis, suggesting the potential function of the ORF11.
30
Harmer, Andrea Leigh. "Mutational analysis of the Rhodobacter Capsulates ORF162B gene." Thesis, 1998. http://hdl.handle.net/2429/7818.
Анотація:
There are several open reading frames (orfs) of unknown function in
the photosynthesis gene cluster of Rhodobacter capsulatus. It is possible
that these orfs, including orfl62b, have a function in bacterial photosynthesis. When R. capsulatus RNA was probed for the presence of an orfl62b transcript, a 4 kb polycistronic mRNA was detected. Strains were then created which possessed either a polar or non-polar deletion mutation of orfl62b. These mutant strains were evaluated on the basis of their light harvesting (LH) and reaction center (RC) complex levels, chromatophore
protein band intensities and their ability to grow photosynthetically. Mutants were complemented with orfl62b in trans. In a non-polar mutant orfl62b strain, decreases in LH and RC complex levels as well as
photosynthetic growth rate were restored by trans complementation.
When a polar mutation was inserted into orfl 62b such that any downstream orfs cotranscribed with 162b would no longer be expressed, a reduction in LH and RC complex levels as well as photosynthetic growth
rate was observed. As with the non-polar mutation, the LH and RC complex
levels were increased upon trans complementation with orfl62b, whereas
the wild type generation time was not restored. It is concluded that
orfl62b and at least one orf found downstream of, and cotranscribed with, orfl62b are genes that encode proteins which are involved in bacterial photosynthesis. A hypothesis that orfl62b functions in the assembly or
interaction of complexes is proposed.
31
Li, Nian Jen, and 李念臻. "Functional analysis of ORF2271/ORF2272 in Staphylococcus aureus." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/665u9q.
32
Lin, Yao-Tang, and 林曜堂. "Characterization of SARS-CoV encoded nonstructural protein ORF10." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/42463768760724588382.
Анотація:
碩士國立陽明大學
微生物及免疫學研究所
93
SARS-CoV has been identified as the agent that caused severe acute respiratory syndrome (SARS). Sequence analysis revealed that the genome organization of SARS-CoV contained replicase and five main structural proteins arranged in the same order as in all other coronaviruses. Apart from that, SARS-CoV has nine predicted open reading frames (ORFs) in the genome, ORFs 3, 4, 7, 8, 9, 10, 11, 13, 14. SARS-CoV-like viruses have been isolated from mammals in China. The most striking difference between animal and most human isolates is that the former retains a 29-nt inframe sequence, thus resulting in the fusion of ORF10 and ORF11 into a single ORF, designated as ORF10’. This additional 29-nt segment was only observed in the human isolate GZ01, a strain that originated from Guangdong. Thus, it is hypothesized that the 29-nt deletion observed in most human isolates could have allowed the virus to adapt and propagate favorably in human hosts. Following this notion, we have set out to study the functionality of ORF10. ORF10 expression was first confirmed by in vitro transcription-translation coupled assay using viral subgenomic RNAs 2.0 as the template, followed by immunoprecipitation with anti-ORF10 antibodies. ORF10 was then shown to localize to mitochondira in Vero-E6 cells by mitochondrial fractionation and immunofluorescence staining. With yeast two-hybrid assay, ORF10 was shown to interact with cellular calcium modulating ligand (CAML) through its N-terminal hydrophobic domain, and this interaction was confirmed in Vero-E6 cells by protein cross-linking followed by immunoprecipitation. Recent reports have shown that KSHV K7 protein was directed specifically to mitochondria and was anti-apoptotic through its interaction with CAML. Interestingly, K7 and ORF10 both share a consensus sequence for directing proteins to mitochondria and interact with CAML. Therefore, ORF10 might very likely be anti-apoptotic.
33
Pogoda, Frank [Verfasser]. "Induktion eines Apoptose-ähnlichen Phänotyps durch ORF21, der Thymidinkinase des Kaposi-Sarkom-assoziierten humanen Herpesvirus-8 / Frank Stefan Pogoda." 2009. http://d-nb.info/998641405/34.
