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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
381

Phagentyp-RNA-Polymerasen in Tabak und Arabidopsis

Sobanski, Johanna 24 February 2014 (has links)
Die Transkription in Plastiden höherer Pflanzen erfolgt durch die plastidärkodierte, bakterienähnliche RNA-Polymerase PEP und die kernkodierten Phagentyp-RNA-Polymerasen RpoTp und RpoTmp (NEP). Da für NEP bislang keine Transkriptionsfaktoren identifiziert wurden, wurden die entsprechenden Enzyme aus A. thaliana und N. tabacum mit verschiedenen, N-terminalen Epitopen in E. coli exprimiert und für pulldown assays zur Identifikation interagierender Proteine eingesetzt. Des Weiteren wurden Epitop-markierte Tabak RpoTp und Arabidopsis RpoTp und -Tmp in vivo exprimiert und zur Co-IP verwendet. In diesen Studien wurden als potentielle Interaktionspartner von RpoTp Ycf1 und Ycf2 gefunden. Des Weiteren konnte mit 3xFLAG-RpoT-exprimierenden Arabidopsis-Mutanten gezeigt werden, dass RpoTp und -Tmp teilweise membranassoziiert sind. Außerdem wurde die duale Lokalisation der Arabidopsis RpoTmp in den Chloroplasten und Mitochondrien nachgewiesen. Mittels RIP-Chip wurden mit RpoTp assoziierte RNAs analysiert und mögliche, bisher unbekannte NEP-Transkripte gefunden. Plastidäre Haushaltsgene besitzen meist sowohl PEP- als auch NEP-Promotoren. Anhand transplastomischer Tabakpflanzen, in denen NEP-Promotoren von accD , rpoB und rrn16 gegen einen PEP-Promotor ausgetauscht bzw. durch Mutagenese ausgeschaltet wurden, sollte die Arbeitsteilung von NEP und PEP in Abhängigkeit vom Entwicklungsstadium beleuchtet werden. Dabei wurde gezeigt, dass die Transkription durch PEP für accD zu einer leichten Überexpression, für rpoB hingegen zu einer verzögerten Entwicklung und verringerten Transkriptmengen führte. Zudem wurden durch RNA-Seq die Aktivierung zusätzlicher TSSs in den Mutanten gezeigt, welche die Effekte auf RNA- und Proteinebene erklärte, und der alternative Promotor PaccD-158 identifiziert, welcher auch im Wildtyp genutzt wird. Es wird diskutiert, inwiefern die Rolle von NEP und PEP individuell für einzelne Gene in Abhängigkeit ihrer jeweiligen Funktion betrachtet werden muss. / The transcription in plastids of higher plants is accomplished by the plastid encoded, bacterial-type RNA polymerase PEP and by the nuclear encoded, phagetype RNA polymerases RpoTp and RpoTmp (NEP). As the identification of transcription factors for NEP failed so far, in this work the corresponding enzymes from A. thaliana and N. tabacum containing different, N-terminally fused epitope tags were expressed in E. coli and used for pulldown assays to identify interacting proteins. Furthermore epitope-tagged tobacco RpoTp and Arabidopsis RpoTp and -Tmp were expressed in vivo and applied for co-immunoprecipitation. In these studies Ycf1 and Ycf2 were found as potential interaction partners of RpoTp. In addition, the 3xFLAG-RpoT-expressing Arabidopsis mutants were used to show, that RpoTp and -Tmp are partly associated with the thylakoid membrane. Further, immunoblot assays confirmed the dual localization of the Arabidopsis RpoTmp in chloroplasts as well as in mitochondria. Moreover, via RIP-Chip analyses RNAs associated with RpoTp were analysed and potential new NEP transcripts were found. Most plastidial housekeeping genes possess PEP as well as NEP promoters. The division of labor between NEP and PEP according to the developmental stage was studied on the basis of transplastomic tobacco plants, in which NEP promoters of accD, rpoB and rrn16 have been exchanged with a PEP promoter or knocked out by mutagenesis. It was shown, that transcription of accD by PEP lead to a slight overexpression, but PEP-dependent transcription of rpoB led to a delayed development and decreased transcript levels. Via RNA-seq an activation of additional TSSs could be shown in the mutants, which explains the effects on RNA and protein level, and the alternative promoter PaccD-158 was identified, that is also used in the wildtype. It is discussed, how the roles and the division of labor of NEP and PEP should be considered individually for each gene according to its function.
382

