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1
Liu, Yuan Yi. "A study of a satellite RNA from arabis mosaic nepovirus." Thesis, University of Oxford, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.335830.
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Lane, Claire Louise. "Structural studies of protein-RNA interactions in Satellite Tobacco Necrosis Virus (STNV) capsids." Thesis, University of Leeds, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485634.
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The protein capsid of an icosahedral virus can be considered a natural scaffold system for the RNA genome which it packages. It is possible to manipulate the nature of this packaging to gain structural information regarding the encapsidated molecule. Satellite Tobacco Necrosis Virus (STNV), is a small T=1 plant satellite virus. It is possible to express and purify intact virus like particles recombinantly. The refined structure of these particles has been solved crystallographically to a resolution of 1.4 A, however no RNA density was visible within this structure. This structure has been re-solved within this thesis to low resolution, 6 A, revealing distinct density in addition to that of protein, attributable to RNA. The recombinant capsid is packaging non-self RNA and doing so with a degree of icosahedral order. The recognition sequence allowing STNV to package its own RNA genome specifically has not been identified. In an effort to achieve this, the process of SELEX has been implemented. Several sequences matching those of the STNV-1 genome have been identified indicating that this procedure has succeeded in selecting RNA sequences which mimic those expected to bind in nature. These matching sequences span the length of the genome, indicating that packaging may not be a result of binding to a single site on the RNA. As the STNV capsid is already designed to function as a scaffold for genomic RNA, it provides an obvious start point for the design of a more complex scaffold. The development of the STNV capsid to be of use as a macromolecular scaffold system is addressed within this thesis. Three hurdles associated with such a system have been identified and addressed by designing mutant STNV coat protein monomers. Upon expression and purifiCation differences in the biochemical properties between the wild type protein and the mutants have been identified.
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Ge, Xin. "Characterization of the Genome of Maize Chlorotic Dwarf Virus and an Associated Satellite RNA." The Ohio State University, 1990. http://rave.ohiolink.edu/etdc/view?acc_num=osu1391600232.
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Lamprecht, Renate Luise. "Molecular characterisation of South African isolates of grapevine fanleaf virus and a new, associated satellite RNA." Thesis, Stellenbosch : Stellenbosch University, 2013. http://hdl.handle.net/10019.1/85600.
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Thesis (PhD)--Stellenbosch University, 2013.ENGLISH ABSTRACT: Grapevine fanleaf virus (GFLV) is one of the oldest, most widespread and devastating viruses infecting grapevine, and occurs globally where Vitis vinifera is grown. In South Africa (SA) GFLV is predominant in the Breede River Valley, one of the highest wine producing regions in SA. To date, only three GFLV isolates have been completely sequenced internationally, and limited sequence information is available for SA GFLV isolates. In this study, the first full-length GFLV genome sequence from a South African isolate, GFLV-SAPCS3, was determined. Full-length sequences were used for phylogenetic analysis and revealed that the SA isolates are separate from other sequenced GFLV isolates. Full-length sequences were also used to investigate putative intra- and interspecies recombination events involving GFLV-SAPCS3 RNA1 and RNA2 between GFLV and Arabis mosaic virus (ArMV) isolates. Using two different recombination analysis software packages, the most notable of the putative recombination events involving GFLV-SAPCS3 indicated that the GFLV-SAPCS3 RNA2 5’ UTR might have evolved from an interspecies recombination event between GFLVF13- type and ArMV Ta-type isolates. The presence of satellite RNAs (satRNA) associated with South African GFLV isolates was also investigated. In a collaborative study (see Chapter 4 for details), more than a 100 GFLV- infected grapevine plants were screened for satRNAs. SatRNAs were present in only two plants, containing isolates GFLV-SACH44 and GFLV-SACH47. The full-length nucleotide sequences of the GFLV-SACH44 genomic RNAs 1 and 2, and the associated satRNA were determined. No significant sequence variation could be detected between the GFLV isolates that had the presence of a satRNA and those that had not. The GFLV-SACH44 RNA2 5’ UTR also had the same conserved sequence that was found in GFLVSAPCS3, which suggests that GFLV-SACH44, like GFLV-SAPCS3, may have arisen from a common ancestor, which may have originated from an interspecies recombination event. The GFLV-SACH44 satRNA was found to be more closely related to the ArMV large satRNA than to the satRNA associated with GFLV-F13. A full-length cDNA clone of GFLV-SACH44 satRNA was constructed and its replication and systemic spread in herbaceous hosts, when mechanically co-inoculated with two GFLV isolates as helper viruses, was demonstrated. Replication of the GFLV-SACH44 satRNA cDNA clone was however abolished when co-inoculated with an ArMV helper virus, even though it is phylogenetically more closely related to ArMV satRNAs. The full-length satRNA clones were modified to be used as vectors for expression and/or silencing of foreign genes, by inserting the green fluorescence protein (GFP) full-length or partial sequences downstream of the open reading frame of the satRNA. These constructs were cloned into a binary vector to allow for agro-infiltration into plants. Full-length cDNA clones of GFLV-SAPCS3 RNA1 and RNA2 were constructed to be used in conjunction with modified GFLV-SACH44 satRNA full-length clones. The full length GFLV-SAPCS3 RNA1 and RNA2 clones were however not infectious in Nicotiana benthamiana after agro-infiltration and therefore the evaluation of the modified satRNA expression and silencing constructs had to be aborted. Attempts to understand this failure revealed that, among other point mutations, four frameshifts had occurred in the RNA1 full-length clone, rendering the transcripts untranslatable, and hence noninfectious. Strategies to correct the mutations are discussed. Once these mutations have been corrected this study can continue in evaluating the use of the satRNA component for expression and silencing analysis.
AFRIKAANSE OPSOMMING: Grapevine fanleaf virus (GFLV) is een van die oudste, mees wydverspreide en mees verwoestende virusse wat wingerd affekteer en word wêreldwyd waar Vitis vinifera verbou word, gevind. In Suid Afrika (SA) kom GFLV veral in die Breederivier vallei, een van die mees produktiewe wyn-produserende areas in SA, voor. Tot dusver is daar net drie GFLV isolate waarvan die volledige nukleïensuurvolgorde internasionaal bepaal is. Die nukleïensuurvolgorde informasie vir SA GFLV isolate is redelik beperk. In hierdie studie was die eerste volledige nukleïensuurvolgorde van ‘n SA GFLV isolaat, GFLVSAPCS3, bepaal. Die volledige nukleïensuurvolgordes was vir filogenetiese analise gebruik, en vermeende intra- en interspesie rekombinasie gebeurtenisse, wat GFLVSAPCS3 RNA1 en RNA2 betrek, tussen GFLV en Arabis mosaic virus (ArMV) isolate was ondersoek. Twee verskillende rekombinasie-analise sagteware programme was gebruik en die noemenswaardigste van die vermeende rekombinasie gebeurtenisse, met betrekking tot GFLV-SAPCS3, het aangedui dat die GFLV-SAPCS3 RNA2 5’ ontransleerde streek (UTR) waarskynlik van ‘n interspesie rekombinasie gebeurtenis tussen ‘n GFLV-F13-tipe en ‘n ArMV-Ta-tipe isolaat ontwikkel het. Die teenwoordigheid van satelliet RNAs (satRNAs), wat met SA GFLV isolate geassosieer is, was ook ondersoek. Meer as ‘n 100 GFLV ge-infekteerde wingerd plante was in ‘n samewerkingsprojek (sien Hoofstuk 4 vir besonderhede) getoets vir die teenwoordigheid van satRNAs. SatRNAs was net in twee plante teenwoordig, in isolate GFLV-SACH44 en GFLV-SACH47. Die vollengte nukleïensuurvolgordes van GFLVSACH44 RNA1, RNA2 en geassosieerde satRNA was bepaal. Geen beduidende volgorde variasie tussen die GFLV isolate wat satRNAs bevat het, en die GFLV isolate sonder satRNA was waargeneem nie. Die GFLV-SACH44 RNA2 5’ UTR het ook die gekonserveerde volgorde, wat in GFLV-SAPCS3 teenwoordig was, gehad en dit dui daarop dat GFLV-SACH44, soos GFLV-SAPCS3, van dieselfde stamvader, wat tydens ‘n vorige rekombinasie gebeurtenis ontstaan het, mag ontwikkel het. Die GFLVSACH44 satRNA was meer naverwant aan die ArMV satRNAs as aan die satRNA, wat met GFLV-F13. ‘n Vollengte cDNA kloon van die GFLV-SACH44 satRNA was ontwikkel en die replisering en sistemiese verspreiding in sagte plante, nadat dit met twee GFLV isolate as helper virusse saam ge-inokuleer was, was gedemonstreer. Replisering van die GFLV-SACH44 satRNA cDNA kloon was egter ontwrig toe dit saam met ‘n ArMV helper virus saam ge-inokuleer was, al is dit filogeneties meer verwant aan ArMV satRNAs. Die vol-lengte satRNA klone was gemodifiseer om as vektore vir uitdrukking en/of uitdowing van transgene te dien, deur om vol-lengte of gedeeltelike groen fluoressensie proteïen (GFP) nukleïensuurvolgordes aan die einde van die satRNA leesraam te koppel. Hierdie konstrukte was in ‘n binêre vektor gekloon om agroinfiltrasie in plante toe te laat. Vol-lengte cDNA klone van GFLV-SAPCS3 RNA1 en RNA2 was ontwikkel om in samewerking met die gemodifiseerde GFLV-SACH44 satRNA konstrukte gebruik te word. Die vol-lengte GFLV-SAPCS3 RNA1 en RNA2 klone het egter nie in Nicotiana benthamiana gerepliseer na agro-infiltrasie nie, daarom was die evaluasie van die gemodifiseerde satRNA konstrukte gestaak. Pogings om die mislukking te verstaan, het daarop gewys dat, behalwe punt mutasies, vier leesraam versteurings in die RNA1 vollengte kloon voorgekom het, wat ontransleerbare transkripte, en dus nie-repliserende konstrukte tot gevolg gehad het. Strategieë om die mutasies te korrigeer is bespreek. Sodra die mutasies gekorrigeer is, kan die studie voortgaan om te evalueer of die satRNA komponent vir uitdrukking en uitdowing analise gebruik kan word.
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Fisher, John R. "Partial characterization of a Cucumber Mosaic Virus Isolate, and its associated Satellite RNA, from Ajuga Reptans /." The Ohio State University, 1999. http://rave.ohiolink.edu/etdc/view?acc_num=osu1488191124570172.
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Ferri, Frederica. "Role of non-coding murine centromeric RNA in the assembly and function of centromere." Paris 7, 2009. http://www.theses.fr/2009PA077129.
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Le centromère est une structure chromosomique spécialisée comportant un type unique de chromatine, nécessaire pour la ségrégation correcte des chromosomes pendant la mitose. L'assemblage et la fonction des centromères exigent l'association dynamique d'une grande variété de protéines au cours du cycle cellulaire. Par ailleurs, des données de plus en plus abondantes montrent que les ARNs non-codant sont des composants intégraux de la chromatine et contribuent à son organisation structurale. Les ARNs centromériques que nous avons récemment caractérisés sont transcrits à partir des répétitions des centromères murins. Leur accumulation sur les chromocentres nous a permis de les proposer comme facteur d'assemblage ou de maintien des protéines centromériques, nécessaires à la ségrégation correcte des chromosomes. Nos résultats montrent que les ARNs centromériques s'accumulent principalement en phase G2/M, au cours de laquelle est assemblé le complexe passager du centromère. Ces ARNs sont des composants intégrales de la chromatine centromérique: ils interagissent avec CENP-A et font partie du complexe passager au début de la mitose. De plus, l'association des protéines passagères Aurora B et Survivin au niveau de la chromatine centromérique et l'activité de la kinase Aurora B dépendent d'un ARN. Enfin, des tests fonctionnels indiquent que l'activité enzymatique de Aurora B dépend de la présence des ARNs centromériques. Ces données suggèrent que les transcrits centromériques pourraient jouer un rôle important dans l'assemblage et la fonction du centromère en recrutant et/ou stabilisant Aurora B au niveau de la chromatine associée à CENP-A et en réglant son activité enzymatiqueThe centromeres of eukaryotic chromosomes are genomic regions featuring a unique, specific chromatin architecture that is necessary for proper chromosome segregation during mitosis. While there is evidence that the assembly and highly specialized fonction of centromeric chromatin domains require the dynamic association of a large variety of proteins during various stages of the cell-cycle, it is now becoming clear that non-coding RNA are integral components of chromatin and contribute to its structural organization. We recently described new RNA, transcribed from murine centromeric minor satellite repeats, which localize on chromocenters. We now considered the implication of these RNA in recruiting and/or stabilizing rfconucleoprotein complexes located at centromeric regions and their functional dynamics during cell cycle. We reported that levels of minor satellite RNA vary during cell cycle progression, accumulating in G2/M phase, concomitantly with the localization of the chromosomal passenger complex to the centromere. We showed that minor satellite RNA are components of CENP-A chromatin and interact with proteins of the chromosomal passenger complex at the onset of mitosis. Both interactions between endogenous passenger proteins Aurora B and Survivin within centromeric chromatin and Aurora B kinase activity are sensitive to RNaseA. More importantly, this Aurora B kinase activity can be specifically rescued by restitution of minor satellite RNA. Together, our data provide new insights into thé implication of minor satellite RNA in the establishment of a functional centromere, by regulating Aurora B association with CENP-A-associated domains and enzymatic function
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Jeffries, Alex Craig. "The study at the molecular level of the New Zealand isolate of Lucerne transient streak sobemovirus and its satellite RNA." Title page, contents and summary only, 1993. http://web4.library.adelaide.edu.au/theses/09PH/09phj47.pdf.