34
Su, Jyun-Min, and 蘇峻民. "Functional analyses of holin (orf74) and lysin (orf169) genes of Xanthomonas fragariae bacteriocin." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/66191161620404455154.
Анотація:
碩士國立中興大學
分子生物學研究所
99
Xanthomonas fragarae (Xf) is a plant pathogenic bacterium that mainly infected strawberry and secreted a high-molecular-weight bacteriocin that classify to phage tail-like bacteriocin. In this study, the complete nucleotide sequences of Xf bacteriocin has been sequenced. The Xf bacteriocin gene cluster is composed of 22,921 bp and predicted to have 32 putative orfs which reveal high similarity to the Pantoea ananatis LMG 20103 prophage except some putative hypothetical proteins. The biological functions of the putative holin (orf74) and the putative lysozyme (orf169) were investigated in this study. The gene organization shows that orf74 is located upstream of orf169 and the translated protein has a highly charge C-terminal region. ORF74 has two predicted transmembrane domains and is classified as Class II holin. According to result of RT-PCR analyses, orf74, orf169 and the downstream orf 151 and orf 106 were transcribed as a single mRNA. This indicates that orf74, orf169, orf 151 and orf 106 of Xf bacteriocin may play the same role as holin, endolysin, Rz and Rz1 as lambda phage. When orf74 and orf169 were co-expressed in the same plasmid, cell growth or death of the E. coli host were observed. Co-expression of pLysS, which encoded T7 lysozyme, and orf74 showed the same effect but no significant effect was found when expressed with orf74 or orf169 alone. The beta-galactosidase activity in the supernatant of the pLysS-orf74-co-expressed transformant, suggesting that ORF74 formed non-specific holes in the cell membrane and the size is sufficient for beta-galactosidase to pass the membrane. Furthermore, the same morphology of lytic phage can be observed in the modified M13 phage when orf74 and orf169 were cloned into M13mp18 plasmid. Overexpression of ORF74 and ORF169 recombinant proteins in E. coli result in death or stop growing of the host. The location of the expressed ORF74 was detected in the inner membrane fraction. The oligomerization of ORF74 in cell membrane was also analyzed by native PAGE. All of the described characteristics of ORF74 is similar to lambda phage holin. ORF169 was found to locate at the host outer membrane fraction. ORF169 was predicted to have a transmembrane domain at N-terminal region and this region may also function as signal peptide. The amino acid sequence of ORF169 is similar to T4 lysozyme family which contain SAR (signal-arrest-release) domain at N-terminal region. From this point of view, ORF169 differ from lysozyme that located in the typical lysis cassette. In order to determine the biological function of Xf lysozyme, ORF146 which elimination of ORF169 N-terminal region but still retain muramidase activity region was used. ORF146 showed bactericidal activity to chloroform-treated Xanthomonas species such as X. fragarae.
35
Yu, Hiseh Wen, and 謝文毓. "Characterization, and Expression Analysis of ORF32R, ORF40L, ORF66L and ORF77L of Grouper Iridovirus." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/41917536181386128231.
Анотація:
碩士國立宜蘭大學
生物技術與動物科學系
104
Iridoviruses, belonging to the family Iridoviridae, are large, icosahedral, double-stranded DNA viruses with a viral particle diameter of 120–350 nm. Grouper iridovirus (GIV), which was isolated from Epinephelus spp. (also called groupers) in southern Taiwan, belongs to the Ranavirus genus and has caused significant economic losses in the grouper aquaculture industry. Although the GIV genome sequence is now known, molecular mechanisms underlying iridovirus pathogenicity are not well understood, mainly owing to insufficient viral genetic information. The aim of this study was to investigate the expression and subcellular localization of GIV-32R, GIV-40L, GIV-66L, and GIV-77L during GIV infection in vitro. RT-PCR analyses revealed that during GIV infection in grouper kidney cells, GIV-40L is detected as early as 3 hours post infection (hpi), and GIV-32R and GIV-40L at 9 hpi, GIV-77L was not detected at any time-point post infection. Analyses with cycloheximide (a protein synthesis inhibitor) or cytosine arabinoside (a DNA synthesis inhibitor) revealed that GIV-40L is expressed immediately after infection, whereas GIV-32R and GIV-66L are expressed at a later stage. Western blot analysis using 4 mouse polyclonal antibodies against GIV-32R, GIV-40L, GIV-66L, and GIV-77L proteins in grouper kidney cells during GIV infection detected GIV-32R, GIV-40L, and GIV-66L at 12 hpi, while GIV-77L was not detected at any time-point post infection. The localization of GIV-32R, GIV-40L and GIV-66L in GIV-infected cells was further characterized by immunofluorescence microscopy with the corresponding mouse polyclonal antibodies. In the infected cells, GIV-66L was mainly aggregated in the cytoplasm, while GIV-32R and GIV-40L were distributed in both the nucleus and cytoplasm. The results of this study will enable investigators to better understand the mechanism underlying iridovirus infection.