The regulatory potential of marine cyanobacteria

Axmann, Ilka Maria 16 March 2007 (has links)
Das Leben auf der Erde wird maßgeblich durch die Kraft der oxygenen Photosythese bestimmt, die Sonnen- in chemische Energie umwandelt. Cyanobakterien wie Prochloro- und Synechococcus zählen zu den wichtigsten primären Produzenten der Ozeane und werden zunehmend als Modelle für photosynthetische Organismen genutzt. Um die Regulationsmechanismen dieser Picocyanobakterien besser zu verstehen, wurde hier die Information von vier Genomen hochgradig verwandter aber dennoch ökologisch unterschiedlich angepasster mariner Stämme genutzt in einer Kombination aus computer-gestützten und experimentellen Untersuchungen. Sequenzsignale und RNA-kodierende Gene wurden als neuartige Regulationselemente identifiziert und entlang des phylogenetischen Gradienten verglichen. Mittels ''phylogenetic footprinting'' konnte ein minimales, konserviertes Set möglicher Transkriptionsfaktoren, deren Bindestellen und Regulons aufgedeckt werden. NtcA-, LexA- und ArsR-ähnliche Motive wurden ebenso gefunden wie neue regulatorische Elemente. Mit Hilfe von RACE Experimenten wurden einige der vorhergesagten Bindestellen Promotorregionen zugeordnet. Eine Suche nach konservierten Sekundärstrukturen detektierte mehrere nicht-kodierende RNAs, benannt Yfr für cYanobacterial Functional RNA. Eine vergleichende Analyse von Yfr7 innerhalb der cyanobakteriellen Linie ergab, dass diese RNA wahrscheinlich ein Homolog der E. coli 6S RNA ist. Zwei verschiedene Yfr7 Transkripte mit einem zirkadianen aber zeitversetzten Akkumulationsmuster lassen eine Verknüpfung ihrer Expression mit dem zirkadianen Rhythmus oder der Lichtintensität vermuten. Experimente in Synechocystis deckten einen neuartigen Regulationsmechanismus durch eine antisense RNA auf, welche die Menge der isiA mRNA kontrolliert und die Assemblierung von IsiA-Superkomplexen beeinflusst. Die funktionelle Zuordnung dieser neuen Elemente wird zu einem besseren Verständnis regulatorischer Netzwerke in marinen Cyanobakterien und darüber hinaus führen. / Life on Earth is driven by the power of oxygenic photosynthesis transforming solar into chemical energy. Cyanobacteria such as Prochlorococcus and Synechococcus belong to the most important primary producers within the oceans and increasingly serve as models for photosynthetic organisms. To better understand the regulatory mechanisms in these picocyanobacteria, here the information from four genomes of closely related and even so ecologically divergent marine strains was used in a combined computational and experimental approach. Sequence signals and RNA-coding genes as novel elements in the regulation of gene expression were identified and their distribution along the phylogenetic gradient compared. Phylogenetic footprinting revealed a minimal conserved set of putative transcription factors, their binding sites and regulons. Sites for NtcA, LexA and ArsR-like regulators were found as well as new cis elements. RACE experiments verified several of these predicted sites belonging to the promoter region. A search, focussing on conserved secondary structures, detected several non-coding RNAs named Yfr for cYanobacterial Functional RNA. A comparative analysis of Yfr7 structures, transcript types and accumulation throughout the cyanobacterial radiation indicated this RNA as the likely homologue of the E. coli 6S RNA. Two distinct Yfr7 transcripts with a circadian but time-shifted expression pattern suggested a coupling of their expression to the circadian rhythm or light intensity. Experiments in Synechocystis discovered a novel antisense RNA-mediated regulatory mechanism that controls isiA mRNA abundance and assembly of IsiA-photosystem I supercomplexes. Functional assignments of these new elements in the future will contribute to a deeper understanding of the regulatory network of marine cyanobacteria and promote new studies on bacterial ncRNAs.
383

Analyse von Komponenten der organellären Transkriptionsmaschinerien aus Arabidopsis thaliana und Nicotiana tabacum

