The glycine riboswitch is a structured RNA found upstream of genes in mRNA transcripts in many bacteria, functioning as a biofeedback gene regulator. Upon binding glycine, a complete RNA transcript including gene sequences is transcribed, effectively turning on gene expression. In an effort to understand the intricacies of its functioning, many mutants of the riboswitch were made and characterized during Ph. D. work, resulting in discovery of a P0 duplex/kink-turn motif involving a few nucleotides upstream of the established glycine riboswitch sequence which changed its ligand binding characteristics (Chapter 1). Previously, the two aptamers of the riboswitch were thought to cooperatively bind glycine, but with the inclusion of this leader sequence which forms a kink turn motif with the linker between the two aptamers, glycine binding in one aptamer no longer requires glycine binding in the other. Furthermore, the Kd from three species tested are now a similar, lower value of about 5 µM, indicating authenticity of this new consensus sequence. Glycine binding and interaptamer interaction both enhanced one another in trans aptamer assays. Another discovery from this was a shortened construct including all of aptamer II but only part of aptamer I in which a few specific nucleotides prevented glycine binding in aptamer II (Chapter 2). This may provide insight into the nature of interaptamer interactions in the full switch; addition of an oligonucleotide complimentary to these nucleotides restored glycine binding ability to aptamer II. With future development, this could also be a useful molecular biology tool, using two signals, glycine and an oligonucleotide, to allow gene expression. To precisely understand how any macromolecule functions, a 3D structure, obtainable by x-ray crystallography, is vital. A new technique to accomplish that for RNA, precedented in the protein world, is Fab chaperoned crystallography, which has advantages compared to RNA alone. A phage displayed library of Fabs with reduced codon diversity designed for RNA was created, the YSGR Min library (Chapter 3). Its Fabs had specificities and affinities equal to or greater than previous libraries which were originally created for phage displayed selection against proteins. Fab chaperoned RNA crystallography is currently in progress for the glycine riboswitch; the best resolution thus far is 5.3 … (Chapter 4). In addition to providing molecular insight into its gene regulation mechanism, a structure of the glycine riboswitch could be applied for use in structure based drug design of novel antibiotics targeting the riboswitch to disrupt important downstream carbon cycle genes in pathogenic bacteria.

Non-coding RNAs (ncRNAs) account for a higher percent of the genome than coding mRNAs, and are implicated in human disease such as cancer, neurological, cardiac and many others. While the majority of ncRNAs involved in disease were originally attributed to a class of RNAs called micro RNAs (miRNAs) with a small size of only about 19 -24 base pairs, emerging research has now demonstrated a class of long non-coding RNAs (lncRNAs) that have a size of over 200 base pairs to be responsible for gene regulation and other functional roles and have also found to contribute to pathogenesis in humans. The increased size and structural complexity require novel tools to study their interactions beyond RNA interference. Synthetic antibodies are classic tools and therapeutics utilized to study and treat proteins involved in human disease. Likewise we hypothesize that structured RNAs can also take advantage of synthetic antibodies to probe their functions and be utilized as therapeutics. Currently, antibodies have been raised against microbial riboswitches and other structured RNAs of single-celled organisms, and only one human structured RNA to the best of our knowledge. However, no one has yet to create a synthetic antibody capable of behaving as a therapeutic against a structured RNA. We therefore sought to raise an antibody Fab against a structured RNA, human initiator tRNA, a model oncogenic non-coding RNA and demonstrate its efficacy in vitro. We then characterized the antibody and explored delivery options in cancer cells including the use of nanoparticle delivery systems. With the emerging transcriptome revealing new ncRNAs implicated in human disease, our research has begun to address a new therapeutic strategy, laying down the foundation for the future of structured RNA-targeted therapies.

No description available.