36
"The trans effect of non-functional L1 loci on retrotransposition of functional L1 elements." Tulane University, 2017.
37
Wu, Ting-Yun, and 吳亭昀. "Monoclonal Antibody Preparation and Cellular Distribution Analysis of Koi Herpesvirus ORF72 Protein." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/8a7b5r.
38
Tzeng, Yi-Chih, and 曾奕之. "Expression,purification and activity of the ORF2p endonuclease from TART retrotransposon of Drosophila telomere." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/29544772337613747656.
Анотація:
碩士淡江大學
生命科學研究所碩士班
96
The Drosophila melanogaster is easy to culture and propagation. In gene studies, the Drosophila melanogaster is the most common experimental animal. It has four pairs of chromosome, and have much genetic variation, so it is often used in basic research. The length of the telomere is closely correlated with aging process. We hope that by studying the Drosophila melanogaster''s telomere we can understand the evolution of telomere and the aging process in general. Drosophila melanogaster telomere is composed of three non-LTR retrotransposons, HeT-A, TART and TAHRE, which are repeated sequences, and TART can translate two kinds of proteins, ORF1p and ORF2p. TART ORF1p has already proved its function in shifting the telomere-associated structures only when coexpressed with HeT-A ORF1p. And ORF2p might have endonuclease (EN) and reverse transcriptase (RT) activity. In this study, we investigate TART-EN expression and purification. We have already over-expressed Endop, point-mutant Endop, and two truncated mutant Endop in suitable E.coli. We Confirmed Endop have endonuclease activity by cutting supercoiled form DNA into open circular form DNA. We can also keep endonuclease activity of purified Endop effectively by storing it in 30％ glycerol and -20℃. After the enzyme activity affirmation, we can study the cutting hot spots of endonuclease and identify their sequences further in order to understand the function of TART.
39
Wang, Shie Shan, and 王世憲. "Transcriptional regulation of the ORF61, ORF60 and ORF46 genes of Kaposi's sarcoma-associated herpesvirus." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/95933108931858843276.
Анотація:
博士長庚大學
臨床醫學研究所
100
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus closely associated with all forms of Kaposi’s sarcoma (KS) as well as lymophoproliferative disorders, including primary effusion lymphoma and multicentric Castleman’s disease. An important step for viral propagation and pathogenesis is the switch from latency to the lytic cycle. The viral gene expression in the lytic cycle program is controlled tightly and expressed in an orderly fashion. In the study, we characterized the transcriptional regulation of three viral genes ORF61, ORF60 and ORF46 during the lytic induction. The ORF61 and ORF60 genes encode the large and small subunits of ribonucleotide reductase, respectively, while the ORF46 gene encodes the uracil DNA glycosylase involved in DNA repair. Our findings reveal that ORF50 protein, a latent-lytic switch transactivator, activates the transcription of these three early-lytic genes through different mechanisms. Activation of the ORF61 promoter by ORF50 protein is dependent on an intact RBP-Jκ-binding site within the identified responsive element and the expression of RBP-Jκ protein in cells. However, the critical element of the ORF60 promoter in response to ORF50 protein is located to a 40-bp region that contains YY1 and Sp1/Sp3 binding sites. Binding of YY1, Sp1/Sp3 proteins to this region may contribute to repression or activation of the ORF60 promoter. Although ORF50 protein did not directly bind to the ORF61 and ORF60 promoters in vitro, we showed the association of ORF50 protein with these two promoters in vivo. For the regulation of the ORF46 gene, three consensus RBP-Jκ binding sites in the ORF46 promoter are critical for the binding of RBP-Jκ protein and conferring the ORF50 responsiveness. Intriguingly, a negative regulatory region is present in the ORF46 promoter, which comprises multiple Sp1-binding sites. Transferring these Sp1-binding sequences to an ORF50-responsive promoter suppressed the ORF50 responsiveness. Particularly, sodium butyrate, a pleiotropic inducing agent for the KSHV lytic cycle, was able to relieve the negative regulation of the ORF46 promoter in the latently KSHV-infected cells. Understanding the diverse regulatory mechanisms of these viral lytic genes provides further insights into the control program of the lytic cycle.