Bohne, Alexandra-Viola 21 August 2009 (has links)
Die Gesamtheit mitochondrialer Gene sowie ein Teil der plastidären Gene photosynthetischer Eukaryoten wird durch kernkodierte Phagentyp-RNA-Polymerasen transkribiert. In der vorliegenden Arbeit wurden unter Verwendung eines homologen in vitro-Transkriptionssystems, die spezifischen Funktionen der Phagentyp-RNA-Polymerasen RpoTm, RpoTp und RpoTmp aus Arabidopsis untersucht. Während RpoTmp keine Präferenz für die angebotenen Promotoren zeigte, transkribierten RpoTm und RpoTp eine überlappende Gruppe mitochondrialer und plastidärer Promotoren vielfältiger Architektur. RpoTm und RpoTp präsentierten eine Kofaktor-unabhängige Fähigkeit zur Promotorerkennung bei Angebot superhelikaler DNA-Matrizen. Eine selektive Promotornutzung sowie die Unfähigkeit zur spezifischen Transkription linearer Promotormatrizen in vitro implizieren die Assoziation zusätzlicher, in die Promotorerkennung und/oder DNA-Aufschmelzung involvierter Kofaktoren in vivo. Die in vitro-Erkennung mitochondrialer Promotoren durch eine plastidäre Phagentyp-RNA-Polymerase (und umgekehrt) sowie weitere Ähnlichkeiten der Transkriptionsapparate der Mitochondrien und Plastiden, wie die strukturelle Organisation ihrer Promotoren und die phylogenetische Herkunft ihrer kernkodierten Transkriptasen inspirierte in planta Studien zur spezifischen Transkription eines mitochondrialen Promotors in den Plastiden. Hierzu wurde die Expression des nptII-Reportergens unter Kontrolle des mitochondrialen PatpA-Promotors aus Oenothera in transplastomischen Tabakpflanzen analysiert. Die durchgeführten Studien belegen eine korrekte Transkription des mitochondrialen PatpA-Promotors durch eine plastidäre Phagentyp-RNA-Polymerase in in vitro-Transkriptionsassays sowie in transplastomischen Tabakpflanzen. Diese Resultate enthüllen weitere unerwartete Ähnlichkeiten der organellären Genexpression, die aufschlussreiche evolutionäre Einblicke erlauben und verbesserte Anwendungen zur Manipulation plastidärer Genome ermöglichen könnten. / All mitochondrial and a subset of plastidial genes of photosynthetically active eukaryotes are transcribed by nuclear-encoded, phage-type RNA polymerases. In this study, a homologous in vitro transcription system was used to define the specific functions of Arabidopsis phage-type RNA polymerases RpoTm, RpoTp and RpoTmp in organellar transcription. RpoTmp displayed no significant promoter specificity, whereas RpoTm and RpoTp were able to accurately initiate transcription from overlapping subsets of mitochondrial and plastidial promoters of diverse architecture. RpoTm and RpoTp thereby demonstrated an intrinsic capability to recognize promoters on supercoiled DNA templates without the aid of protein cofactors. A selective promoter recognition by the phage-type RNAPs in vitro and the inability to recognize promoters on linear templates imply that auxiliary factors are required for efficient initiation of transcription and/or DNA melting in vivo. Crosswise recognition of organellar promoters by the phage-type RNA polymerases in vitro as well as other similarities of the mitochondrial and plastidial transcription machineries such as promoter structures and the phylogenetic origin inspired in planta studies to investigate specific transcription of a mitochondrial promoter in plastids. Therefore, the expression of an nptII reporter gene under control of the mitochondrial PatpA promoter from Oenothera was analyzed in transplastomic tobacco plants. The data presented here demonstrate the faithful recognition of the mitochondrial PatpA promoter by a plastid RNA polymerase both in in vitro transcription assays and in transplastomic tobacco plants. These findings disclose further unexpected similarities of the organellar gene expression systems which deliver interesting evolutionary insights and might facilitate improved applications for chloroplast genome engineering.
384

Analysis of components of the mitochondrial transcription machinery in Arabidopsis thaliana

Kühn, Kristina 11 April 2006 (has links)
In der vorliegenden Arbeit wurde die Transkription mitochondrialer Gene durch die kernkodierten Phagentyp-RNA-Polymerasen RpoTm und RpoTmp der Pflanze Arabidopsis untersucht. Im Mitochondriengenom von Arabidopsis wurden f r 12 Gene Promotoren bestimmt. Diese zeigten verschiedene Sequenzelemente und wichen meist von der f r Dikotyle publizierten Konsensussequenz ab. F r die Mehrheit der Gene wurden multiple Promotoren identifiziert. Es wurden weiterhin Promotoren nachgewiesen, welche die Transkription vermutlich nicht funktioneller Sequenzen aktivieren. Architektur, Lokalisation und Nutzung mitochondrialer Promotoren implizieren eine wenig stringente Kontrolle der Transkriptionsinitiation in Arabidopsis-Mitochondrien. Zur Analyse der Funktionen von RpoTm und RpoTmp wurde ein in vitro-Transkriptionssystem entwickelt. Da RpoT-Enzyme m”glicherweise Kofaktoren ben”tigen, wurde in Arabidopsis nach Genen potentieller mitochondrialer Transkriptionsfaktoren gesucht. Als mitochondriales Protein mit Žhnlichkeit zu mtTFB, einem essentiellen Transkriptionsfaktor in Hefemitochondrien, wurde MetA identifiziert. In in vitro-Assays initiierte RpoTm an verschiedenen Promotoren die Transkription, w„hrend RpoTmp keine signifikante Promotorspezifit„t zeigte. Die spezifische Promotornutzung durch RpoTm erforderte superhelikale DNA. Weder RpoTm noch RpoTmp wurde durch MetA stimuliert. Eine mtTFB-„hnliche Funktion von MetA ist daher unwahrscheinlich. F r MetA wurde ausserdem eine engere phylogenetische Beziehung zu nukle„ren rRNA-Dimethylasen als zu mtTFB ermittelt. Die hier vorgestellten Studien belegen die Transkription mitochondrialer Gene in Arabidopsis durch RpoTm; f r RpoTmp ist eine nicht-redundante Transkriptionsfunktion denkbar. Die Kofaktor-unabh„ngige Spezifit„t von RpoTm f r verschiedene Promotoren und die wenig stringente Initiationskontrolle in vivo legen nahe, dass eine individuelle Regulation mitochondrialer Gene in Arabidopsis auf Transkriptionsebene nicht erfolgt. / Mitochondria depend on a nucleus-encoded transcription machinery to express their genome. The present study examined the transcription of mitochondrial genes by two nucleus-encoded phage-type RNA polymerases, RpoTm and RpoTmp, in the plant Arabidopsis. For selected mitochondrial genes in Arabidopsis, transcription initiation sites were determined. Most genes were found to possess multiple promoters. The identified promoters displayed diverse sequence elements and mostly deviated from a nonanucleotide consensus derived previously for dicot mitochondrial promoters. Several promoters were detected that activate transcription of presumably non-functional sequences. Promoter architecture, distribution and utilization suggest a non-stringent control of transcription initiation in Arabidopsis mitochondria. An in vitro transcription system was set up to elucidate the roles of RpoTm and RpoTmp. Since RpoT enzymes possibly require auxiliary factors, the Arabidopsis genome was screened for potential cofactors of phage-type RNA polymerases. A mitochondrial protein (MetA) with similarity to mtTFB, an essential transcription factor in yeast mitochondria, was identified. In in vitro transcription studies, RpoTm recognized various promoters whereas RpoTmp displayed no significant promoter specificity. Promoter recognition by RpoTm depended on supercoiled DNA templates. Transcription initiation by RpoTm or RpoTmp was not affected by MetA, indicating that MetA is not functionally equivalent to mtTFB. Besides, MetA was found to be more closely related to non-mitochondrial rRNA dimethylases than to mtTFB. The present study establishes RpoTm to transcribe mitochondrial genes; RpoTmp may have a non-overlapping transcriptional role in mitochondria. The cofactor-independent promoter specificity of RpoTm and the apparently non-stringent control of transcription initiation in vivo imply that mitochondrial genes in Arabidopsis may not be regulated individually at the transcriptional level.
385