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Meyer, Michel. "Contribution a l'etude de la structure et de l'organisation du rna-2 (isolat s) et des rna satellites de 5 isolats du tbrv." Université Louis Pasteur (Strasbourg) (1971-2008), 1986. http://www.theses.fr/1986STR13091.
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La sequence de l'arn-2 de l'isolat s comporte une seule phase de lecture ouverte qui correspond a une proteine de p. M. 150 k. La proteine de la coque est localisee dans la region c terminale de la proteine 150 k. Une proteine "de diffusion" se situerait en amont de la proteine de la capside. Les sequences des arn satellites issus d'isolats appartenant aux serotypes s (s et l) et g (g, e et c) comportent toutes une seule phase de lecture ouverte correspondant a une proteine de 48 k. La comparaison des sequences permet de distinguer 2 groupes: les arn s et l d'une part, les arn g, e et c, d'autre part. A l'interieur de chaque groupe, les arn presentent plus de 90% d'homologie de sequence tandis que 63% des nucleotides sont communs aux 5 arn etudies
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Fuchs, Marc. "Le grapevine fanleaf virus, agent du court-noue de la vigne : approche moleculaire de la premunition, structure et expression du rna satellite." Université Louis Pasteur (Strasbourg) (1971-2008), 1989. http://www.theses.fr/1989STR13179.
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Parmi les nombreuses maladies virales qui affectent la vigne, la maladie du court-noue est l'une des plus graves et des plus repandues. Le grapevine fanleaf virus (gflv) et l'arabis mosaic virus (armv) sont deux nepovirus responsables de cette maladie. Les moyens de lutte actuels contre cette maladie font essentiellement appel a l'utilisation de plantes certifiees indemnes de virus (selection sanitaire) et a des traitements chimiques des sols pour eliminer les nematodes vecteurs, mais les nematicides employes sont couteux, polluants et pas toujours efficaces. Disposant de plusieurs pathotypes de court-noue qui se distinguent par leur virulence sur vigne et sur hotes herbacess, nous avons envisage d'utiliser les caracteres d'hypovirulence comme nouvelles methodes de lutte. C'est pourquoi, une etude moleculaire de la protection croisee entre isolats hypo- et hypervirulents a ete realisee sur chenopodium quinoa. Cette plante herbacee permet des experiences rapides sur vigne. Elles ont montre que dans les plantes premunies la synthese de la capside et des rna de l'isolat hyperagressif est diminuee ou meme parfois inhibee. Une application agronomique de la premunition a la vigne est prevue. Une autre approche est liee a la presence de rna satellite dans certains isolats. La structure et l'expression du rna satellite de l'isolat gflv-f13 ont ete etudieses. Ce rna a ete clone sous forme cdna et sa sequence comparee a celle d'autres rna satellites: il presente de fortes homologies avec un rna satellite de l'armv. L'etude des proprietes biologiques du rna satellite associe au gflv a ete realisee avec des transcrits synthetises apres clonage de son cdna dans un vecteur d'expression. L'expression des transcrits, analysee in vitro dans un systeme de proteosynthese et in vivo par co-inoculation sur c. Quinoa avec un isolat de gflv naturellement depourvu de rna satellite, a montre que ces transcrits sont fonct
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GREIF, CHARLES. "Contribution a l'etude des relations structure-fonction du genome du virus des anneaux noirs de la tomate et de son rna satellite." Strasbourg 1, 1989. http://www.theses.fr/1989STR13133.
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Determination de la sequence nucleotidique du rna-1 du virus des anneaux noirs de la tomate (tbrv). Etude de la sequence de la polyproteine correspondante. Mise en evidence de cette polyproteine par traduction in vitro. Realisation de transcripts par clonage du rna. Les transcripts induisent la synthese d'une polyproteine mais ne sont pas infectieux. Synthese d'un rna satellite biologiquement actif
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Alkan, Can. "Computational Studies on Evolution and Functionality of Genomic Repeats." Case Western Reserve University School of Graduate Studies / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=case1120143436.
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ONCINO, CELINE. "Etude du role de la proteine codee par le rna satellite associe au virus des anneaux noirs de la tomate ou tomato black ring nepovirus (tbrv)." Université Louis Pasteur (Strasbourg) (1971-2008), 1996. http://www.theses.fr/1996STR13020.
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Le virus des anneaux noirs de la tomate ou tomato black ring virus (tbrv) est un nepovirus, dont le genome est constitue de deux rna de polarite positive, necessaires a la replication, l'encapsidation et la diffusion du virus dans la plante. Le tbrv peut contenir un rna satellite, qui n'est pas necessaire a la multiplication du virus helper, mais ne peut pas se multiplier en son absence. Ce rna satellite, constitue de 1372 a 1376 nucleotides selon les isolats, code pour une proteine non structurale de 48 k (la proteine p48). Mon travail de these a consiste a etudier le role de cette proteine et se partage en trois parties: 1) l'etude de la multiplication de transcrits correspondant au rna satellite associe au tbrv l ou c, sauvages ou mutants, dans les plantes et les protoplastes a permis de montrer que la proteine est necessaire a la replication du rna satellite. L'impossibilite de complementer un rna satellite deficient dans la synthese de proteine en le co-inoculant avec un rna satellite infectieux suggere que la proteine p48 n'agit qu'en cis. 2) l'analyse de la multiplication de transcrits, correspondant aux rna satellites l et c, a montre que la replication du rna satellite est specifiquement liee a celle du virus helper. La multiplication de rna satellites chimeres suggere que les determinants de cette specificite pourraient etre localises dans une region centrale de la proteine p48. 3) une forte affinite de la proteine p48 pour les acides nucleiques a ete mise en evidence in vitro. Aucune specificite de fixation vis-a-vis d'une sequence ou d'une structure particuliere n'a ete observee. Apres deletion de plusieurs regions de la proteine, l'affinite pour le rna est conservee, il est probable que plusieurs domaines proteiques pourraient se lier au rna
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Petrany, Michael J. "Consequences of Cell Fusion and Multinucleation for Skeletal Muscle Development and Disease." University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1595847866440328.
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Gimenez, Octavio Manuel Palacios [UNESP]. "Padrões de evolução de sistemas de cromossomos sexuais em grilos: uma abordagem integrada entre citogenética e genômica." Universidade Estadual Paulista (UNESP), 2017. http://hdl.handle.net/11449/152458.
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Os cromossomos sexuais se originam independentemente de um par de homólogos autossômicos e em várias linhagens apresentam características comuns, tais como acúmulo de vários tipos de DNA repetitivo, restrição da recombinação e perda ou ganho de genes devido á diferenciação morfológica e genética entre os cromossomos sexuais X e Y ou Z e W. Estas características representam um exemplo fascinante de convergência evolutiva. Em Orthoptera, o sistema cromossômico sexual comumente encontrado na maioria das espécies estudadas é do tipo X0♂/XX♀. Entretanto, sistemas cromossômicos sexuais derivados dos tipos neo-XY♂/XX♀ e neo- X1X2Y♂/X1X1X2X2♀ são também observados, surgindo repetidamente por fusões cêntricas e em tandem, inversões e dissociações envolvendo cromossomos sexuais ancestrais e autossomos. O presente trabalho teve três objetivos. Primeiro, entender o possível papel dos DNAs repetitivos na estrutura/diversificação dos cromossomos sexuais simples e derivados, a partir do isolamento e mapeamento físico de sequências, tais como, famílias multigênicas, DNA satélite (DNAsat) e microssatélites, nas espécies Gryllus assimilis, Cycloptiloides americanus e Eneoptera surinamensis. Segundo, testar e comparar transcrição diferencial de DNAsat entre diferentes tecidos, sexos e espécies a partir de transcriptomas de Gryllus assimilis, G. bimaculatus, G. firmus e G. rubens, com o objetivo de entender os possíveis papéis funcionais destas sequências na regulação gênica, modulação da cromatina e como componentes funcionais de importantes estruturas como telômeros, centrômeros e cromossomos sexuais. Terceiro, a partir de transcriptomas de espécies de grilos (Gryllus assimilis, G. bimaculatus e G. firmus) prospectar genes codificadores de proteínas relacionados com a determinação sexual, envolvidos com o fitness reprodutivo e genes enviesados do sexo, responsáveis pelas diferenças fenotípicas entre machos e fêmeas, e tentar elucidar de uma maneira comparativa os fatores evolutivos atuando nestes loci. Origem de novo de cromossomos sexuais mediante rearranjos cromossômicos, assim como acúmulo de DNA repetitivo que levaram a diferenciação entre cromossomos sexuais são relatados em C. americanus (X1X20) e E. surianmensis (neo-X1X2Y). Estas características observadas em grilos representam outro caso notável de convergência evolutiva devido os cromossomos sexuais não relacionados compartilharem muitas propriedades entre táxons distantes. Acúmulo surpreendente de loci de DNAsat foi encontrado no neo-Y altamente diferenciado de E. surinamensis, incluindo 39 DNAsat representados em excesso neste cromossomo, que é a maior diversidade de DNAsat até agora relatada para cromossomos sexuais. Foi documentado que, particularmente os DNAsat, contribuíram grandemente para o aumento de tamanho genômico entre G. assimilis e E. surinamensis. Um achado interessante foi a identificação de DNAsat conservados entre espécies de grilos (Gryllus assimilis, G. bimaculatus e G. firmus), mas transcritos diferencialmente. Os dados relativos à presença de DNAsat no genoma de G. assimilis foram discutidos em um contexto evolutivo, com dados transcricionais permitindo comparações entre os sexos e entre os tecidos quando possível. Foram discutidas hipóteses para a conservação e transcrição de DNAsat em Gryllus, que podem resultar do seu papel na diferenciação sexual no nível da cromatina, na formação da heterocromatina e na função centromérica. Outra descoberta foi a identificação de genes determinantes do sexo e outros genes relacionados ao fitness reprodutivo, como a biossíntese de hormônios de insetos e ritmo circadiano entre espécies de Gryllus. Os efetores e os alvos downstream das vias de determinação do sexo foram previamente identificados em outros insetos, mas nunca em Orthoptera. Usando G. assimilis como modelo para estudar genes enviesados do sexo foi possível identificar um conjunto de genes altamente expressos que podem explicar diferenças fenotípicas entre os sexos. Estimou-se que os genes codificadores de proteínas relacionadas com a diferenciação sexual e com o fitness reprodutivo evoluem mais rapidamente do que os genes não reprodutivos (genes housekeeping) como resultado de uma forte seleção positiva nos primeiros. Além disso, foi encontrado que as espécies estudadas apresentam níveis excepcionalmente elevados de duplicações gênicas. As descobertas sugerem que as duplicações gênicas podem desempenhar um papel na expressão de genes enviesados do sexo no grilo de campo G. assimilis, uma espécie que no futuro provavelmente irá fornecer informações sobre genômica funcional e epigenética da determinação do sexo.
Sex chromosomes have arisen independently from an ordinary autosomal pair and in several lineages they present common characteristics, such as accumulation of distinct classes of repetitive DNAs, restriction of the recombination and loss or gain of genes due to the morphological and genetic differentiation between the sexual chromosomes X and Y or Z and W. These characteristics represent a fascinating example of evolutionary convergence. In Orthoptera, the X0♂/XX♀ sex-determining system is considered modal but eventually, diverse sex chromosome systems evolved several times, such as neo-XY♂/XX♀, X1X20♂/X1X1X2X2♀ and even neo- X1X2Y♂/X1X1X2X2♀. It was found that particularly centric fusions (i.e., Robertsonian translocations) and tandem fusions with autosomes, dissociations and inversions contributed to the formation of neo-sex chromosomes in Orthoptera. The present work had three objectives. First, get insights of the role of repetitive DNAs in the structure/diversification of simple and derivative sex-chromosomes by isolation and physical mapping of repetitive DNA sequences, such as multigene families, satellite DNA (satDNA) and microsatellites using Gryllus assimilis, Cycloptiloides americanus e Eneoptera surinamensis, as models. Second, looking at differential satDNA transcription between different tissues, sexes, and species from transcriptomes of Gryllus assimilis, G. bimaculatus, G. firmus and G. rubens, I tried to understand the possible functional roles of these sequences in gene regulation, chromatin modulation and as functional components of important structures such as telomeres, centromeres and sex chromosomes. Third, using transcriptomes from cricket species (Gryllus assimilis, G. bimaculatus and G. firmus), I searched for genes encoding proteins related to sexual determination, reproductive fitness and sex-biased genes which are responsible for the phenotypic differences between males and females. I also tried to elucidate in a comparative way the evolutionary factors acting at these loci. De novo origin of sex chromosomes by chromosomal rearrangements, as well as repetitive DNA accumulation that led to the differentiation between sex chromosomes are reported for C. americanus (X1X20) e E. surianmensis (neo-X1X2Y). These features observed in crickets represent another remarkable case of evolutionary convergence because unrelated sex chromosomes share many common properties among distant taxa. Especially astonishing accumulation of satDNAs loci was found in the highly differentiated neo-Y, including 39 satDNAs over-represented in this chromosome, which is the greatest satDNAs diversity yet reported for sex chromosomes. It has been documented that, particularly the satDNA, contributed greatly to the increase in genomic size between G. assimilis and E. surinamensis. An interesting finding was the identification of satDNA conserved among species of crickets (Gryllus assimilis, G. bimaculatus and G. firmus), but differentially transcribed. The data regarding satDNA presence in G. assimilis genome was discussed in an evolutionary context, with transcriptional data enabling comparisons between sexes and across tissues when possible. I discussed hypotheses for the conservation and transcription of satDNAs in Gryllus, which might result from their role in sexual differentiation at the chromatin level, heterochromatin formation, and centromeric function. Another finding was the identification of sex-determining genes and other genes related to reproductive fitness, such as biosynthesis of insect hormones and circadian rhythm among Gryllus species. The effectors as well as downstream targets of sex-determination pathways have been previously identified in other insects but never in Orthoptera. Using G. assimilis to study sex-biased genes I identified a set of highly expressed genes that might account for phenotypic differences between sexes. Furthermore, I estimated that proteinencoding reproductive genes evolve faster than non-reproductive genes as result of strong positive selection at those loci. It was documented that the species studied harbor exceptionally high levels of gene duplications. The findings suggest that gene duplications may play a role in sex-biased genes expression in the field cricket G. assimilis, a species likely to yield insights into the functional genomics and epigenetics of sex determination.