40
Grillhösl, Christian [Verfasser]. "Mutagenese von Herpesvirus Saimiri Bacmiden und nähere Charakterisierung einer orf51-Insertionsmutante / vorgelegt von Christian Grillhösl." 2010. http://d-nb.info/1008539902/34.
41
Kawach, Oliver [Verfasser]. "Charakterisierung des Slr1649 aus Synechocystis sp. PCC 6803, ein Homolog zu Orf222 aus Guillardia theta / vorgelegt von Oliver Kawach." 2007. http://d-nb.info/983040494/34.
42
Gan, Ya-Wen, and 甘雅文. "Identification and characterization of ORF162-MPT, a putative gene on the male and female sex chromosomes, from Marchantia polymorpha in Taiwan." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/67490310395428879420.
Анотація:
碩士國立臺灣大學
植物學研究所
91
Marchantia polymorpha L. is a liverwort with heteromorphic sex chromosomes. Its gametophyte is therefore dioecious. The sex chromosome in a female gametophyte is defined as the X chromosome and in a male gametophyte is defined as the Y chromosome. Previous studies demonstrated that the M. polymorpha genome has many unique repetitive sequences in the Y chromosome containing an open reading frame, ORF162, which is specifically expressed in the male gametophores. However, in this study, we demonstrated that there are several homologues of ORF162 in the female genome of the same species in Taiwan. Further analyses suggested these homologues were very likely located on the X chromosome. In this study, by PCR, cloning and sequence analysis, we found two different copies in the male genome in Taiwanese M. polymorpha, named as ORF162-MPT1 and ORF162-MPT2. We also found twelve different copies in the female genome, named as ORF162-MPT3, ORF162-MPT4, ORF162-MPT5, ORF162-MPT6, ORF162-MPT7, ORF162-MPT8, ORF162-MPT9, ORF162-MPT10, ORF162-MPT11, ORF162-MPT12, ORF162-MPT13 and ORF162-MPT14. The former three copies were identified from the female genomic DNA, and the latter nine were identified from its cDNA. Analysis of the promoter sequences demonstrated remarkable differences among the fourteen copies and the published ORF162, including 14~27 substitutions and insertions or deletions. In the open reading frame, there are also 4~59 substitutions and insertions or deletions. The RT-PCR results showed that ORF162-MPT1/2 are expressed at all developmental stages of male gametophores, and a weak signal was detected from ORF162-MPT6/7/8/9/10/11/12/13/14 at early stages of female gametophores. Since the cDNA sequences of ORF162-MPT6/7/8/9/10/11/12/13/14 contain several stop codons, we expect that there will not be translated products. In comparison, there are also several stop codons in the sequences of ORF162-MPT3/5, thus ORF162-MPT3/5/6/7/8/9/10/11/12/13/14 are all likely to be pseudogenes. The only exception is ORF162-MPT4, which has an identical open reading frame with ORF162-MPT1/2, but no expression was detected in our analysis, and awaits for further studies.