Erkennung von Regulationssequenzen für die Transkription in heterologen Systemen

Jacob, Daniela 15 July 2003 (has links)
Während der Evolution entwickelten sich Promotorelemente von Prokaryonten, Eukaryonten und Plastiden in Sequenz und Struktur unterschiedlich. Ein Transfer von Promotorsequenzen zwischen Prokaryonten, Eukaryonten und Plastiden sollte daher zu keiner effizienten Genexpression führen. Mit dieser Arbeit sollte die Spezifität von Promotoren analysiert und ihre Funktionalität über `kingdom´-Grenzen hinaus untersucht werden. Hierfür wurden zwölf pflanzenspezifische Promotoren, welche in der Gentechnik zur Herstellung von transgenen Pflanzen eingesetzt werden, auf Genexpression in fünf Bakterienarten untersucht. Weiterhin wurden drei bakterielle und sechs Plastiden Promotoren auf Genexpression in Nicotiana tabacum untersucht. Die Frequenz, mit der die pflanzenspezifischen Promotoren (P) in den untersuchten Bakterienarten zu einer Expression führten, lag bei 50 % der getesteten Kombinationen. Für den P ST-LS1 aus Solanum tuberosum wurde eine Expression in den fünf untersuchten Bakterienarten nachgewiesen. Zwei der getesteten pflanzenspezifischen Promotoren, P RolC aus Agrobacterium rhizogenes und P 247 aus N. tabacum zeigten keine Expression in den untersuchten Bakterienarten. Die Charakterisierung der mRNA-Transkripte ausgewählter Fusionen der pflanzenspezifischen Promotoren mit den Reportergenen zeigten eindeutig, dass für die Transkriptionsinitiation durch die bakterielle RNA-Polymerase Sequenzelemente der pflanzenspezifischen Promotoren genutzt wurden. Mit einer ortsspezifischen Mutagenese des P ST-LS1 konnte die von der bakteriellen RNA-Polymerase genutzte -10-Region in Escherichia coli und Acinetobacter sp. ermittelt werden. Die `down-Mutanten´ führten in E. coli und Acinetobacter sp. zu einer Reduktion der Expression, wohingegen sie nach stabiler Integration in das Pflanzengenom von N. tabacum eine unverminderte Expression in bezug auf den Wildtyp-Promotor zeigten. Die Untersuchung der bakteriellen und Plastiden Promotoren ergab nur in einem einzigen Fall von neun getesteten Promotoren eine sehr geringe transiente Expression. / During evolution the promoter elements from prokaryotes, eukaryotes and plastids have developed differently with regard to their sequence and structure, implying that in general a transfer of promoter sequences between prokaryotes, eukaryotes and plastids will not cause an efficient gene expression. The aim of this study was to investigate the specificity of promoter sequences and their functionality in different kingdoms. Therefore 12 different plant-specific promoters, all used for the construction of genetically modified plants, were tested for their capability to direct a gene expression in various bacteria. Furthermore three bacterial and six plastid promoters were tested with respect to their ability to direct a gene expression in Nicotiana tabacum. The frequency of plant-specific promoters (P) directing gene expression in bacteria was 50 % of the combinations analysed. The promoter P ST-LS1 of Solanum tuberosum was functional in all bacteria tested. Two of the plant-specific promoters, P RolC of Agrobacterium rhizogenes and P 247 of N. tabacum, caused no gene expression in the bacteria tested. The characterisation of mRNA-transcripts of fusions between the plant-specific promoter sequences and the reporter genes proved that sequence elements of the plant-specific promoters themselves were used for transcription initiation by the bacterial RNA polymerase. By site-directed mutagenesis of the P ST-LS1 the -10 region used by the bacterial RNA polymerase of E. coli and Acinetobacter sp. was identified. The generated `down-mutants´ of the P ST-LS1 promoter showed a reduction of expression in E. coli and Acinetobacter sp. while these mutants were still fully active after stable integration in the genome of N. tabacum compared to the wildtype promoter. The investigation of the bacterial and the plastid promoters showed a very low transient expression of one out of nine promoters tested.
386