FAPESP: 2014/02038-8
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HANS, FABIENNE. "Replication du rna satellite du virus du court-noue de la vigne (gflv) dans des protoplastes de chenopodium quinoa. Action antivirale de la proteine de capside du gflv exprimee par des tabacs transgeniques." Université Louis Pasteur (Strasbourg) (1971-2008), 1992. http://www.theses.fr/1992STR13147.
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Le grapevine fanleaf virus ou gflv est un nepovirus responsable de la maladie du court-noue de la vigne. Il comporte deux rna genomiques de 7342 et 3774 nt, de polarite positive, polyadenyles en 3 et lies a une proteine vpg en 5. La souche f13 du gflv contient en plus un rna satellite de 1114 nucleotides, appele aussi rna3. Il code pour une proteine de mr=37k dont la fonction est inconnue. Un protocole de preparation de protoplastes de c. Quinoa a ete mis au point. La methode d'infection par le peg permet la replication des rna viraux dans ces protoplastes et la methode d'infection par electroporation, la replication des transcrits du rna satellite en presence d'un virus assistant. Des transits du rna satellite contenant 33 nucleotides extra-viraux a leur extremite 5 ne se multiplient pas dans les protoplastes de c. Quinoa alors que des transcrits avec 1 nucleotide extra-viral en 5 se multiplient. La presence de nucleotides extra-viraux en 3 n'a que peut d'effet sur la replication des transcrits. Des transcrits du rna satellite contenant des mutations dans les parties non codantes ne se multiplient pas dans les protoplastes de c. Quinoa. Des mutations introduites dans la partie codante du rna3 suppriment l'activite biologique des transcrits correspondant, a l'exception du transit mute au niveau du second aug en phase avec le codon d'initiation. Ces resultats suggerent un role probable des extremites non codantes du rna satellite du gflv ainsi que de la proteine p3 et/ou de la region codante dans la replication du rna satellite. Le gene de la proteine de cpside du gflv-f13 a ete transfere dans des tabacs n. Benthamiana par agrotransformation. Apres infection, les rna du gflv se multiplient moins bien dans les feuilles inoculees des plantes transgeniques que dans des plantes temoin. Certaines plantes transgeniques sont protegees contre le gflv-f13 car aucun rna viral n'est detecte dans les feuilles superieures alors qu'il le sont dans les plantes temoin
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Gaspar, Helena Isabel Dias. "Estudo de um isolado de Olive Latent Virus 1: I. caracterização molecular e II. resposta à presença de bactérias promotoras de fitossanidade." Master's thesis, Universidade de Évora, 2014. http://hdl.handle.net/10174/12957.
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Este trabalho incidiu na caracterização molecular de um isolado de Olive latent virus 1 (OLV-1), denominado G1A, obtido de Olea europaea L.. O isolado G1A foi transmitido mecanicamente a Nicotiana benthamiana na qual produziu necroses locais e sistémicas. A sequência parcial do genoma do isolado G1A tem 3668 nucleótidos e apresenta elevada identidade com outras sequências de OLV-1 existentes na base de dados. O isolado G1A apresenta a encapsidação de uma pequena molécula de RNA com cerca de 600 nucleótidos, previamente identificada como um RNA satélite (satRNA).
Paralelamente, foi analisada a resposta de N. benthamiana à infeção por parte do isolado G1A, na presença de um isolado bacteriano termofílico 18UE/10, este obtido a partir de solo de olival do Alentejo. Esta bactéria é capaz de produzir sulfato e amónio, nutrientes importantes para o crescimento das plantas. As plantas previamente regadas com uma suspensão de 18UE/10 apresentaram sintomas retardados de infeção viral em comparação a plantas controlo. Este resultado foi um exemplo de resistência induzida pelo enxofre, devido ao sulfato produzido pelo isolado bacteriano; ABSTRACT:
Study of an isolate of Olive latent virus 1: I. Molecular characterization and II. Response to the presence of plant health-promoting bacteria
This work focused on the molecular characterisation of an isolate of Olive latent virus 1 (OLV-1), G1A, obtained from Olea europaea L.. Isolate G1A was mechanically transmitted to Nicotiana benthamiana plants producing local and systemic necrotic symptoms. The partial genome sequence of G1A isolate is 3668 nucleotides and has high identity with the OLV-1 sequences available in GenBank. The G1A isolate shows the encapsidation of a small RNA molecule of about 600 nucleotides, previously identified as a satellite RNA (satRNA). In addition, the response of N. benthamiana plants to G1A infection, in the presence of a bacterial thermophilic isolate 18UE/10 obtained from an olive orchard soil in Alentejo, was evaluated. This bacterial isolate produces sulphate and ammonium, important nutrients for plant growth. The plants that were watered with the bacterial suspension prior to virus inoculation showed delayed symptoms of viral infection when compared to control plants. This result was an example of the resistance induced by sulphur due to the sulphate produced by the bacterial isolate.
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Vautrot, Valentin. "Recherche des mécanismes impliqués dans les dérégulations de l'épissage alternatif à l'origine de la progéria et étude du rôle de l'étape d'épissage dans les changements globaux d'expression des gènes en réaction au choc thermique." Thesis, Université de Lorraine, 2013. http://www.theses.fr/2013LORR0321/document.
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Le syndrome de Hutchinson-Gilford, ou progéria, est une pathologie génétique rare qui se caractérise par des symptômes assimilés à un vieillissement prématuré. Les mutations à l'origine de la progéria affectent le gène LMNA, codant la lamine A, qui joue un rôle majeur dans la formation, la maintenance et la résistance du noyau. Ces mutations activent l'utilisation de sites 5' alternatif ou cryptique d'épissage présents dans l'exon 11 du pré-ARNm LMNA en amont du site normalement utilisé. Nous avons révélé un effet des mutations sur la structure secondaire de l'ARN aux alentours des mutations, qui permet l'augmentation de l'utilisation des sites d'épissage mutants. De plus, nous avons montré l'implication de plusieurs protéines SR (SRSF1, SRSF5 et SRSF6) dans la régulation de l'utilisation des différents sites d'épissage. D'autre part, il a déjà été observé que les noyaux des cellules des patients atteints de progéria contiennent des granules de stress, les nSB, situés dans les régions péricentromériques des chromosomes et contenant des ARN dits satellite III et des facteurs d'épissage. Des nSB similaires sont formés dans les cellules saines suite à divers stress, comme le stress thermique. Il est possible que ces nSB séquestrent ces facteurs d'épissage afin de réguler le profil d'épissage alternatif des cellules pendant la régénération après un stress. Nous avons purifié les protéines associées aux ARN satellite III in vitro afin de trouver de nouveaux composants des nSB et analysé, par emploi de puces jonction-exon, le transcriptome de cellules soumises à un choc thermique, pour mieux comprendre à terme comment la formation des nSB peut affecter l'épissage alternatifThe Hutchinson-Gilford syndrome, also called progeria, is a rare genetic disease, characterized by symptoms that can be assimilated to accelerated natural ageing. Mutations that cause progeria affect the LMNA gene, which codes the lamin A that plays a major role in the shaping, maintenance and resistance of the nucleus. These mutations lead to the activation of alternative or cryptic 5' splice sites located within the exon 11 of LMNA pre-mRNA upstream from the normal 5' splice site. Our work revealed an effect of the mutations on the 2D RNA structure of the splice sites, which contributes to the increased use of the mutant sites. On top of it, we showed the impact of several SR proteins, (SRSF1, SRSF5 and SRSF6) on the regulation of the use of the exon 11 5' splice sites. On the other hand, it was previously observed that cells from progeria patients contain nuclear stress bodies (nSB), located in chromosomal pericentromeric regions and containing satellite III RNAs and several splicing regulatory proteins. Similar bodies are formed in healthy cells submitted to various stresses such as heat shock. A work hypothesis is that those nSBs sequester splicing factors in order to regulate the global alternative splicing profile in cells during the recovery period after stress. We purified proteins associated with satellite III RNAs in vitro, to find new components of the nSBs, and analyzed the transcriptome of cells subjected to heat shock using exon junction microarrays, in order to eventually understand how nSB formation can affect alternative splicing
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Gossett, John Jared. "Analysis of macromolecular structure through experiment and computation." Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/51925.
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This thesis covers a wide variety of projects within the domain of computational structural biology. Structural biology is concerned with the molecular structure of proteins and nucleic acids, and the relationship between structure and biological function. We used molecular modeling and simulation, a purely computational approach, to study DNA-linked molecular nanowires. We developed a computational tool that allows potential designs to be screened for viability, and then we used molecular dynamics (MD) simulations to test their stability. As an example of using molecular modeling to create experimentally testable hypotheses, we were able to suggest a new design based on pyrrylene vinylene monomers. In another project, we combined experiments and molecular modeling to gain insight into factors that influence the kinetic binding dynamics of fibrin "knob" peptides and complementary "holes." Molecular dynamics simulations provided helpful information about potential peptide structural conformations and intrachain interactions that may influence binding properties. The remaining projects discussed in this thesis all deal with RNA structure. The underlying approach for these studies is a recently developed chemical probing technology called 2'-hydroxyl acylation analyzed by primer extension (SHAPE). One study focuses on ribosomal RNA, specifically the 23S rRNA from T. thermophilus. We used SHAPE experiments to show that Domain III of the T. thermophilus 23S rRNA is an independently folding domain. This first required the development of our own data processing program for generating quantitative and interpretable data from our SHAPE experiments, due to limitations of existing programs and modifications to the experimental protocol. In another study, we used SHAPE chemistry to study the in vitro transcript of the RNA genome of satellite tobacco mosaic virus (STMV). This involved incorporating the SHAPE data into a secondary structure prediction program. The SHAPE-directed secondary structure of the STMV RNA was highly extended and considerably different from that proposed for the RNA in the intact virion. Finally, analyzing SHAPE data requires navigating a complex data processing pipeline. We review some of the various ways of running a SHAPE experiment, and how this affects the approach to data analysis.
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Lemaire, Olivier. "Contribution a l'etude des proprietes biologiques des rna du virus de la rhizomanie (beet necrotic yellow vein virus) et de leur role dans l'etiologie de la maladie." Université Louis Pasteur (Strasbourg) (1971-2008), 1988. http://www.theses.fr/1988STR13115.
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Tsao, Theresa Tsun-Hui. "Towards the development of transgenic banana bunchy top virus (BBTV)-resistant banana plants : interference with replication." Queensland University of Technology, 2008. http://eprints.qut.edu.au/17031/.
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Banana bunchy top virus (BBTV) causes one of the most devastating diseases of banana. Transgenic virus resistance is now considered one of the most promising strategies to control BBTV. Pathogen-derived resistance (PDR) strategies have been applied successfully to generate plants that are resistant to numerous different viruses, primarily against those viruses with RNA genomes. BBTV is a circular, single-stranded (css) DNA virus of the family Nanoviridae, which is closely related to the family Geminiviridae. Although there are some successful examples of PDR against geminiviruses, PDR against the nanoviruses has not been reported. Therefore, the aim of this thesis was to investigate the potential of BBTV genes to interfere with virus replication when used as transgenes for engineering banana plants resistance to BBTV. The replication initiation protein (Rep) of nanoviruses is the only viral protein essential for viral replication and represents an ideal target for PDR. Therefore, this thesis focused on the effect of wild-type or mutated Rep genes from BBTV satellite DNAs or the BBTV integral genome on the replication of BBTV in banana embryogenic cell suspensions.
A new Rep-encoding satellite DNA, designated BBTV DNA-S4, was isolated from a Vietnamese BBTV isolate and characterised. When the effect of DNA-S4 on the replication of BBTV was examined, it was found that DNA-S4 enhanced the replication of BBTV. When the replicative capabilities of DNA-S4 and the previously characterised Rep-encoding BBTV satellite, DNA-S1, were compared, it was found that the amount of DNA-S4 accumulated to higher levels than DNA-S1. The interaction between BBTV and DNA-S1 was also examined. It was found that over-expression of the Rep encoded by DNA-S1 using ubi1 maize polyubiquitin promoter enhanced replication of BBTV. However, when the Rep-encoded by DNA-S1 was expressed by the native S1 promoter (in plasmid pBT1.1-S1), it suppressed the replication of BBTV. Based on this result, the use of DNA-S1 as a possible transgene to generate PDR against BBTV was investigated.