43
Tanner, Tricia Lynn. "Analysis of the function of two varicella-zoster virus proteins involved in gene regulation IE63 and ORF29 /." 2007. http://proquest.umi.com/pqdweb?did=1240709031&sid=6&Fmt=2&clientId=39334&RQT=309&VName=PQD.
Анотація:
Thesis (Ph.D.)--State University of New York at Buffalo, 2007.Title from PDF title page (viewed on July 18, 2007) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Ruyechan, William T. Includes bibliographical references.
44
Shen, Shu-Yu, and 沈姝瑜. "Genetic Variation of ORF1b, 5 and 7 of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in Taiwan." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/05889507640379427059.
Анотація:
碩士國立嘉義大學
獸醫學系研究所
95
To investigate the genetic variation and serological prevalence of porcine reproductive and respiratory syndrome virus (PRRSV) in Taiwan, 630 blood samples were collected from 21 pig farms from July to August in 2005. Serological tests of PRRSV antibodies were performed using IDEXX HerdChek PRRS ELISA Kits. Virus isolation was confirmed by indirect immunofluorescent antibody test using SDOW17 monoclonal antibody. Genetic variation of partial ORF1b, entire ORF 5 and 7 performed by RT-PCR and sequencing was analyzed by DNASTAR software. Serological results showed all farms were PRRS positive with prevalence of 40.48 % (85/210), 83.81 % (176/210) and 95.24 % (200/210) at ages of 6, 10, and 16 week old pigs respectively. Totally, forty-two isolates collected from 6 week old pig sera were confirmed to be all belong to the North American-type. Compared with VR2332, genetic divergence of ORF 1b, 5 and 7 was 0.7 % (0 %) ~ 8 % (11.4 %), 11.6 % (12.4 %) ~ 14.8 % (17.4 %), and 5.4 % (4.8 %) ~ 9.1% (7.3 %) respectively at nucleotide (amino acid) level. While compared with MD001, the divergence of ORF5 and 7 was 1.8 % (1.5 %) ~ 11.9 % (11.9 %) and 0.3 % (0.8 %) ~ 8.9 % (7.3 %) respectively. Also, phylogenetic analyses of ORF5 of these 42 isolates indicated that Taiwan PRRSVs formed a clade distinct from reference North American PRRSV (VR2332). The clade was at least divided into 5 major genetic clusters randomly distributing in Taiwan pig farms. These isolates in the Cluster 1 dominated in Taiwan pig farms during the time studied, while the other isolates separately evolved into the cluster 2 to 5 in different geographical areas. In conclusion, PRRSV was widely prevailed in Taiwan pig farms and revealed distinct genetic variation.
45
Chen, Yu-San, and 陳裕森. "Cloning, Expression, and Antigenesity Analysis of the PCV2 Coat Protein Gene (ORF2)." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/99558729710906776501.
Анотація:
碩士國立宜蘭大學
生物技術研究所碩士班
94
Porcine circovirus type 2 belongs to the Circoviridae family. PCV2 is a small, nonenveloped virus with a single-stranded circular DNA genome of about 1.76 kb. The open reading frame ORF2 of the PCV2 genome encodes the immunogenic structural capsid protein. The expression for this immunogenic protein using full length ORF2 DNA was difficult to obtain in large quantity in prokaryotic expression system. Therefore, an alternative approach was to divide ORF2 DNA into three segments for expression, F1, F2, and F3. It was discovered that expression in E.coli was obtained in segments other than F1, which contained the N-terminal sequences. When F1+F2 (named F6) and F2+F3 (named F5) fragments were cloned and expressed, F6, similar to F1, was not expressed due to the N-terminal sequences. On the other hand, F5 was still expressed in large quantity. When dividing the full length ORF2 into half, F4 with C-terminal sequences was again expressed in large quantity as predicted. Analysis on PCV2 ORF2 full length sequence showed that within F1, there were 21 arginine codons, far more than the 9 arginine codons in F2 and F3 combined. Therefore, five additional constructs with the first 9, 21, 36, 48, 111 nucleotides missing of ORF2 (named F10, F22, F37, F49, and F112 respectively) were cloned and subsequently expressed. It was discovered that PCV2 ORF2 recombinant protein expression increased as arginine codons decreased. When immunizing rat with purified F5 recombinant protein, high antibody titer against PCV2 was induced, as confirmed by the IPMA method in PCV2 infected PK-15 cells. Currently, since PCV2 vaccine is not commercially available, this research project was also aimed for the development of a PCV2 subunit vaccine. Rats were used as an experimental animal model to evaluate the various recombinant proteins, F2, F3, F5, and F49, for their abilities to induce antibodies against PCV2. In addition, F5 and F49 recombinant proteins, in conjunction with ORF1 and/or ORF2 recombinant eukaryotic expression plasmids, were injected in rats to evaluate their abilities to induce antibodies against PCV2. Other than using IPMA for the detection of neutralizing antibodies, I used ELISA to measure the levels of antibodies induced in the collected rat serum of different experimental groups. The result of ELISA indicated that F2 recombinant protein induced the highest level of IgG antibodies in rat. As for neutralizing antibodies, neither the recombinant proteins nor when used in conjunction with recombinant eukaryotic expression plasmids were induced when using rat as an animal model.