Promotores específicos para expressão gênica no floema na transformação genética de citros / Specific promoters for gene expression in the phloem in citrus genetic transformation

Miyata, Luzia Yuriko 10 February 2010 (has links)
O Huanglongbing (HLB) é uma das doenças mais ameaçadoras para citricultura mundial e, até o momento, não foi encontrada resistência na base genética do gênero Citrus. A doença é causada pela bactéria Candidatus Liberibacter spp., endêmica de floema. Portanto, na busca por uma planta transgênica resistente ao HLB é desejável avaliar construções gênicas em que o gene de interesse se expresse preferencialmente na região em que a bactéria coloniza a planta, ou seja, no floema. Assim, o objetivo deste trabalho foi a obtenção de plantas transgênicas via Agrobacterium tumefaciens, de citrange Carrizo [Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osbeck] e de laranja doce [Citrus sinensis (L.) Osbeck] cultivares Hamlin, Valência e Pêra, contendo o gene uidA (GUS) sob o controle dos promotores Citrus pholem protein 2 (CsPhP2), Arabidopsis thaliana pholem protein 2 (AtPhP2) e Arabidopsis thaliana sucrose transporter 2 (AtSuT2), para verificar se esses promotores regulam a expressão do gene repórter na região do floema. Foram utilizados segmentos de epicótilo de plântulas germinadas in vitro e como agente de seleção de regeneração de plantas transgênicas foi utilizado o gene nptII, que confere resistência ao antibiótico canamicina. Dos brotos regenerados foi coletada uma amostra de material para a realização do ensaio histoquímico com X-GLUC. Os brotos que formaram coloração azulada confirmaram a integração do transgene, sendo esses enxertados em porta enxertos previamente germinados e estiolados in vitro. A partir do número de explantes introduzidos, número explantes responsivos, número de brotos regenerados e número de brotos regenerados GUS positivos calculou-se a eficiência de transformação genética dos experimentos. Para a confirmação da transformação genética de laranja Hamlin foram realizadas análises de PCR e Southern blot de três plantas GUS positivas aclimatizadas. Também foram feitos cortes histológicos manuais para melhor visualização da reação histoquímica de GUS das plantas de laranja Hamlin Southern blot positivas. Todos os experimentos de transformação regeneraram pelo menos um broto GUS positivo. As plantas de laranja Hamlin analisadas por PCR e Southern blot analisadas foram confirmadas como transformadas com uma inserção do transgene. Plantas nas quais foram realizados cortes histológicos indicaram expressão diferencial das construções gênicas no floema. / Huanglongbing (HLB) is one of the most threatening diseases to worldwide citriculture and till the present moment, no resistance has been found in citrus genetic basis. This disease has Candidatus Liberibacter spp. as the pathogenic agent, an endemic phloem bacterium. Therefore, in the search for a transgenic plant resistant to HLB, it is desirable to evaluate constructions in which the gene of interest is expressed, preferentially, in tissues where the bacteria grow, i.e., in the phloem. Therefore, this work aimed to obtain transgenic plants of Carrizo citrange [Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osbeck], and of Hamlin, Valencia, and Pera sweet oranges [Citrus sinensis (L.) Osbeck], via Agrobacterium tumefaciens, containing uidA (GUS) gene, controlled by the promoters Citrus pholem protein 2 (CsPhP2), Arabidopsis thaliana pholem protein 2 (AtPhP2) and Arabidopsis thaliana sucrose transporter 2 (AtSuT2), in order to check if these promoters drive the reporter gene expression in the phloem. For the transformation, in vitro germinated seedlings epicotil segments were used. The gene nptII, which confers resistance to the antibiotic kanamycin, was used as the selective system. From the regenerated shoots, a tissue sample was collected to perform the X-GLUC histochemical analysis. The regenerated shoots colored in blue were considered transgenic, and these were grafted on in vitro grown rootstocks. The transformation efficiency of each experiment was calculated based on the number of introduced explants, responsive explants, number of regenerated shoots, and number of GUS positive regenerated shoots. In order to confirm the genetic transformation of Hamlin sweet orange, PCR and Southern blot analyses of three positive GUS acclimatized plants were performed. Anatomic slices for better visualization of blue color formed by GUS reaction were also made. All transformation experiments regenerated at least one GUS positive shoot. Plants of Hamlin sweet orange analyzed by PCR and Southern blot are transformed and have one transgene insertion. Anatomical analyses indicated preferential expression of the transgenes in the phloem.
387