The roles of the Rep-encoding and U5 genes of BBTV DNA-R, and the effects of over-expression of these two genes on BBTV replication were also investigated. Three mutants of BBTV DNA-R were constructed; plasmid pUbi-RepOnly-nos contained the ubi1 promoter driving Rep expression from DNA-R, plasmid pUbi-IntOnly-nos contained the ubi1 promoter driving expression of the DNA-R internal gene product (U5), while plasmid pUbi-R.ORF-nos contained the ubi1 promoter driving the expression of both Rep and the internal U5 gene product. The replication of BBTV was found to be significantly suppressed by pUbi-RepOnly-nos, weakly suppressed by pUbi-IntOnly-nos, but strongly enhanced by pUbi-R.ORF-nos.
The effect of mutations in three conserved residues within the BBTV Rep on BBTV replication was also assessed. These mutations were all made in the regions in the ATPase motifs and resulted in changes from hydrophilic to hydrophobic residues (i.e. K187→M, D224→I and N268→L). None of these Rep mutants was able to initiate BBTV replication. However, over-expression of Reps containing the K187→M or N268→L mutations significantly suppressed the replication of BBTV.
In summary, the Rep constructs that significantly suppressed replication of DNA-R and -C in banana embryogenic cell suspensions have the potential to confer resistance against BBTV by interfering with virus replication. It may be concluded that BBTV satellite DNAs are not ideal for conferring PDR because they did not suppress BBTV replication consistently. Wild-type Rep transcripts and mutated (i.e. K187→M and N248→L) Rep proteins of BBTV DNA-R, however, when over-expressed by a strong promoter, are all promising candidates for generating BBTV-resistant banana plants.
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Meyer, Michel. "Contribution à l'étude de la structure et de l'organisation du RNA-2 (isolat S) et des RNA satellites de 5 isolats du TBRV." Grenoble 2 : ANRT, 1986. http://catalogue.bnf.fr/ark:/12148/cb37599635j.
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Flinders, Jeremy Cole. "Nuclear magnetic resonance studies of catalytic RNAs : the Varkud satellite ribozyme /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2004. http://uclibs.org/PID/11984.
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Vittorazzi, Stenio Eder 1984. "Caracterização citogenética e molecular do DNAr 5S e sua forma variante de DNA satélite em espécies do grupo de Physalaemus cuvieri (Anura, Leptodactylidae) = Cytogenetic and molecular analysis of the 5S rDNA and its variant form of satellite DNA in Physalaemus cuvieri species group (Anura, Leptodactylidae)." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317964.
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Orientadores: Shirlei Maria Recco Pimentel, Luciana Bolsoni LourençoTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Os genomas dos eucariotos são ricos em sequências repetitivas, as quais compreendem sequências dispersas e em tandem. Entre as sequências em tandem, incluem-se os DNA satélites, os DNA ribossomais e cópias parálogas. Os DNA satélites são os principais constituintes da heterocromatina constitutiva e os genes ribossomais transcrevem os RNAr para compor os ribossomos, que dividem-se em duas famílias, DNAr 45S e 5S. Em um mesmo organismo, diferentes tipos de DNAr 5S já foram reconhecidos, mostrando a existência de uma diversidade quanto a esse tipo de sequência. No anuro Physalaemus cuvieri, uma forma de DNA satélite denominada PcP190 foi caracterizada e teve sua origem atribuída a uma derivação do DNAr 5S em uma espécie ancestral. Em diferentes populações de P. cuvieri estudadas previamente com as ferramentas da citogenética convencional, uma acentuada variação interpopulacional nos cromossomos portadores da NOR pôde ser observada, e nas demais espécies do grupo de P. cuvieri, peculiaridades interespecíficas foram evidenciadas, porém, os cariótipos entre essas espécies são muito semelhantes. O objetivo nessa tese foi ampliar o estudo citogenético de espécies do grupo de P. cuvieri que possuíam carência na descrição cromossômica, como P. kroyeri e P. cicada. Objetivou-se também analisar o DNA satélite PcP190 em nível cromossômico e molecular, assim como estudar a organização molecular e a diversidade de sequências do DNAr 5S no genoma das espécies de Physalaemus e espécies de gêneros relacionados. Com os resultados da pesquisa, três capítulos são apresentados, sendo eles: (i) "Long-time evolution and highly dynamic satellite DNA in leptodactylid and hylodid frogs", o qual mostra que o DNA satélite PcP190 é amplamente conservado e pode ser reconhecido em representantes de duas famílias de anuros, Leptodactylidae e Hylodidae; além disso, mostra também que essas sequências são altamente dinâmicas nos cromossomos das espécies do grupo de P. cuvieri. (ii) "Diversidade do DNAr 5S em Leiuperinae (Anura)", os resultados mostram que no genoma desses anuros existe uma diversidade dessa classe de família multigênica maior do que proposto até então, e que essas sequências do DNAr 5S permanecem conservadas desde a divergência evolutiva entre os Actinopterigio e Sarcopterigio. (iii) "Cariótipo e mapeamento de DNA repetitivo em Physalaemus kroyeri e P. cicada (Anura Leptodactylidae)", onde é apresentado que a banda 5p de P. kroyeri tem se mostrado um bom marcador cromossômico para as espécies do grupo de P. cuvieri, o que indica se tratar de uma sinapomorfia cromossômica. Contrariamente, a ausência dessa banda 5p em P. cicada não fornece evidência para a inclusão de P. cicada no grupo de P. cuvieri ou em qualquer outro grupo de espécies
Abstract: The genomes of eukaryotic organisms are rich in repetitive DNA sequences, which can be dispersed or arrayed in tandem. The tandem repeat sequences include the satellite DNA, the ribosomal DNA, and paralogous copies. Satellite DNA is the main component of constitutive heterochromatin, while the ribosomal genes encode the rRNAs that make up the ribosomes; they are divided into two families, the 45S and 5S rRNA. Different types of 5S rDNA have been identified in a single organism, proving that there is diversity in this type of DNA sequence. In the anuran Physalaemus cuvieri, a satellite DNA family called PcP190 has been identified, which is thought to have derived from the 5S rDNA of an ancestor species. In different populations of P. cuvieri that were previously studied with conventional cytogenetic tools, an accentuated interpopulational variation among chromosomes harboring the NOR was observed, while in other species of the P. cuvieri group, interspecific traits were found. However, the karyotypes in these species are very similar. The aim of this thesis was to expand the cytogenetic studies on P. cuvieri species that needed further chromosomal description, such as P. kroyeri and P. cicada. Another objective was to analyze the PcP190 satellite DNA at the chromosomal and molecular level, as well as to study the molecular organization and the diversity of 5S rDNA sequences in the genomes of the Physalaemus species and other species of related genera. We present three chapters as a result of this research: (i) "Long-time evolution and highly dynamic satellite DNA in frogs," which demonstrates that the PcP190 satellite DNA is widely conserved and was recognized in representatives of two anurans families, Leptodactylidae and Hylodidae. Moreover, it also demonstrates that these sequences are highly dynamic within the chromosomes of the P. cuvieri species group. (ii) "5S rDNA in Leiuperinae (Anura): new insights on its evolution." The results show that in the genomes of these anurans there is wider diversity among this multigenic family than previously assumed and that these 5S rDNA sequences have remained conserved since the evolutionary divergence of Actinopterygii and Sarcopterygii. (iii) "Karyotype and repetitive DNA mapping of the Physalaemus kroyeri and P. cicada (Anura Leptodactylidae)," which demonstrates that the 5p chromosomal band of P. kroyeri is a good chromosomal marker for species from the P. cuvieri group, indicating that it is a chromosomal synapomorphy. Conversely, the absence of this 5p band in P. cicada does not provide evidence for the inclusion of this species in the P. cuvieri group or any other species group
Doutorado
Biologia Celular
Doutor em Biologia Celular e Estrutural
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Richard, Manon. "Clusters de gènes de résistance aux maladies chez le haricot commun : bases moléculaires, régulation et évolution." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA112401.
Full textAbstract:
Le haricot commun est la légumineuse à graine la plus consommée au monde en alimentation humaine. Le génome du haricot possède plusieurs énormes clusters de gènes de résistance (R) qui ont la particularité de se cartographier en extrémité de groupes de liaison. Le génome du haricot commun (génotype Andin G19833) a été récemment séquencé et nous avons participé à ce projet en annotant la famille des NB-LRR (NL), classe prépondérante des gènes de résistance. Ces données génomiques nous ont permis de réaliser les 3 études suivantes. (i) L’identification des bases moléculaires de Co-x un gène R vis-à-vis d’une souche très virulente de C. lindemuthianum chez JaloEEP558 a été initiée. La cartographie fine de Co-x suivie du séquençage de la région cible chez JaloEEP558 (Co-x) a permis d’identifier un gène candidat codant une kinase atypique qui pourrait être la cible d’un effecteur fongique, gardée par un gène R. (ii) Des études récentes ont mis en évidence l’implication de petits ARNs (miRNAs induisant la production de phased siRNAs) dans la régulation de l’expression des NL. Le séquençage et l’analyse de banques de sRNAs de haricot nous ont permis d’identifier ce mécanisme et de mettre le doigt sur un nouveau mécanisme de régulation des NL impliquant des sRNAs de 24 nt. (iii) Des ADN satellites ont été étudiés à l’échelle du génome du haricot. L’étude des centromères de haricot a permis de mettre en évidence l’existence de 2 ADN satellites différents, Nazca et CentPv2. Nous avons également étudié un ADN satellite subtélomérique khipu précédemment identifié au niveau de 2 clusters de gènes R du haricot. L’étude de khipu à l’échelle du génome suggère l’existence d’échanges fréquents de séquences entre subtélomères de chromosomes non homologues. Ces résultats nous ont amenés à proposer que des éléments structuraux et une combinaison de mécanismes de régulation (TGS et PTGS) permettent la prolifération des NL sans effet néfaste pour la plante, conduisant à l’obtention de très gros clusters de NL dans le génome du haricotCommon bean is the main source of protein for human consumption in many developing countries. Several huge disease resistance (R) gene clusters have been mapped at the end of common bean linkage groups. The common bean genome (Andean genotype G19833) has recently been sequenced. Access to the complete genome sequence of common bean allowed us to annotate the Nucleotide Binding-Leucine Rich Repeat (NL) encoding gene family, the prevalent class of disease R genes in plants, and to perform the 3 following studies: (i) We have investigated the molecular basis of Co-x, an anthracnose R gene to a highly virulent strain of C. lindemuthianum, previously identified in the Andean cultivar JaloEEP558. Fine mapping of Co-x and sequencing of the target region in JaloEEP558, allowed us to identify a candidate gene encoding an atypical kinase. We hypothesised that this atypical kinase is a fungal effector target. (ii) Several recent studies have highlighted the role of small RNA (miRNAs that triggered phased siRNAs production) in the regulating of NL gene expression. Analyses of small RNAs libraries of common bean led to the identification of this mechanism in common bean and also allowed us to propose a new NL regulation pathway involving 24 nt sRNAs. (iii) We have studied centromeric and subtelomeric satellite DNAs at common bean genome level. We have identified 2 different satellite DNAs in common bean centromeres, Nazca and CentPv2. We have also conducted the analyze of the subtelomeric satellite khipu, previously identified in common bean R clusters and confirmed that frequent sequence exchange occurs between non-homologous chromosome ends in common bean genome. Together, these results led us to propose that both structural elements and a combination of regulatory mechanisms (TGS, PTGS) allow the amplification of NL sequences without detrimental effect for the plant leading to the large NL clusters observed in common bean
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Barbosa, Patrícia. "ELEMENTOS GENÔMICOS REPETITIVOS NO COMPLEXO Astyanax scabripinnis (TELEOSTEI, CHARACIDAE)." UNIVERSIDADE ESTADUAL DE PONTA GROSSA, 2013. http://tede2.uepg.br/jspui/handle/prefix/982.
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The most part of the eukaryote genomes is constituted for repetitive DNA or multiple copies DNA, which has already been considered as “junk”, may be associated to the heterochromatin. In this study three Astyanax scabripinnis populations from Pindamonhangaba and Guaratinguetá (SP, Brazil) rivers and stream and one population from Maringá (PR, Brazil) were analyzed about the nucleolar organizing region (NORs), As51 satellite DNA, 18S and 5S rDNA location. Moreover, repetitive sequences were isolated and mapped through Cot-1 technique, which showed homology with UnaL2, a LINE type retrotransposon. The fluorescent in situ hybridization (FISH), with the isolated built retrotransposon probe, evidenced disperse labeled and stronger in centromeric and telomeric chromosomes regions, co-located and interspersed with the 18S DNAr and As51, proven by the fiber-FISH technique. The B chromosome of those populations showed very conspicuous labeled with the LINE probe, also co-located with the As51 sequences. The NORs were actives in a single site of a homologue pair in all three populations, with no evidence that the transposable elements and repetitive DNA have influence in its regulation at the performed analyzes level.