46
Li, ChiWei, and 李誌偉. "A novel mechanism to regulate IS1550 putative transposase production by ORF1 protein." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/22403527173198471760.
Анотація:
碩士國立陽明大學
醫學生物技術研究所
90
We have previously identified a transposable element, IS1550 from Mycoplasma fermentans. This element encodes two open reading frames, ORF1 and ORF2. Two proteins were detected with the molecular weight of 16 kDa and 50 kDa, respectively, when expression of the IS1550 genes was examined in E. coli T7 expression system. The 16-kDa protein was determined to be encoded by ORF1, whereas the 50-kDa protein was produced by forming a fusion of ORF1 and ORF2, ORF12’, through an efficient —1 translational frameshift mechanism. Our previous studies have confirmed a framseshift signal in IS1550. It contains an essential shifty site AAAAAAG (A6G) which is located near the 3’ end of ORF1, and an enhancing element, the internal inverted repeat (IIR) to form a RNA stem-loop structure, which is located 5 bp downstream of the essential shifty site. In this study, we examined the effect of ORF1 protein on the -1 translational frameshift product by using our previously established dual-reporter gene (lacZ and luc) system. The results showed that ORF1 protein could repress the -1 frameshift product both in vivo and in vitro with an inhibitory effect of about 60 % and 80 %, respectively. We also showed the ORF1 protein could bind to the frameshift signal DNA region. Furthermore, we proved that a truncated mRNA was formed when the ORF1 protein was present. Thus, we proposed that the repression of ORF1 protein on the -1 frameshift product was possibly caused by ORF1 protein binding to the frameshift signal DNA region, blocking the RNA polymerase proceeding, and resulting in the decreasing of the fusion protein (a putative transposase) production.
47
Chiu, Jui-Yu, and 邱芮瑜. "Molecular Cloning of Recombinant ORF2 and Infectious Clone of Porcine Circovirus Type II." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/77399778243414145956.