Promoter and Enhancer Chromatin Dynamics during Direct Cell Fate Programming

Ibrahim, Mahmoud 09 August 2017 (has links)
Die Beschreibung genregulatorischer Ereignisse ist entscheidend um Zelldifferenzierung und -entwicklung zu verstehen. Dynamische Vernderungen der Chromatinstruktur, Histonmodifikationen und das Binden von Transkriptionsfaktoren an Enhancer und Promotoren, koennen mit Hilfe von genomweiten Hochdurchsatz-Sequenziertechniken wie ChIP-Seq, DNase-Seq, ATACSeqund RNA-Seq untersucht werden. In dieser Arbeit entwickele ich mehrere probabilistische Modelle fuer die Analyse von genomweiten Sequenzierungsdaten. Diese umfassen 1. einen Peak-Finder fuer ChIP-/DNase-/ATAC-Seq-Daten, der sich Replikate zunutze macht und praezise Peak-Weiten berechnet, 2. eine Pipeline um das Genom in hoher Aufloesung in eindeutige Klassen von Kombinationen von Histonmodifikationen zu segmentieren, 3. ein Bayes-Netzwerk-Modell welches multiple zeitlich aufgelste Histonmodifikations-ChIP-seq-Daten kombinatorisch clustert Klassen von regulatorischen Elementen zu identifizieren. Mit Hilfe dieser Modelle untersuchen wir die Promotorumgeben und zeigen einen Zusammenhang zwischen Chromatinstruktur und Promotordirektionalitaet. Darueber hinaus verwenden wir ein Modell zur direkten Reprogrammierung von Stammzellen in Motorneuronen durch die gezielte Expression von Transkriptionsfaktoren und analysieren die dadurch induzierten zeitlichen Vernderungen der Chromatinstruktur und Transkriptionsfaktorbindedynamik. Wir beobachten, dass Promotoren verschiedenen Chromatin-Dynamiken zur Aktivierung und Repression folgen, die mit den Chromatin-Dynamiken von Enhancer-Elementen korrelieren. Enhancer hingegen werden durch kooperatives Verhalten direkt induzierter Transkriptionsfaktoren und anderen Faktoren, die in den Stammzellen zu Beginn vorhanden waren oder im Verlaufe der Differenzierung aktiviert wurden, kontrolliert. Diese Arbeit zeigt wie wichtig Chromatin-Dynamik und ihre Beziehung zur Logik von Transkriptionsfaktoren ist, um die Veraenderungen der Genexpression zu verstehen. / Delineating transcription regulatory events is crucial to understand cell differentiation and development. Dynamic changes of chromatin structure, histone modifications and transcription factor binding to enhancers and promotors can be investigated with the aid of genome-wide high-throughput sequencing technologies such as ChIP-Seq, DNase-Seq, ATAC Seq and RNA Seq. In this thesis, I develop several probabilistic models for the analysis of genome-wide sequencing data. These include: 1. a peak finder for ChIP-Seq, DNase-Seq and ATAC Seq data, which exploits biological replicates and accurately demarcates peak widths, 2. a pipeline for high-resolution genome segmentation into unique classes of combinations of histone modifications and 3. a Bayesian network model that can co-cluster multiple time-course histone modification ChIP-Seq data sets into distinct classes of regulatory elements. With the aid of these models we investigate the promoter chromatin environment and show a link between chromatin state and transcription initiation directionality. In addition, we use a system for direct reprogramming of stem cells in motor neurons by the targeted expression of transcription factors to analyse changes in chromatin state and transcription factor dynamics during differentiation. We observe that promoters follow different chromatin dynamics for activation and repression that correlate with the chromatin dynamics of enhancer elements. Enhancers are controlled by cooperative behavior of directly induced transcription factors and other factors present in the stem cells initially, or activated in the course of differentiation. Overall, this work demonstrates the importance of understanding chromatin dynamics and their relationship to transcription factors logic in order to better explain changes in gene expression.
388

Gene supressor de tumor RECK: clonagem e caracterização do promotor e regulação de sua expressão / Gene suppressor tumor RECK: cloning and characterization of the promoter and regulation of its expression