A maior parte do genoma dos eucariotos é constituída por DNA repetitivo ou DNA de múltiplas cópias, o qual já foi considerado “lixo”, podendo estar associado à heterocromatina. Neste estudo foram analisadas três populações de Astyanax scabripinnis provenientes de rios e córregos de Pindamonhangaba e Guaratinguetá (SP, Brasil) e uma população da cidade de Maringá (PR, Brasil) quanto a localização das regiões organizadoras de nucléolo (RONs), DNA satélite As51, DNA ribossomal (DNAr) 18S e DNAr 5S. Ainda, foram isoladas e mapeadas sequências repetitivas por meio da técnica de Cot-1, que mostrou homologia com UnaL2, retrotransposon do tipo LINE. A hibridação in situ fluorescente (FISH), com sonda construída para o retrotransposon isolado, evidenciou marcações dispersas e mais concentradas em regiões centroméricas e teloméricas dos cromossomos, co-localizadas e interespaçadas com DNAr 18S e As51, comprovada pela técnica de fiber-FISH. O cromossomo B das populações mostrou marcações bastante conspícuas com a sonda LINE, também co-localizada com sequências As51. As RONs apresentaram-se ativas em sítios únicos de um par homólogo nas três populações, não havendo indícios de que elementos transponíveis e DNA repetitivo tenham influência na sua regulação ao nível das análises realizadas.
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Tsao, Chang-Chi, and 曹昌麒. "Bamboo mosaic potexvirus satellite RNA encoded P20 potentiates satellite RNA movement." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/47118094066401263582.
Full textAbstract:
碩士國立中興大學
農業生物科技學研究所
89
A satellite RNA of 836 nt depends on the bamboo mosaic potexvirus (BaMV) for its replication and encapsidation. The BaMV satellite RNA (satBaMV) contains a single open reading frame encoding a 20-kDa nonstructural protein (P20). The N-terminal arginine-rich motif of P20 is the RNA binding domain, which preferentially kinds at the 5’ and 3’ untranslated regions of satBaMV RNA. When the P20 open reading frame was replaced with sequence encoding chloramphenicol acetyltransferase (CAT), the accumulation level of BSCAT in systemic leaves of infected plants was only about 1/40-1/100 of that in inoculated leaves. Even though P20 is not required for satBaMV replication, the N-terminal or internal mutations of P20 caused a 21-50% decrease in the level of satBaMV accumulation in inoculated protoplasts when compared with the wild-type satBaMV. These results indicate that P20 may assist satBaMV replication, movement, or other functions. In this study, transgenic Nicotiana benthamiana plants that express the P20 were produced to investigate the effects of P20 on satBaMV movement. Complementary DNA of satBaMV that contains nt 59 to 836 was cloned in a plant expression vector, plasmid pKyLx7, and transformed to N. benthamiana by Agrobacterium-mediated transformation. R0 transformants selected were based on kanamycin resistance and verified by the PCR, Southern blot, Northern blot, and Western blot analyses. The ratio of kanamycin resistance in the F1 progenies was 3:1 which followed the Mendelian law. Transgenic lines which have homozygous satellite cDNA genotype were selected after analysis of F2 progenies. We have selected seedlings which have homozygous satellite cDNA genotype. The homozygous transgenic lines were challenged with BaMV and the virus titers were analyzed by indirect ELISA and western blots. The results showed that virus concentration in the transgenic lines was similar with the untransformed plants and the P20 level was not amplified by BaMV infection. Coinoculation of pCB and pCBSCAT onto untransformed plants, the BSCAT could move cell-to-cell in the inoculated leaves but failed to spread systemically. However, BSCAT could be detected in 16.6~80% of the upper uninoculated leaves of P20 transgenic plants 10 or 15 days after inoculation. Thses results indicat that the P20 transgenic plants can trans-complement the systemic movement of BSCAT. The ability that BSCAT spread from inoculated leaves to non-inoculation leaves was dependent upon the P20 protein in the transgenic plants. These results conclusively demonstrate that the P20 protein of satBaMV potentiates the movement of the satellite RNA.
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Huang, Ying-Wen, and 黃纓雯. "Mechanism of Bamboo mosaic virus satellite RNA replication." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/52501867289919365024.
Full textAbstract:
博士國立中興大學
生物科技學研究所
96
Bamboo mosaic virus satellite RNA (satBaMV RNA) is the only example of satellite RNA present in the potexvirus group. It is a single-stranded RNA molecular of 836 nucleotide (nt) (excluding the 3′ poly(A) tail) flanked by 159 nt of the 5′-untranslated region (UTR) and 125 nt of the 3′-UTR and shares no significant homology with BaMV RNA. SatBaMV RNA depends on BaMV for replication, encapsidation, and spread between the host cells. In vitro replication assays of satBaMV RNA were performed with RNA-dependent RNA polymerase (RdRp) complexes isolated from BaMV-infected Nicotiana benthamiana. The BaMV RdRp complexes not only correctly recognize promoter sequence of full-length positive- (+) or negative- (-) strand satBaMV RNA transcript for synthesis of complementary RNA but also complete the satBaMV RNA replication cycle. This revealed that the satBaMV and BaMV might use the same replication complexes for RNA synthesis. The same procedure was used for partial purification of Foxtail mosaic virus (FoMV) and Potato virus X (PVX) RdRp complexes and then for analysis of satBaMV RNA synthesis in vitro. Both of them could catalyze (+)- and (-)- satBaMV RNA for complementary RNA synthesis. Nevertheless, the in vivo inoculation tests revealed that besides BaMV, the replication, encapsidation, and movement of satBaMV were only supported by FoMV; however, the RNA accumulation efficiency is much lower than that by BaMV supporting. One possibility was that the ability of encapsidation of satBaMV RNA by FoMV coat protein was not sufficient for stabilizing satBaMV RNA in cells. FoMV infects at least 56 plant species in the Gramineae as well as a number of species in 35 dicotyledonous families. The satellite-supporting ability of FoMV was exploited in an attempt to extend the host range of satBaMV as a vector system. The 3′-UTR of satBaMV contains the cis-acting elements for replication: 1. secondary structure of satBaMV 3′-UTR comprises three stem-loops, design as SLA, SLB, and SLC. 2. conserved hexamer motif, ACCUAA, and polyadenylation signal AAUAAA. Disruptions of the secondary structures or deletions of the conserved sequences significantly decrease satBaMV RNA accumulation in plants. Analysis of natural satBaMV isolates revealed the conservation of the 3′-UTR structures which are crucial in the life cycle of satBaMV. BaMV and satBaMV 3′-UTRs share similar secondary structures and conserved elements; however, satBaMV 3′-UTR dose not contain the pseudoknot structure, which locates in the BaMV 3′-terminus and is required for BaMV RdRp binding and replication. Replacement of the pseudoknot structure or whole 3′-UTR of BaMV to satBaMV 3′-UTR interfered with the function of satBaMV RNA replication, implying that the recognition motif in the 3′-UTR and the replication mechanism of satBaMV might be different from that of its helper, BaMV RNA, and perhaps involve different host factors specifically for BaMV and satBaMV RNA replication.
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Chernysheva, Olena. "Modular arrangement of viral cis-acting RNA domains in a tombusvirus satellite RNA /." 2005. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR11767.
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Thesis (M.Sc.)--York University, 2005. Graduate Programme in Biology.Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR11767
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LIAO, FANG-MING, and 廖芳明. "Effects of satellite RNA on translation of cucumber mosaic virus RNAs in vitro." Thesis, 1988. http://ndltd.ncl.edu.tw/handle/65340866783298291951.
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Masuta, Chikara. "STUDIES ON SATELLITE PANICUM MOSAIC VIRUS (SPMV) AND CUCUMBER MOSAIC VIRUS (CMV) SATELLITE RNA (STRAIN Y)." Doctoral thesis, 1989. http://hdl.handle.net/2115/32777.
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Lin, Meng-Chi, and 林盟旗. "The sequence of bamboo mosaic virus satellite RNA involved in." Thesis, 1996. http://ndltd.ncl.edu.tw/handle/37080804087158660932.
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碩士國立中興大學
農業生物科技學研究所
84
RNA病毒的包被 (encapsidation) 在病毒的生命週期中扮演著重要的角 色，包被是否完整直接影響病毒的穩定性，自然也就影響到此病毒的移 動及感染力，若能了解包被過程的機制，在病毒防治及病毒載體構築方 面都會有所幫助。自1950年，菸草嵌紋病毒 (tabacco mosaic virus) 包 被機制的研究開始後，陸續有許多病毒包被的模型被發表。而在病毒包 被研究的方法上，除了直接在核酸或鞘蛋白上作突變外，隨病毒產生的 小片段核酸亦可提供重要的訊息，如衛星核酸(satellite RNA) 及DI RNA (defective interfering RAN)，都是被研究的對象。這篇報告所使用的 材料 －竹嵌紋病毒衛星核酸 (bamboo mosaic virus satellite RNA, SatBaMV)， 為一段長度836核甘酸 (不含poly (A) tail) 的單股RNA， 在1994年由 林納生及徐堯煇博士自感染竹嵌紋病毒 (bamboo mosaic virus) 的泰山 竹 (Bambusa vulgaris) 中分離所得，是目 前已知的potexvirus群中第一 株衛星核酸，它在感病植物中由竹嵌 紋病毒外鞘蛋白 (coat protein) 包 被，但是其核酸中除了在5'端 有零星的核甘酸和竹嵌紋病毒相似外，並 無其它同源性，這自然 提供一個研究竹嵌紋病毒包被機制的系統。本篇 研究的主要目的 是確定此衛星核酸包被訊息區的位置，故以膠體阻滯實 驗為方法 (gel retardation assay)，先用不同來源的病毒鞘蛋白和 SatBaMV全長的RNA結合，包括自竹嵌紋病毒-L (BaMV-L)、竹嵌紋 病毒-S (BaMV-S)、馬鈴薯X病毒 (PVX)、菸草嵌紋病毒 (TMV) 及胡 瓜嵌紋病毒 (CMV) 純化的鞘蛋白，亦利用由大腸菌系統表現的竹嵌紋 病毒-O、-L及-S鞘蛋白，其中以竹嵌紋病毒-L鞘蛋白的結合力最強， 以此證明包被的專一性。接著將不同大小的衛星核酸3'端及5'端片段和 竹嵌紋病毒-S鞘蛋白反應，試圖找出包被訊息區 (signal region) 的位 置，結果顯示3'端的結合力較強；這和以往potexvirus的報告不同，故 再將3'及5'端的RNA作競爭力分析，但是每一片段之間的結和力並無 明顯差異，或許在植物體中，有其它因子參與此衛星核酸的包被。 For RNA viruses, the encapsidation is an essential step in their life cycle. It provides a direct protection of viral RNA and relates greatly to viral infection and movement. Since the first report of tobacco mosaic virus (TMV) assembly was carried out in 1950's, many models of virus encapsidation were proposed. In the study of encapsidation mechanism, although direct deletion and mutation of viral RNA and coat protein are the most straight forward strategy, some small viral RNA fragments can provide a better system for this research, such as DI RNAs (defective interfering RNAs) and satellite RNAs. The RNA fragment used in this study, bamboo mosaic virus satellite RNA (SatBaMV) isolated with BaMV-V from infected Bambusa vulgaris (Lin and Hsu., 1994), was the first satellite RNA identified among potexviruses. This SatBaMV, in the length of 836 nucleotides excluding ploy (A) tail, can be encapsidated by BaMV coat protein. However, except the 5' GAAAAC and some identical short stretches scattered on the very 5' end, there is no sequence homology between SatBaMV and BaMV RNAs. Therefore SatBaMV provides a good system to study the encapsidation mechanism of BaMV. In this study, we aimed to define the signal region of SatBaMV involved in coat protein binding. First at all, the binding capacity assays of several viral coat proteins to SatBaMV full length RNA were carried out to confirm the specificity of the SatBaMV RNA binding. The coat proteins used were either directly isolated from purified virus particles, including BaMV-L, BaMV-S, potato virus X (PVX), cucumber mosaic virus (CMV), or purified from E. coli overexpression system, including coat proteins of BaMV-O, -L and -S. The coat protein from BaMV-L presented the strongest binding capacity among them. Gel retardation assay of the binding of 3' end and 5' end SatBaMV RNA fragments to BaMV-S coat protein was followed to elucidate the location of binding signal region. A different result from that of PVX was obtained. RNA fragments of the 3' end seem to provide a better affinity than that of the 5' end. To verify this, competition assays of these two regions were performed with several truncated RNA fragments. As the results shown, the affinity of these fragments binding to BaMV-S coat protein was the same. It might be that, the encapsidation of this SatBaMV needs factors other than BaMV coat protein in infected plant cells.
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Rogalska, Tetyana. "Requirement(s) for the Replication of Lucerne Transient Streak Virus Satellite RNA." Thesis, 2012. http://hdl.handle.net/1807/33510.
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The satellite RNA of Lucerne Transient Streak Virus (LTSV) is a 322-nucleotide, single-stranded circular RNA that has a rod-like structure very similar to that of viroids. As it does not encode any translation products and cannot replicate independently of a helper virus, the satellite RNA is proposed to rely on viral-encoded proteins for the replication and/or cell-to-cell movement that facilitate its systemic infection in a host. To investigate the requirements for replication of the LTSV satellite RNA, transgenic plant systems were generated to express the viral RNA-dependent RNA polymerase and predicted viral transport protein independently as well as in combination. Results of infectivity assays of these transgenic lines demonstrated for the first time that the viral-encoded RNA-dependent RNA polymerase is necessary and sufficient for the replication of LTSV satellite RNA, and that no additional viral proteins are required for its cell-to-cell or systemic transport.