Анотація:
碩士國立臺灣大學
獸醫學研究所
94
Post-weaning multisystemic wasting syndrome (PMWS) becomes a major problem in many pig-producing countries throughout the world. Porcine circovirus type 2 (PCV2) has been identified as an essential component of PMWS. Approximately 83.5% of the pigs in Taiwan were seropositive to PCV2. The open reading frame 2 (ORF2) encoding the major capsid protein with epitopes to induce neutralizing antibodies is the potential target gene for vaccine development in against PCV2 infection. However the difficulty to effectively express ORF2 seems to limit the development of PCV2 vaccine. In this study, we expressed full-length, N-terminal, middle and C-terminal ORF2 covering high antigenic epitopes by E. coli. Only ORF2M and ORF2C could be overexpressed and purified. The yield of purified ORF2M or ORF2C was about 20 mg/L. Mouse antisera and monoclonal antibodies produced by ORF2M or ORF2C recognized PCV2 under Western blotting and immunofluorescence assay. The neutralization titer of ORF2M-induced serum was about 32X, suggesting that ORF2M is a potential candidate of PCV2 subunit vaccine. We also tried but failed to express ORF2 in protozoa expression system. It is hard to isolate PCV2 from field sample and to propagate it in cell culture. We constructed the infectious clone of PCV2 to generate a biologically pure and homogeneous infectious virus stock to study viral behaviors. In PCV2-infected PK-15 cells ORF2’ was detected at 12 hours post inoculation (h.p.i.) by Western blotting, but ORF1 was detected at 24 h.p.i.. The expression of ORF2 seemed to be earlier than that of ORF1. On the other hand, overexpression of ORF1, ORF2 or ORF3 had no effect on viral propagation. However, overexpression of ORF2 advanced, but ORF1 delayed the appearanceof cytopathic effect (CPE). The ORF1-delayed CPE increased the life span of PCV2-infected cells and then viral propagation. The results provide useful information for efficiently producing PCV2 virus stock in PK-15 cell model.
48
"Analysis Of The Line-1 Orf2 Protein Using An Evolutionarily-informed Genetic Approach." Tulane University Digital Library, 2016.
Анотація:
Long Interspersed Element 1(LINE1 or L1), along with the parasitic Short Interspersed Element (SINE), are the only two currently active retrotransposons in the human genome. These genetic elements are capable of causing DNA damage through their mobilization and the enzymatic activities of the L1-encoded Open Reading Frame 2 (ORF2) protein (ORF2p). The L1 ORF2p contains four annotated domains important to retrotransposition. These include the endonuclease (EN) and reverse transcriptase (RT) domains, as well as the Z domain and the cysteine rich domain (Cys). While much is known about the enzymatic activities of the EN and RT domains, and individual amino acids important to retrotransposition have been identified in the Z and Cys domains, more than 50% of the 150kDa ORF2p amino acid sequence serves no known function. I hypothesized that the unannotated areas of the ORF2p, specifically the sequence C-terminal to the EN domain and N-terminal to the Z domain as well as the sequence C-terminal to the Cys domain, contained amino acids important to the retrotransposition process. Specifically, I hypothesized that they contained amino acids involved in the activity of the EN domain, RT domain, or interaction with the L1 ORF1p. To test this hypothesis, I developed a technique termed Bipartile Alu Retrotransposition (BAR) that utilized EN and RT-containing ORF2 fragments combined with the Alu retrotransposition reporter construct. This system allowed me to define a new ORF2p region, which I termed Cryptic. This region contains an essential WD pair important for cDNA syntheses by the ORF2p. This WD pair is also involved in the regulation of the EN domain activity. I also discovered a putative PCNA binding domain that is essential for retrotransposition. Additionally, I identified the region in Cryptic that is involved in the previously reported differences in subcellular localization and cytotoxic potential of EN-containing ORF2p fragments. Using truncated ORF2p fragments generated for use in BAR, I also made several discoveries concerning the extreme C-terminus of the ORF2p. Notably, I discovered that the extreme C-terminal end of the ORF2p is dispensable for retrotransposition. I also identified a human-specific Y residue that is important for Alu retrotransposition driven by the ORF2p.1
Claiborne Magnant Christian
49
Shyu, Duan-Liang, and 徐敦良. "Antigenic Analysis of ORF1, OR2 and ORF3 of Porcine Circovirus Type 2 Expressed Viral Peptides." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/76665853457948582976.