Sasahara, Regina Maki 10 January 2000 (has links)
A expressão do gene RECK é ubíqua em tecidos normais e não detectável nas linhagens celulares tumorais testadas e em fibroblastos transformados por diversos oncogenes. Inicialmente isolado como um gene indutor da reversão fenotípica tumoral→normal em fibroblastos transformados por v-Ki-ras, o gene RECK codifica uma glicoproteína de membrana que suprime a invasão tumoral e metástase através da regulação da metaloprotease de matriz-9. Para entender os mecanismos de inibição da expressão do gene RECK, mediada por oncogenes, isolou-se e caracterizou-se a região 5\'- flanqueadora do gene RECK de camundongo (mRECK). Ensaios de atividade promotora utilizando mutantes de deleção da região 5\' - flanqueadora e o gene repórter luciferase, revelaram que a sequência de 52 pb, imediatamente \"upstream\" ao gene, possui uma atividade promotora que é suprimida pelo produto do oncogene Ha-ras (V12). Esta sequência contém dois sítios de ligação a Sp1 (SplA e SplB), um sítio de ligação a cEBPb e um CAAT box. EMSAs e ensaios de atividade promotora, utilizando mutantes pontuais nestes sítios, revelaram que as proteínas Spl e Sp3 se ligam a ambos os sítios Spl, e que a resposta a Ras é mediada apenas pelo sítio SplB. Análise por Southern blot utilizando uma enzima de restrição sensível à metilação e por Northern blot de mRNA extraído de células tratadas com um agente desmetilante, sugeriram que o mecanismo de metilação de DNA não está envolvido na regulação transcricional do gene RECK. O envolvimento de RECK em diversos sistemas de proliferação, invasão e reversão celular foi analisado através de Northern blot. Os resultados sugerem que a expressão de RECK é regulada por soro na linhagem A3l de fibroblastos normais de camundongo. / The RECK gene is ubiquitously expressed in normal human tissues, but is downregulated both in tumor cell lines and in oncogenically transformed fibroblasts. Initially isolated as a tumor→normal phenotypic reversioninducing gene in v-ki-ras-transformed fibroblast, RECK encodes a membrane-anchored glycoprotein that suppresses tumor invasion and metastasis by regulating the matrix metalloproteinase-9. In order to understand the mechanism of oncogene-mediated suppression of RECK gene expression, we have isolated and characterized the 5\'-flanking region of the mouse RECK gene (mRECK). Deletion mutants constructs of this 5\' -flanking region with the luciferase reporter gene, revealed that the 52 base pairs upstream displays promoter activity which is suppressed by the Ha-ras (V12) oncogene. This region contains two Spl-binding motifs (SplA and SplB), one cEBPb-binding motif, and one CAAT box. Gel shift analysis and reporter gene assays, in combination with site-directed point mutations in these elements, revealed that both Spl sites associate with Spl as well as Sp3 proteins, although Ras- responsiveness seems to be mediated only by the downstream Spl site (SplB). Southern blot analysis using a methylation-sensitive restriction enzyme and Northern blot analysis with mRNA extracted from cells treated with a demethylating agent, indicated that the mechanism of DNA methylation is not involved in the regulation of RECK gene transcription. The role of RECK in several systems of cell proliferation, invasion and reversion, analysed by Northern blot, suggested that RECK expression is cell cycle regulated by serum in normal A3l mouse fibroblast cell line.
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Estrutura da comunidade de Bacteria do trato intestinal de frangos suplementados com promotores de crescimento. / Bacteria community structure of the intestinal tract of chickens supplemented of growth promoters.

Pedroso, Adriana Ayres 28 July 2003 (has links)
O trabalho objetivou avaliar o efeito de probióticos e antibióticos utilizados como promotores do crescimento sobre o desempenho de frangos de corte e a capacidade dos agentes de alterar o ecossistema intestinal de aves criadas em baterias e sobre piso. Adicionalmente foi estudado o efeito dos probióticos sobre a presença de oocistos na cama das aves. Os antibióticos tiveram sua eficácia, como promotores de crescimento, comprovada para aves criadas sobre piso, mas não em bateria. Foram observadas alterações na estrutura da comunidade de Bacteria no trato intestinal de frangos criados em baterias e sobre piso e suplementados com antibióticos. Não houve evidência de efeito favorável dos probióticos sobre o desempenho e incidência de oocisto na cama das aves. Os probióticos não tiveram a capacidade de colonizar o epitélio intestinal de frangos de corte. Foram observadas discretas modificações na estrutura da comunidade de Bacteria de frangos criados em bateria e sobre piso e suplementados com dietas contendo probióticos. A estrutura da comunidade de Bacteria do intestino delgado de frangos foi modificada em função do ambiente no qual frangos, suplementados com probióticos e antibióticos, foram criados. Frangos isentos de qualquer tipo de promotor de crescimento apresentaram 15 unidades taxonômicas operacionais distintas na microbiota intestinal aderida ao epitélio, predominantemente Lactobacillus e Pseudomonas. / This study was conducted to evaluated the effects of growth promoter probiotic and antibiotics on the perfomance and organ morphometry of broiler chickens raised in floor pens and in batteries and the ability of the additive to promote changes in the intestinal ecosystem. Additionally, the effect of probiotics on the presence of coccidia oocysts in the litter was evaluated. The efficacy of antibiotics as growth promoters was observed when the chickens were raised in floor pens but not in batteries. Antibiotic supplementation caused changes in the structure of Bacteria community of the intestinal tract of chicken raised in floor pens or in batteries. The probiotic additives tested did not result in improvement in performance in both environmental or in oocyst incidence in the litter. Also, the probiotic did not have the ability to colonize the intestinal epithelium of the birds. Discrete changes in the structure of Bacteria community were observed when probiotics were supplemented to chickens raised in floor pens or in batteries. Bacteria community structure in the small intestine of chicken was modified as a function of the environmental in which the birds were raised. Chicken fed diets devoid of growth promoters had 15 distinct phylogenetic groups in the microbiota adhered to the intestinal epithelium.
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Sistema agroalimentar da avicultura fundada em princípios da Agricultura Natural: multifuncionalidade, desenvolvimento territorial e sustentabilidade / Agrifood System of chicken meat and egg production based on principles of Nature Farming method: multifunctionality, local development and sustainability