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Chu, Allen Wing Ho. "Preparation of a Multi-Part Varkud Satellite Ribozyme Variant for Kinetics Studies." Thesis, 2009. http://hdl.handle.net/10012/4710.
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The Varkud Satellite (VS) ribozyme is the largest of the “small” nucleolytic
ribozymes and is the only one for which there are no high resolution crystal structures
available. The VS ribozyme comprises a catalytic domain and a substrate domain. The
catalytic domain includes five helices that interact with the stem-loop substrate.
The substrate is docked within a cleft that is formed by helices II and VI. This
naturally brings the cleavage site in close proximity to the A730 loop in helix VI. The
adenines within the A730 loop are very crucial to the cleavage reaction and any
substitution causes a major decrease in the cleavage activity of the ribozyme.
This study is aimed at designing and producing a variant of the Varkud Satellite
ribozyme that consists of multiple parts that can be used for detailed studies of
ribozyme kinetics and assembly.
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Chang, Chih-Hao, and 張志豪. "Long-distance trafficking of Bamboo mosaic virus satellite RNA in plant." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/3aczvd.
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Wang, Guan-Zhi, and 王冠智. "Association of Bamboo mosaic virus satellite RNA encoded P20 with mitochondria." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/72801568774076713448.
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碩士國立中興大學
生物科技學研究所
96
Bamboo mosaic virus (BaMV), is a single-stranded positive-sense RNA virus with flexuous rod-shaped morphology. The satellite RNA (full-length 836 nt) associated with BaMV, designated as satBaMV RNA, encodes a 20 kDa nonstructural protein (P20). Using several programs to predict the subcellular locatizaion of P20, we found transmembrane domain and mitochondrial targeting signal (MTS), typical viral proteins that can localize on mitochondria. We used discontinuous percoll gradient to isolate mitochondria of Nicotiana benthamiana. The mitochondrial fraction was confirmed by a mitochondrial specific dye Mitotracker Orange CMTMRos and antibody of cytochrome c. Using western blot analysis P20 was detected in mitochondrial enriched fraction from satBaMV transgenic line 2-6 that was inoculated with BaMV. Protoplasts were prepared from inoculated leaves of N. benthamiana co-infected with BaMV-S viral RNA and in vitro transcripts of pBSGFP, pBSGFPP20 and pBSP20GFP, respectively. By Confocal microscopy, protoplasts from pBSGFPP20 infected leave, similar to pBSGFP displayed the green fluorescence which scattered in cytoplasm. The protoplasts contained P20GFP had obvious GFP signal that co-localized and surrounded with mitochondria of diameter at least 2μm at low expression, but the fluorescence were too strong to recognize if P20GFP had transported into mitochondria. When P20GFP gradual accumulated, the phenomenon which green fluorescence co-localize with mitochondria was lost and the 2μm diameter mitochondria were disappeared. The Protease K protection assay showed the mitochondrial membrane could be preserved completely 7 days after isolation. However P20 within mitochondrial fraction from N. benthamiana line 2-6 challenged with BaMV-S could not be protected by mitochondrial membrane after Protease K treatment. No appearance of products once MTS predicted by MitoProt II were cleaved as most of mitochondrial import mechanism. Our data support a hypothesis that P20 of satBaMV interacted with mitochondria that may be just at early phase of viral life cycle.
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Tsai, Ming-shiun, and 蔡明勳. "Characterization of Bamboo Mosaic Potexvirus Satellite RNA-encoded Non-structural Protein." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/70455099042047193334.
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博士國防醫學院
生命科學研究所
88
A satellite RNA of about 836 nucleotides in length (excluding poly (A) tail) depends on the bamboo mosaic potexvirus (BaMV) for its replication, movement and encapsidation. The BaMV satellite RNA (satBaMV) contains a single open reading frame (ORF) encoding a 20 kDa nonstructural protein (P20). Although P20 is not essential for satellite RNA replication (Lin et al., 1996), it plays assistant roles in replication and systemic movement of satellite RNA itself. The ORF of P20 is conserved in all satBaMV variants that we have sequenced (Liu et al., 1997). We chose the BSF4 (Lin and Hsu, 1994) and BSL6 (Liu et al., 1997) satBaMV variants as the satBaMV sequences and P20 sources for biochemical studies. These two satBaMV variants display distinctly biological properties in satBaMV systemic movement, repression of BaMV accumulation, and attenuation of symptom expressed by BaMV (Hsu et al., 1998). Recombinant P20 was over-expressed in Escherichia coli cells and purified to be nearly homogenous. Experiments of gel-retardation, UV cross-linking, and Northwestern hybridization identified that P20 was a nucleic acid binding protein. The binding of P20 to nucleic acids was strong, highly cooperative, and with preferential sequences. P20 preferred binding satBaMV or BaMV-related rather than non-related sequences. The P20 binding sites on satBaMV RNA were mainly located in the 5'' and 3'' untranslated regions (UTRs), and the RNA-protein interactions could be competed with poly(G) RNA, less efficiently with poly(U) RNA, but not with poly(A) and poly(C) RNAs. The N-terminal arginine-rich motif was proven to be the RNA binding domain of P20. This is the first report that a plant viral satellite RNA-encoded nonstructural protein binds nucleic acids in vitro (Tsai et al., 1999). Pre-incubation with P20 but not P18, N-terminal 15 amino acid trucation of P20, significantly inhibited the in vitro translation of satBaMV RNA. The efficiencies of translational repression by P20 were correlated with the affinities of P20 to RNAs, ie. more efficient on satBaMV RNA than the RNA templates containing the 5’ and 3’ UTRs of satBaMV RNA, and the least on other unrelated RNAs. P20 showed no significant influence on the stabilities of interacting RNAs. Therefore, the inhibition of RNA translation was due to the cooperative binding of P20 on RNAs. The strong protein-protein interactions of P20 itself were identified by yeast two-hybrid system analysis, but P20 seemed to have no direct interaction with BaMV-encoded proteins, including BaMV capsid protein. By deletion analysis, the strong P20-P20 interaction needed the predicted eight-stranded b—sheet structures of P20. The C-terminal 33 amino acid residues played no role in P20-P20 interaction, but the deletions that influenced or abolished the formation of predicted b—sheet structure greatly reduced or abolished the P20-P20 interaction. The results of Far-western hybridization also showed the strong P20-P20 interaction. When the purified P20 was concentrated to approximately 1 ug/ul, the protein bands representing the dimer, trimer, and multimer forms of P20 were found by SDS-PAGE and Western blotting analysis. Furthermore, the in vitro translated P20 was chemically cross-linked, and then the protein suggested to be the dimer form of synthesized P20 was found. The high molecular weight protein complexes containing P20 were also identified. The expressed P20 in infected young bamboo leaves was detected with three major degraded products. The accumulation of P20 in petiole exudates of BaMV and satBaMV co-infected N. benthamiana systemic leaves was detected at around 12 days post-inoculation. The detected amounts of P20 were correlated with the movement efficiencies of co-infected satBaMV variants. Thus, the amount of detected P20 in BSF4 infected plants was much higher than in BSL6 infected plants. These results provided indirect evidence to support the role of satBaMV-encoded P20 in the systemic movement of satBaMV RNA. The results of biochemical assays that provide in this thesis may help to explore the physiological functions of satBaMV-encoded P20. 緒言(Introduction)………………………………………………...….1 材料與方法(Materials and Methods)……………………………..13 I. BaMV and satBaMV isolates……………………...…………………………13 II. BaMV and satBaMV cDNA clones………………………………………....13 III. Over-expression of satBaMV-encoded protein in E. coli…………………..13 IV. Preparation of 32P-labeled nucleic acid probes for nucleic-acid-binding assays……………………………………………………………………….16 V. Preparations of viral RNAs and RNA transcripts…………………………...19 VI. Assays of protein-RNA interactions………………………………………..20 VII. Effects of P20 and P18 on RNA translations and stabilities in vitro..……..22 VIII. Identification of P20 interacting proteins and the functional domain on P20-P20 interaction……………………………………………………….22 IX. Analysis of P20 and its degraded products in infected plants……………...26 結果(Results) Section I. The nucleic acid binding properties of P20……………..28 I. Construction, expression, and purification of recombinant proteins……………………………………………………..28 II. Examination of RNA binding activity of purified BSF4 P20 ……………………………………………………………...30 III. The interacting mode of BSF4 P20 with nucleic acids..….30 IV. The movement of purified P20 in agarose gel…………….31 V. The binding strength of BSF4 P20 with BSF4 riboprobe….32 VI. The studies of P20 binding sequences.………………..….34 VII. The main binding sites of P20 on satBaMV RNA……….33 VIII. The nucleic acid binding domain of BSF4 P20…………35 IX. Comparison of binding affinities of P20 from BSF4 and BSL6 satBaMV variants with 5’ positive-sense sequences of BSF4 and BSL6 RNAs……………………………………36 Section II. The effects of P20 binding on RNA translations and stabilities in vitro…………….………………………..39 I. Inhibition of BSF4 RNA translation by P20 binding in vitro ……………………………………………………………..39 II. The sequence-dependent inhibitions of RNA translations by P20 pre-incubation………………….……..………………39 III. The effects of P20 binding on RNA stabilities in vitro…...40 Section III. Identification of P20-P20 interaction and detection of P20 in infected plants……………..………………………..42 I. Identification of P20-P20 interaction by yeast two-hybrid system……………………………………………………42 II. Identification of protein-protein interaction domain on P20 …………………………………………………………….42 III. Analysis of P20-P20 interaction by Far-western hybridization ……………………………………………………………43 IV. Detection of P20 multimer in vitro………………………44 V. Analysis of protein-protein interaction of P20 by chemically cross-linking………………………………………………45 VI. Detection of P20 in vivo…………………………………45 討論(Discussion)…………………………………………………...48 Purification of His-Tag fusion P20……………………………….48 Purified P20 indeed to be an RNA binding protein in vitro………49 The nucleic-acid binding domain of P20…………………………50 The P20 binding sequences………………………………………50 Comparison of the nucleic acid binding properties of P20 with plant viral movement proteins and capsid proteins…………………….52 The specific inhibition of satBaMV RNA translation by P20 binding in vitro…………………………………………………………….53 Differentiation of P20 affinities on the 5’ ends of BSF4 and BSL6 satBaMV RNAs…………………………………………………..55 Comparison of the biochemical properties of P20 with plant viral MPs and CPs………………………………………………………56 Detection of P20 in infected plants……………………………….57 參考文獻(References)……………………………………………..59
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LIN, FANG-RU, and 林芳如. "Satellite RNA of cucumber mosaic virus:its effects on replication and cDNA cloning." Thesis, 1988. http://ndltd.ncl.edu.tw/handle/93520395337991761535.
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Yen, Sih-Min, and 顏思敏. "Molecular evolution and sequence analyses of populations ofBamboo mosaic virus satellite RNA." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/07354761704619427794.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
96
Satellite RNA associated with Bamboo mosaic virus (satBaMV) are subviral agents depending on BaMV for replication, encapsidation and systemic movement. SatBaMV is a linear RNA molecule of 836 nucleotides (nt) which contains an ORF for a protein of 20 kDa (P20) flanked by a 5'' untranslated region (UTR) of 159 nt and a 3'' UTR of 125 nt. The secondary structures of the 5'' and 3'' UTR were earlier predicted by the mfold program and confirmed by enzymatic probings and mutational analyses. Natural satBaMV isolates were classified into two major phylogenetic groups, in which one hypervariable (HV) region was identified in the 5’UTRs of those satBaMV isolates, but most of all could fold into a conserved apical stem loop (AHSL) structure. Some interfering satBaMVs significantly reduce BaMV RNAs replication and suppress the BaMV-induced symptoms, whereas others do not. The integrity of the AHSL structures and the specific nucleotides, C60 and 81UGC83 in the internal loops are the major determinants of the downregulation of BaMV replication. P20 is an RNA-binding protein, which facilitates the long-distance movement of satBaMV in Nicotiana benthamiana, but is dispensable for satBaMV replication. Thus, the satBaMVs could serve as excellent materials for studying the interactions among hosts and helper viruses, and the molecules could be developed into vectors for foreign gene expression. In order to understand the stability of the RNA molecules and the evolutionary models, naturally infected bamboo leaves were collected from five species in different locations: Bambusa oldhamii Munro (Bo) in National ChungHsing University, Taichung; B. ventricosa McClure (Bv) in Hainai, China; B.vulgaris Schrad. ex Wendl. var. vulgaris (Bv.v) in Taipei Botanial Garden; Dendrocalamus latiflorus Munro (Dl) in Taipei Botanial Garden and D. latiflorus Munro cv “Meinung” (Dl.M) in Pingtung. The cDNAs of the satBaMVs isolates were cloned and sequenced, which came from the same source were gathered for one population. The control sequences of satBaMVs isolated from N. benthamiana (Nb) leaves were artificially co-inoculated with pBSF4 RNA transcripts and BaMV-S at 7 dpi. Sequence analyses revealed that 67 defective satBaMVs were found in total 479 sequences, among these, 63 defective satBaMVs were derived from the same single internal deletion in the P20 ORF of satBaMV RNA. This defective type was detected in the five populations except for Dl in Taipei Botanical Garden. The satBaMV populations of Dl in Taipei Botanical Garden were identified as the greater sequence diversity. Those constituted one major phylogenetic group, while the other one group was consisted of the other five populations of various host. In addition, six satBaMV populations clustered independently reflecting the strong host specific characteristics on the basis of the sequence. Base frequencies per site in six populations were calculated, host specific marker nucleotides was detected for determining the host source of an unknown satBaMV RNA. Besides, nucleotide and amino acid variations were used to study for the effect upon the structures or the functions of the RNA and protein. Most variations of the 5’UTR occurred in the HV region, concurrent covariations were also found in the stem and loops in the AHSL. However, the sequences of the HV region were conserved within single satBaMV population, and the variation degree of the 5’UTR were not significantly greater than the degree of the P20 coding region and the 3’UTR. 12% satBaMV isolates from N. benthamiana evolved into interfering mutants while their U82 were changed to C82. Variations in the P20 coding regions almost belonged to synonymous substitutions, and there were no significant codon usage bias in the P20. The three-dimensional structure of P20 were predicted by homology modeling, and amino acid substitutions which occurred in the population of Dl in Taipei Botanical Garden locate on different strands of beta-sheets but spatially near with each other. Variations of the 3’ UTR identified from the satBaMV populations Bv and Dl revealed that loops and the linear regions were variable, but not the stems. Approximately, differential selective pressures affected satBaMVs at RNA and protein level in different hosts during the evolutionary processes. The distribution of variation sites in the RNA secondary structure of satBaMV population of Nb suggested that earlier mutations took places in the UTRs but not in the coding region at 7 dpi. It is expected that through the analyses of evolutionary extents of the satBaMV RNA populations, the mechanisms and relationships governing the interactions among host plants and satBaMVs will be elucidated.