Анотація:
碩士國立臺灣大學
獸醫學研究所
92
Porcine circovirus type 2 (PCV2)cause PMWS in weaning piglets, and was found highly infectious in pug farms. Polymerase chain reactions (PCR) was used to amplify ORF1, ORF2 and ORf3 of PCV2 from infected lymph node emulsion by using specific primers. E. coli expression vector pRSET A and DNA vaccine vactor pcDNA3 were used as the plasmid to clone the 3 ORFs of PCV2, respectively. Recombinant proteins of ORF1, ORF2 and ORF3 of PCV2 were expressed in E. coli were analyzed by SDS-PAGE and western blotting. The DNA vector vaccine were used to immulized rabbits by intramuscular injection. MagicShuttle protein(MGT), a kind of DNA binding protein, were also used to increase the efficiency of DNA transfection. PCV2 antibody and viral neutralizing antibody of immunized rabbits serum were tested by indirect immunofluorescent assay (IFA). The result of E. coli expression showed that the soluble form of ORF1 and ORF2 recombinant proteins, and the inclusion form of ORF3 recombinant protein could be recognized by PCV2 polyclonal antibody but not by PCV2 ORF2 monoclonal antibody. The size of ORF1 and ORF2 recombinant proteins were correct, but that of ORF3 was bigger than the expected size. Sera from DNA vector vaccine immunized rabbits showed that ORF2 and ORF3 immunized sera had PCV2 antibody titers, but not found in ORF1 immunized sera. Viral neutralizing antibody activities were only detected in sera collected in ORF2 and PCV2 infected lymoh node emulsion immunized sera：the neutralizing activities was only found in using low amount of PCV2 (10 TCID50).
50
Li, Yi-Ija, and 李宜佳. "Functional analysis of the capping enzyme and helicase like domain of the ORF1 of bamboo mosaic potexvirus." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/68667673059521006118.
Анотація:
博士國立中興大學
農業生物科技學研究所
90
Bamboo mosaic virus (BaMV), a member of the potexvirus group, infects primarily members of the Bambusoideae. Open reading frame 1 (ORF1) of BaMV encodes a 155-kDa polypeptide that has long been postulated to be a replicase involved in the replication and formation of the cap structure at the 5¢ end of the viral genome. Analysis of the amino acid sequences reveals several conserved motifs suggesting that three functional domains, methyltransferase, helicase, and RNA dependent RNA polymerase on the order of N to C termini, are organized in the 155-kDa polypeptide. In our previous study, we have demonstrated the RdRp activity of BaMV. In this study, we went on exploring the activities associated with the putative methyltransferase and helicase domains. We used the putative methyltransferase domain expressed in yeast to investigate the capping mechanism of BaMV. In the regard of methyltransferase domain, both methyltransferase and guanylyltransferase activities were identified. The methylyltransferase activity transfers the methyl group from AdoMet to GTP, subsequently, the guanylyltransferase activity catalyzes the mGTP to mGMP and forms the intermediate mGMP-enzyme complex. Therefore, the putative methyltransferase domain is indeed a RNA capping enzyme. The capping activity of BaMV is AdoMet dependent and follows a similar role as the cap formation of Semiliki Forest virus that differs from eukaryotic mRNA cap formation. Regarding the putative helicase domain, we demonstrated that it could hydrolyze nucleoside triphosphate to nucleoside diphosphate in the presence of Mg2+. In addition to nucleoside triphosphatase, the helicase-like domain also possesses the RNA-5¢ triphosphatase activity. The g-phosphate of nascent RNA is removed by helicase-like domain, which facilitates the capping enzyme forming the cap structure mGpppG. This is the first report of the helicase domain of a plant virus involved in cap formation. Further, we found that the helicase Walker A motif (GxGKS) mutation abolishes not only nucleoside triphosphatase but also RNA 5¢-triphosphatase activity. This result suggests that the catalytic sites of nucleoside triphosphatase and RNA 5¢-triphosphatase are overlapping. At the last, we looked into the duplex unwinding activity of the helicase-like domain of BaMV. To date only few cases of superfamily 1 viral helicase, to whom the BaMV helicase belongs, have been demonstrated its duplex unwinding activity such as nsP2 of Semiliki Forest virus (SFV). We used duplex-RNA with 3¢ or 5¢ single strand tails as substrates. Marginal activity was found when using a 5 ¢-to-3¢ polarity RNA as substrate. However, no significant differences between the wild type and Walker A motif or motif V mutants. From these results, we assume that the putative helicase domain might need cellular factors to help processing the duplex unwinding activity, or follow the role of duplex unwinding in a different way from others RNA virus in virus replication.