Demattê Filho, Luiz Carlos 14 August 2014 (has links)
O presente trabalho de pesquisa se refere às atividades e às relações desenvolvidas em torno da agroindústria brasileira Korin Agropecuária Ltda. Essa empresa possui uma origem peculiar uma vez que foi fundada com o propósito de colocar em prática os princípios, conceitos e métodos originados da Agricultura Natural, sistema agrícola preconizado por Mokiti Okada (Japão, 1882-1955). Okada enfatizou a necessidade de um perfeito equilíbrio entre as atividades humanas e as forças da natureza, para se alcançar bons resultados na produção, privilegiando a segurança dos alimentos, a interação e o respeito à natureza e a saúde e o bem estar socioeconômico dos agentes participantes deste sistema. Segundo Okada, a Agricultura Natural é um dos pilares de sustentação de uma civilização e sociedade ideais, onde a saúde, a prosperidade e a paz são predominantes. Assim, enquanto método produtivo enquadra-se perfeitamente na ideia de uma agricultura sustentável, pelo não uso de adubos químicos solúveis, agrotóxicos, antibióticos, melhoradores de desempenho e outros insumos industriais, veiculando uma abordagem socioambiental da atividade agrícola, com enfoque na qualidade diferenciada dos alimentos. Especificamente sobre a produção avícola de corte e postura convencional discutimos os problemas decorrentes do uso excessivo de antibióticos e promotores de crescimento, apresentando a evolução técnico-produtiva deste sistema de integração vertical alternativo no qual estas substâncias não são utilizadas e o bem estar animal é considerado. Prioritariamente abordamos, neste trabalho, a sustentabilidade sob o ponto de vista da multifuncionalidade da agricultura, tratando também do desenvolvimento da empresa sob a ótica dos Sistemas Agroalimentares Localizados - SIAL. Considerando essas abordagens, realizamos pesquisa qualitativa junto a 28 produtores predominantemente familiares integrados à empresa, aplicando questionário semiestruturado para explorar e analisar suas atitudes, percepções e experiências em relação aos conceitos e métodos da empresa, ao meio ambiente, segurança alimentar e suas condições socioeconômicas. No que diz respeito à sede da empresa, apresentamos um estudo, que contou com nossa colaboração, sobre a sustentabilidade de seu sistema de produção através de método que utiliza indicadores socioambientais, desenvolvido pela Embrapa Meio Ambiente. Trata-se do APOIA - Novo Rural, voltado para análise de unidades produtivas rurais. A análise integrada de sustentabilidade avalia a gestão ambiental do estabelecimento rural, tendo como base o contexto local e as práticas de manejo adotadas. A agroindústria em questão obteve um excelente índice integrado de sustentabilidade (0,87), justificado pelo seu histórico de mais de 20 anos de práticas relacionadas à Agricultura Natural. Peculiarmente, este complexo em torno da Agricultura Natural está inserido na confluência das divisas de duas unidades de conservação do estado de São Paulo, as Áreas de Proteção Ambiental - APA Corumbataí e a APA Piracicaba, o que promove sinergias com vistas a alcançar modelos ambientalmente adequados de produção agroalimentar. Os dados obtidos neste estudo junto aos avicultores integrados nos permitiram verificar que a prática da Agricultura Natural tem contribuído sinergicamente para que as dimensões da multifuncionalidade da agricultura sejam reconhecidas no território em questão, dinamizando e consolidando um sistema agroalimentar localizado sob uma perspectiva de sustentabilidade na produção agropecuária. / This research study refers to the activities and relationships developed around a Brazilian agrifood company named Korin Agropecuária Ltda. With a peculiar origin, this company was founded with the purpose of putting into practice the principles, concepts and methods originated from the Nature Farming system. NF is an agricultural approach created and advocated by Mokiti Okada (Japan, 1882-1955). Okada emphasized the necessity of a perfect balance between human activities and the nature forces in order to achieve good results in the production, favouring food safety, nature interaction and respect, health, and the social-economic well-being of all players of this system. According to Okada, Nature Farming is one of the pillars of true civilization and an ideal society where health, prosperity and peace are predominant. Thus, while a productive method, perfectly fits in the idea of sustainable agriculture since its methodological aspects as the non-use of chemical fertilizers, pesticides, antibiotics, growth promoters and other synthetic inputs, leading to a social-environmental approach to agriculture, with a focus on differentiated quality food. Regarding chicken meat and eggs regular production, we discuss the concerns about the overuse of antibiotics and growth promoters, showing the technical evolution of this particularly verticalized system where these substances are not used and the animal welfare is highly take into consideration. As a priority, we address in this work, sustainability from the point of view of the multifunctionality of agriculture and also addressing the company\"s development from the perspective of Localized Agrifood Systems. Considering these approaches, we conducted a qualitative research on twenty eight integrated farmers who are linked to the company, applying semi-structured questionnaire. The objective was to explore and describe their attitudes, perceptions and experiences in relation to the company concepts and methods towards the environment, food safety, and their socio-economic conditions. With regard to corporate headquarters, located in a farm on a rural area, we present a study on the sustainability of the production system through a social-environmental indicator developed by Embrapa, named APOIA - NovoRural. This indicator was designed to analyse rural productive units with focus on the environmental management based on local context. The company have achieved an excellent sustainability index (0.87), due to its history for more than twenty years of Nature Farming\'s handling. Peculiarly this Nature Farming complex is inserted at the confluence of two conservation units of the state of São Paulo, called Environmental Protected Areas of Corumbataí and of Piracicaba, which reinforces the need to search for environmentally suitable models of agrifood production. The data obtained from this study with the integrated farmers have allowed us to verify that the dimensions of multifunctionality of agriculture are being recognized within this particular territory, streamlining and consolidating this agrifood localized system under a sustainability perspective in agriculture.

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