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Lee, Ya-Chien, and 李雅倩. "Development of Transgenic Plants Expressing Bamboo Mosaic Potexvirus(BaMV) and Associated Satellite RNA (satBaMV RNA) Replicon for the Studies of Helper Virus-satBaMV RNA Interactions." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/89568209661828741818.
Full textAbstract:
博士國立中興大學
生物科技學研究所
93
The development and application of BaMV- and satBaMV RNA-based vector systems have been limited by the narrow host range of BaMV and the relative low accumulation level of satBaMV RNA. The aims of this study are to combine the transgenic and viral replicons technologies to overcome the shortages, and to search for the alternative helper virus and satellite variants to improve the efficacy of this satellite vector. Nicotiana benthamiana, an experimental host of BaMV, and two non-host, N. tabacum and potato, were transformed with a full-length cDNA copy of the BaMV or satBaMV genome under the control of the 35S promoter. These transgenic lines were used to investigate the replication and movement of BaMV and satBaMV RNA in plants. Replicative and infectious virus particles were expressed in BaMV-transformed N. benthamiana , N. tabacum and potato. BaMV accumulation in leaves of transgenic N. benthamiana was 4-fold higher than that of transgenic N. tabacum. Only one line of transgenic N. benthamiana developed symptoms similar to that of BaMV infection. The replication of satBaMV RNA and expression of P20 were observed in BaMV transgenic N. benthamiana and N. tobacum lines. For satBaMV RNA transgenic N. benthamiana, the expression of satBaMV encoded P20 protein in transgenic N. benthamiana could be induced by BaMV inoculation, and the accumulation level was up to 50-fold greater than in uninoculated transgenic plants. The results indicated that the combination of transgenic technology and viral replicons may enhance the expression level and expand the host range of the satBaMV-based vector systems. Foxtail mosaic potexvirus (FoMV), also a member of the genus potexvirus, infects at least 56 plant species in the Gramineae as well as a number of species in 35 dicotyledonous families. The satellite-supporting ability of FoMV was exploited in an attempt to extend the host range of satBaMV as a vector system. In all virus-host combinations tested, FoMV could support the replication of satBaMV, but showed no better helper activity than BaMV did. Inoculation of satBaMV transgenic N. benthamiana with FoMV revealed that FoMV could indeed support the replication and expression of satBaMV. These results indicated the replication and encapsidation of satBaMV RNA could be supported by a non-cognate helper virus, FoMV. In addition, two natural occurring defective satBaMV RNAs (satBaMV D-RNA) isolated from FoMV-inoculated transgenic plants expressing satBaMV RNAs were molecularly characterized to unveil their biological functions in this study. One of the satBaMV D-RNA was found to be derived from a single internal deletion in the P20 ORF of satBaMV RNA. The satBaMV D cDNA is 730 nts in length and contains a single point mutation at the nucleotide position 106 that results in an open reading frame (ORF) encoding a putative 18 kDa protein encompassing the C-terminal 137 amino acids of the P20. The other satBaMV D-RNA was found to have similar deletion region in the P20 ORF but could only encode an 18 amino-acid peptide corresponding to the N-terminal sequence of P20. These two D-RNA could be supported by the replication machineries of FoMV and BaMV, and both could move systemically with BaMV in Nicotiana benthamiana. This is the first report D-RNAs of satRNA in the Potexviruses to be generated de novo. With the alignments of sequences of wild type satBaMV RNAs near the junction sites of satBaMV D-RNAs, high degree of sequence similarity adjacent to the junction sites was revealed. This result suggested that common mechanism for viral RNA recombination was not only involved in the generations of the D-RNA of BaMV, but also in D-RNA of satBaMV RNA. The experimental systems developed in this study may be applied in the improvement of satBaMV RNA-based vector systems and in the investigation of satRNA-helper virus interactions.
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Chia-Yuan, Yu. "The Study on the Encapsidation of Bamboo Mosaic Virus Satellite RNA in vitro." 2006. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0005-1407200613581900.
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Kuan-YuLin and 林冠妤. "Investigation and application of satellite RNA-mediated interference with Bamboo mosaic virus replication." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/16747777861176879786.
Full textAbstract:
博士國立成功大學
生物科技研究所碩博士班
100
Bamboo mosaic virus (BaMV), a single-stranded, positive-sense RNA virus, infects more than 90 % of cultivated bamboos having pachymorph rhizomes in Taiwan bamboo plantations and caused great economic loss. Satellite RNAs of BaMV (satBaMVs), a single-stranded, positive-sense RNA molecules, depend on BaMV for replication and encapsidation. Some satBaMV isolates, such as BSL6, collected from the natural field, interfere with BaMV replication whereas BSF4 does not. The determinants of BaMV interference have been mapped to a single nucleotide in the apical hairpin stem-loop structure of the satBaMV 5’ untranslated region. However, the underlying mechanism is still unclear. Because RNA silencing functions in antiviral defense, the role of gene silencing in BaMV and satBaMV replication or in BSL6-mediated BaMV interference remains invedtigated. Mutants with deleted or overexpressed genes involved in RNA silencing, such as dicer-like (DCL), argonaute (AGO) and RNA-dependent RNA polymerase (RDR) were used to test the effects on BaMV and satBaMV replication and BSL6-mediated BaMV interference in Arabidopsis thaliana and Nicotiana benthamiana. BaMV and satBaMV levels were increased in dcl 2/4 and dcl 2/3/4 but reduced in dcl 1 A. thaliana. In rdr6 N. benthamiana, the accumulation and movement of BaMV and satBaMV were enhanced. Despite the increased level of BaMV in rdr6 N. benthamiana, the level of BaMV small RNAs (vsRNAs) were lower than that in BaMV-infected wild-type plants. However, the level of satBaMV small RNAs (sat-sRNAs) were related to the satBaMV level. It was known that AGO1 and AGO2 play important roles in antiviral defense, and we found overexpression of AGO 2, 5 and 9 could downregulate BaMV replication. BSL6 mediated the repression of BaMV replication in all the DCLs, AGO1 and RDR6 knockdown mutants. Global analyses of BaMV and satBaMV small RNA profiles revealed that most vsRNAs and sat-sRNAs are 21 and 22 nt in length from both positive and negative polarities. The vsRNAs hot-spot regions differ in N. benthamiana and A. thaliana but not with satBaMV co-inoculation. Only few amount of vsRNAs were detected from plants co-infected with BaMV and BSL6 demonstrated that BSL6 interferes with BaMV replication may take place before RNA silencing. Moreover, BaMV-resistant transgenic N. benthamiana and A. thaliana plants expressing interfering satBaMV were successfully generated, which confers more resistance to viral RNA than with virion infection. The resistance of BSL6 transgenic plants was positively associated with the transcript level of the transgene. Together with previous results (Chen et al, 2012), our studies suggest that BSL6 interferes in BaMV replication through competition for replication complexes with BaMV. In summary, DCL 2/4 and RDR6 are involved in restricting BaMV and satBaMV replication, and satBaMV achieves preferential replication by avoiding RDR-dependent silencing. I propose a model of BSL6-mediated interference of BaMV through replication complex competition. The interfering satBaMV transgenic plants show BaMV resistance, which could be applied in bamboo biotechnology for control of BaMV.
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Li, Hsiang-Chi, and 李湘琪. "Establishment of an in vivo encapsidation system for Bamboo mosaic virus satellite RNA." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/08399398521042504909.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
100
The Bamboo mosaic virus satellite RNA (satBaMV) genome is a positive-sense RNA molecule of 836 nucleotides encoding a single open reading frame for a 20-kDa protein (P20). The P20 facilitates long-distant movement of satBaMV in the host plant. SatBaMV is encapsidated by the BaMV coat protein (CP) into a rod-shape particle, 50-60 nm in length. To investigate the in vivo satBaMV encapsidation mechanism, BaMV replicase, CP, and satBaMV RNA were transiently expressed by agro-infiltration in Nicotiana benthamiana. BaMV CP and satBaMV RNA were detected in infiltrated leaves, and the satBaMV virus-like particles (VLPs) were purified from infiltrated leaves using sucrose density gradient centrifugation. Transmission electron microscopy (TEM) examination showed that satBaMV VLPs have a similar morphology to that of natural isolates, demonstrating that satBaMV could be packaged in a BaMV-freed satBaMV encapsidation system. Western blot analysis demonstrated that both BaMV CP and P20 were detected in satBaMV VLPs. To clarify if P20 is involved in the encapsidation of satBaMV, two satBaMV mutants, BSF6 and BSG3’P20, were co-infiltrated into N. benthamiana plants, along with ORF1 and CP. TEM micrographs showed that satBaMV mutant VLPs could be assembled, suggesting that P20 is not essential in satBaMV encapsidation. The length distribution of satBaMV mutant VLPs without or with chloroform treatment shows that BSF4 VLPs length ranged from 21-120 nm, with a peak shown at 61-80 nm; BSF6 VLPs length is mostly of about 61-80 nm, and some VLPs with a length above the 120 nm regions are one to three times the 61-80 nm length. The length of BSG3’P20 VLPs came out to be shorter than 120 nm after chloroform treatment, and was found around the 81-100 nm region. To investigate the existence of proteins on the surface of satBaMV VLPs, immunoelectronic microscopy (IEM) was used. The results show that P20 exists on the surface of satBaMV BSF4 VLPs and that most of them are located at the terminal end of the VLPs. In the presence of BaMV TGBp1, TGBp1 protein could also be detected at the terminal end of the VLPs.
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CHEN, WEI, and 陳薇. "Effect of delection Mutation upon replication of satellite RNA of cucumber mosaic virus." Thesis, 1992. http://ndltd.ncl.edu.tw/handle/92315042499248442628.
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Yu, Chia-Yuan, and 游佳原. "The Study on the Encapsidation of Bamboo Mosaic Virus Satellite RNA in vitro." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/18324350326990249274.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
94
Bamboo mosaic virus satellite RNA (satBaMV), a single-stranded RNA of 836 nucleotides [excluding poly(A) tail] and encoding for an open reading frame (ORF), depends on the helper virus for replication, movement and encapsidation. Its ORF can be replaced with non-homologous genes as a vector system for foreign gene expression. The study on an encapsidation signal of satBaMV will be helpful for increasing RNA stability and the expression level of carrying gene. In this study, the location of specific packaging sequence was determined by the analysis of in vitro assembly with truncated satBaMV RNA and Bamboo mosaic virus (BaMV) coat protein. The coat proteins were purified from two sources including E. coli-expressed recombinant coat protein (rCP) and plant-produced viral coat protein (vCP). The assembled particles of satBaMV RNA with BaMV rCP was similar to that with vCP protein under electron microscope. This result confirmed that satBaMV RNA was encapsidated by BaMV coat protein with the morphology as a short rod-shaped particle. Besides, the rCP protein formed longer empty particles in the absence of RNA in the buffer with low pH and the particle length was restricted as the wild type in the presence of satBaMV RNA. Two of the unrelated viral RNA transcripts, including Cucumber mosaic virus satellite RNA and Tobacco mosaic virus coat protein gene coding RNA, were used as negative controls of assembly specificity. Based on the particle length corresponding to the length of packaged RNA, specific packaging sequence was found to be within the nucleotide 439-489 of satBaMV RNA by comparing assembly reactions of satBaMV deletion mutants. Although the condition of in vitro assembly analysis was not specific enough to get a significant result, it implied a vaccine application using epitope-fusion coat protein to produce assembled particles as larger immunogens that can trigger a strong immune response.
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Lin, Choy-Chieng, and 林翠芊. "The Systemic Spread of Bamboo Mosaic Virus and its Associated Satellite RNA in Plants." Thesis, 1997. http://ndltd.ncl.edu.tw/handle/51660988645516495611.
Full textAbstract:
碩士文化大學
生物科技研究所
85
Bamboo mosaic virus (BaMV) has a genome consisting of single-stranded, positive-sense RNA. The virus is a member of the potexvirus group. Most susceptible bamboo species belong to the genera Bambusa and Dendrocalamus. Satellite BaMV (satBaMV) is the only one known to be associated with potexvirus group. Two satBaMVs, BSF4 and BSL6, with a 6.9 % difference in nucleotide sequence, exhibit distinct phenotypes in infected plants. BSF4 has little effect on BaMV RNA replication nor of symptom expression caused by BaMV. Moreover, BSF4 spreads in tobacco systemically and efficiently. In contrast, BSL6 not only reduces replication and delays systemic symptom development but also is inefficient for long distance transport in tobacco (Nicotiana benthamiana). Immunogold-silver staining of the infected tissues was used for localization of BaMV capsid protein in systemically infected bamboo plants. The roll leaves and young stems of common bamboo (Bainbusa vulgaris) infected with BaMV-V (with satellite) and green bamboo {B. oldhamii) infected with BaMV-O (free of satellite) were embedded in paraffin, processed for immuno-staining, reacted with anti-BaMV-O virion serum and labelled with immunogolds followed by silver enhancement for visualization. The localization of silver aggregates was found to be limited in some areas showing a mosaic-like pattern. The virion or coat protein could be detected frequently in the leaf mesophyll, epidermal cells, bundle sheath extension fibers, fusoid cavities and bundle sheaths occasionally observed in the metaxylem, xylem parenchyma cells, xylem fibers, sieve elements and companion cells. However, silver aggregates in stem sections were detected in parenchyma cells surrounding vascular, protoxylem and epidermal cells. Moreover, sections of leaf sheath showed silver aggregates in epidermal cell, bundle sheath extension fibers, parenchyma cells, bundle sheaths, parenchyma cells of xylem, fiber of xylem, sieve elements and companion cells. Preimmune serum was used as control and no labelling was observed. The metaxylem, bundle sheath extension fibers, fusoid cavities, protoxylem and fiber of xylem are dead cells physiologically but labels were frequently observed in these structures. To analyze the difference in systemic movement of the two isolates of satellite RNA, tobacco plants were infected with BaMV-S RNA, BaMV-S RNA with BSL6, BaMV-S RNA with BSF4, respectively. Inoculated and systemic leaves were harvested at different time intervals for the detection of viral RNA and satBaMV using dot blot hybridization. At 16 days post-inoculation (d.p.i.), viral RNA was detectable in systemic leaves of tobacco. Nine out of ten plants coinfected with BSF4 transcripts showed the presence with satBaMV whereas only 1 out of 10 plants coinfected with BSL6 was positive for satBaMV in the upper non-inoculated leaves. At second passage, all of the plants coinfected with BSF4 showed the presence of satellite at 20 d.p.i. wheresa 4 out of 10 plants coinfected with BSL6 were positive for satBaMV. After the third passage, 7 out of 7 and 10 out of 15 plants were positively detected with BSF4 and BSL6, respectively, in their coinfected systemic leaves at 14 d.p.i. . These results indicated that the third passage showed the fastest movement systemically for BSL6 satBaMV. To verify whether the satRNA in the systemic leaves is BSF4 or BSL6 in every passage, restriction enzymes were used to map the cDNA derived from reverse transcription-polymerase chain reaction of total RNAs from each samples. The results indicated that no evident changes in satRNA sequence was found during the whole process.
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Li, Qing-Wei, and 李青蔚. "Establishment and resistance assay of transgenic tobacco plants expressing cucumber mosaic virus satellite RNA." Thesis, 1993. http://ndltd.ncl.edu.tw/handle/83351486599364168676.
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Liou, Ming-Ru, and 劉命如. "Development of Bamboo mosaic virus and its satellite RNA for virus-induced gene silencing vector." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/79895521521130039465.
Full textAbstract:
博士中興大學
生物科技學研究所
99
Virus-induced gene silencing (VIGS) is recently used to analyze gene function in plants since it contains the advantages of rapid, efficient, and specific. The satellite Bamboo mosaic virus RNA (satBaMV) is the only one found in the potexvirus group, which has become a successful plant expression vector. To develop satBaMV-induced gene silencing as a tool for identification of host factors associated with Bamboo mosaic virus (BaMV) life cycles and analysis of gene function in plants, in this study, we have assessed the significance of sulfur gene (SU) essential for chlorophyll synthesis against yellow-photobleaching full-length cDNA by using satBaMV vector. Nicotiana benthamiana plants were co-inoculated with clones of BaMV and chimeric satBaMV by mechanical inoculation using plasmid DNA as inocula. The phenotype of N. benthamiana plants inducing VIGS of endogenous SU gene at 14 days post inoculation, showed silencing areas in the vein vicinity. The satBaMV vector also triggered gene silencing by agro-infiltration method. Compare with Tobacco rattle virus (TRV), the advantage of satBaMV vector could prevent the silencing plant to die, and when essential genes for plant development, such as heat shock protein 90 (Hsp90) and Hsp70 were silenced. In addition, the satBaMV vector could also induce gene silencing in temperate grass model plant of monocot purple false brome (Brachypodium distachyon). It could be used for studies of comparative functional genomics in both dicotyledonous and monocot plants. To compensate some genes targeted for gene silencing without displaying a visible phenotype, we simultaneously developed BaMV-induced gene silencing vector. It could induce gene silencing in tobacco, purple false brome, and barley (Hordeum vulgare). When the green fluorescent protein (GFP) transgenic N. benthamiana were co-inoculated with BaMV- and satBaMV-VIGS vector carrying genes of SU and GFP, respectively, the results demonstrated that simultaneously silencing of both genes can be accomplished. This system provides a genetic tool to investigate two genes in a single test. Further, in order to confirm RNA dependent RNA polymerase 6 (RDR6) effect on efficiency of BaMV- and satBaMV-VIGS vector, the RDR6-deficient N. benthamiana were inoculated with BaMV-VIGS vector or co-inoculated BaMV and satBaMV-VIGS vector. The results showed that RDR6-deficient N. benthamiana plants exhibited severe mosaic and stunting symptoms. Northern hybridization analysis revealed that the viral RNA of BaMV and satBaMV could be accumulated in the top-most leaves of RDR6-deficient N. benthamiana plants. The efficiency of BaMV-induced gene silencing was remarkable reduced, while the efficiency of satBaMV-induced gene silencing was relatively increased. The data supported that RDR6 gene is essential for BaMV and satBaMV to invade apical growth point. To the best of our knowledge, this is the first development of satellite RNA based vector system for virus-induce gene silencing applicable in monocotyledons and dicotyledonous.
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Kong, Qingzhong. "Plant resistance and symptom modulation by a satellite RNA in turnip crinkle virus/Arabidopsis system." 1996. https://scholarworks.umass.edu/dissertations/AAI9709616.
Full textAbstract:
In this dissertation, I report my studies on the resistance of Arabidopsis thaliana ecotype Dijon (Di-0) to turnip crinkle carmovirus (TCV), a simple, single-stranded, positive-sense, plant RNA virus. TCV efficiently replicated in protoplasts prepared from Di-0 callus culture, but it did not move long-distance in Di-0 plants (Simon et al., 1992). Cardamine chlorotic fleck virus (CCFV), the carmovirus most closely related to TCV, infected Di-0 plants systemically (Oh et al., 1995). I found that TCV with the coat protein open reading frame (ORF) from CCFV (TCV-CP$\rm\sb{CCFV})$ and TCV with a single base mutation in the initition codon (AUG to ACG) of the coat protein ORF (TCV-CPm) were infectious on ecotypes Columbia (Col-0, TCV-susceptible) and Di-0 (TCV-resistant). These results indicate that TCV coat protein is the viral determinant for resistance of Di-0 to TCV. In addition, my results suggest that the resistance of Di-0 to TCV is specific to TCV and may involve an RNA degradation activity. I also studied the movement of TCV, CCFV, and TCV-CP$\sb{\rm CCFV}$ in Col-0 and Di-0 plants using a whole plant in situ hybridization technique. The results indicate that TCV was restricted to within small areas around the initial infection sites on inoculated leaves of Di-0 plants, whereas TCV (in Col-0), CCFV and TCV-CP$\sb{\rm CCFV})$ (in Col-0 and Di-0) moved long-distance to metabolic sink tissues (young leaves and roots) at different rates. Many plant RNA viruses are associated with small subviral RNAs, including satellite (sat-) RNAs. Small subviral RNAs require a helper virus for replication and movement, and they often intensify or attenuate the symptoms caused by the helper virus. TCV is associated with sat-RNA C, a sat-RNA that normally intensifies the symptoms of the TCV-M isolate (Simon et al., 1988; Li and Simon, 1990). I will report my work on the mechanism of symptom modulation mediated by sat-RNA C in Arabidopsis plants. My results indicate that symptom modulation by sat-RNA C is mediated by the viral coat protein, and a putative interaction between the 3$\sp\prime$ end of sat-RNA C and the N-terminus of coat protein is involved. Symptom attenuation by sat-RNAs is widely believed to be mediated by inhibition of helper virus replication through competition for replication factors (Roossinck et al., 1992). My study shows that inhibition of virus long distance movement is involved in sat-RNA C-mediated symptom attenuation of TCV-CPm (and probably TCV-CP$\rm\sb{CCFV})$ and inhibition of helper virus replication is not important. In addition, symptom attenuation mediated by sat-RNA C is localized and does not involve the major plant defense pathway. A model that explains sat-RNA C-mediated symptom attenuation is proposed.
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Mao, Yie-Chie, and 毛乙智. "The Study on the Interaction between Coat Proteins and Satellite RNA of Cucumber Mosaic Cucumovirus." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/55327153997726051916.
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碩士國立中興大學
農業生物科技學研究所
89
The interactions between satellite RNAs (satRNAs) and coat proteins of the helper virus, cucumber mosaic cucumovirus (CMV), were studied to elucidate the corresponding regions in coat proteins and satRNAs responsible for recognition. This work is of vital importance for the successful development of satRNA-based transient expression system. To facilitate efficient detection and analysis of interactions between nucleic acids and protein molecules, a system based on enzyme linked immunosorbent assay (nucleoprotein binding-ELISA, NB-ELISA) was developed and applied to the fast and efficient analysis of interactions between satRNAs and coat proteins of CMV. The satRNA from CMV-C strain (C-sat) and coat proteins of CMV-NT9 strain were used to optimize and standardize the NB-ELISA procedures. A positive, linear correlation was observed between the concentrations of coat proteins of CMV and the observed OD405 value. The C-sat showed binding preference for CMV coat proteins over non-related proteins, such as tobacco mosaic virus coat proteins, bovine serum albumin, and healthy plant extracts. Sucrose density gradient centrifugation assay indicated that the satRNA might attach to the outer surface of the virus particles, besides being encapsidated inside. The result of time course study suggested the binding of satRNAs to coat proteins proceeded in a cooperative manner as a function of time. The coat protein genes of CMV-NT9, CMV-Gem and CMV-M48 strains were cloned and sequenced for further investigation of interactions with satRNAs. The N-, C-terminal and internal fragments (from amino acid number 1 to 76, 176 to 218, and 77 to 169, respectively) of CMV-NT9 coat proteins were subcloned into plasmid pETblue2 for efficient expression in Escherichia coli to study the regions on the coat proteins responsible for the interaction with satRNAs. The N-terminal fragment of coat protein was shown to be the sole required region to recognize satRNAs. Terminal and serial internal deletion mutants of C-satRNA were used to investigate the nucleotide sequences that are required for the proper recognition by the CMV coat proteins. The result indicated that CMV coat proteins interacted with both DNA and RNAs, single- or double-stranded, without evident specificity. These results provided further insight into the biological functions of satRNAs to facilitate the better design of efficient gene-transfer vectors and disease management strategies.
50
Liu, Kuang-Yang, and 劉光仰. "Effect of P20/GFP fusion on cell to cell movement of Bamboo mosaic virus satellite RNA." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/94102388791107864334.
Full textAbstract:
碩士國立中興大學
生物科技學研究所
100
Satellite RNAs (satRNAs) are subviral agents which depend on helper viruses and host factors for replication, encapsidation and movement. Bamboo mosaic virus (BaMV), a member of the genus Potexvirus, is the only potexvirus that naturally associates with a satRNA, designated satBaMV, which encodes a 20 kDa RNA-binding protein (P20) involved in efficient long-distance movement and replication of satBaMV. The aims of this study were to investigate the abilities of other non-cognate viruses in supporting the replication of satBaMV and to explore the effects of P20 phosphorylaion on the movement of satBaMV. Dimeric satBaMV transgenic plant lines expressing P20 fused with green fluorescent protein (GFP) either at N- or C- terminus were generated. BaMV and two non-cognate viruses, Potato virus X (PVX) and Foxtail mosaic virus (FoMV), were inoculated onto the transgenic plant to monitor their abilities in supporting satBaMV. Through the examination of GFP expressions in the inoculated transgenic plants, it was confirmed that the non-cognate helper FoMV marginally supports the replication of satBaMV, whereas PVX does not. Plasmids harboring GFP fused to the N- or C-terminus of P20 protein with mutations on the serine at amino acid position 11 (S11) to inhibit or mimic the phosphorylation state of P20 were constructed, and there cell-to-cell movement ability were examined in Chenopodium quinoa by fluorescent microscopy. The results revealed that both mutations of P20 reduced the cell-to-cell movement of satBaMV, corroborating the previous finding that phosphorylation of S11 is important for the expression and movement of satBaMV.