In recent years, the world has faced major health challenges, from the rise of antibiotic resistance to the emergence of new pathogens with pandemic potential. This highlights the importance of considering human health as inextricably intertwined with that of other animals and the environment in which they live. This paradigm is known as “One Health”, which is the integration of environmental sciences, veterinary science, and medicine. Within this perspective, viruses, the most abundant biological entities on Earth, play a central role in connecting different organisms, deeply influencing the health of their hosts. Despite their great importance, most viruses are still poorly understood, mainly because of the technical and economic limitations posed by isolation, cultivation, and single colony sequencing. However, recently developed genomic technologies offer a cheaper and more sensitive alternative to study viruses, allowing a better integration of data from various sources and making it possible to explore how they circulate among different hosts and environments. In this thesis, I hypotesise that, by combining different classes of genomic methods with One Health practices, it is possible to reveal much more of the entire picture of viral diversity and evolution that by simply using them in a separated way. I show that this is the case for each one of the scientific questions addressed in this work, which are organised in three main chapters: - In the second chapter I analysed 22 metatranscriptomes from tick samples from different parts of Italy, obtaining a set of 91 viral contigs for which I reconstructed the phylogeny, with the aim to identify the presence of possible pathogens and characterise the unexplored viral diversity in the country. This analysis not only clarified the molecular epidemiology of well-known pathogens such as Tick-borne encephalitis virus, but also allowed the discovery of at least 10 novel viral species. - In the third chapter, I investigated the origin and spread of West Nile virus, an emerging pathogen causing neurological disease worldwide. The goal was to expand the current knowledge of this virus by increasing the number of sequenced genomes and to reconstruct how the virus moved between Africa and Europe. Phylogeographic and phylodynamic methods showed that this pathogen originated in Africa and repeatedly invaded the European continent, revealing the dynamics of its evolution through space and time. - In the last chapter, I contributed to obtaining a set of DNA phages assembled from human microbiomes and viromes by manual curation and comparative genomics and developed a new approach to study their evolution in relation to their bacterial hosts. I found that the strength of co-phylogenetic patterns between viruses and their hosts is generally weak, suggesting that their ecological relationships emerge on short evolutionary timescales. Taken together, these results show how the integration of viral metagenomics and phylogenomics in One Health is essential to answer fundamental questions about the diversity of viruses and how they spread and evolve. Furthermore, the methods and protocols developed in these studies can be applied to similar cases, allowing a systematic exploration of many other datasets to expand our knowledge of the virosphere. This information can be used to implement containment strategies, public health policies, therapies, and biotechnologies.

Zusätzlich zu der eubakteriellen RNA-Polymerase (RNAP) der Plastiden sind im Zellkern von Arabidopsis thaliana drei weitere, phagentypische RNAP kodiert, die jeweils aus nur einer Einheit aufgebaut sind. Die Enzyme RpoTp und RpoTm werden in die Plastiden, bzw. die Mitochondrien transportiert, während RpoTmp in beiden Organellen zu finden ist. Um die Lichtabhängigkeit der RpoT-Gene zu untersuchen, wurde die lichtinduzierte Akkumulation ihrer Transkripte in 7-Tage alten Keimlingen, sowie 3- bzw. 9-Wochen alten Rosettenblättern mittels quantitativer real-time PCR ermittelt. Die entwicklungsabhängige Regulation der RpoT-Transkript-Akkumulation wurde außerdem während der Blattentwicklung analysiert. Zusätzlich wurde der Einfluss des circadianen Rhythmus untersucht. Es stellte sich heraus, dass die Transkriptakkumulation aller drei RpoT-Gene stark lichtinduziert war und nur marginalen circadianen Schwankungen unterlag. In weiteren Versuchen mit verschiedenen Lichtrezeptor-Mutanten und unterschiedlichen Lichtqualitäten wurde der Einfluss multipler Rezeptoren auf den Prozess der Lichtinduktion gezeigt. In den Zellen höherer Pflanzen finden sich drei Genome. Die Biogenese von Chloroplasten und Mitochondrien, sowie lebenswichtige Prozesse, wie Atmung und Photosynthese setzen oftmals die Aktivität von Genen auf mindestens zwei dieser Genome voraus. Eine intrazelluläre Kommunikation zwischen den verschiedenen Genomen ist daher unumgänglich für einen funktionierenden Stoffwechsel der Pflanze. In dieser Arbeit wurde herausgestellt, dass die Zahl mitochondrialer Genkopien in photosynthetisch inaktiven Arabidopsis-Keimlingen drastisch erhöht ist. Bei der Untersuchung des DNA-Gehaltes in Proben, die Altersstufen von 2-Tage alten Keimblättern bis hin zu 37-Tage alten, seneszenten Rosettenblättern umfassten, fand sich ein deutlicher Anstieg der Kopienzahlen in älteren Rosettenblättern. Außerdem unterschieden sich die Kopienzahlen der untersuchten Gene zum Teil erheblich voneinander. / In addition to eubacterial-like multi-subunit RNA polymerases (RNAP) localized in plastids and the nucleus, Arabidopsis thaliana contains three phage-like single-unit, nuclear-encoded, organellar RNAPs. The enzymes RpoTp and RpoTm are imported into plastids and mitochondria, respectively, whereas RpoTmp shows dual targeting properties into both organelles. To investigate if expression of the RpoT genes is light-dependent, light-induced transcript accumulation of RpoTm, RpoTp and RpoTmp was analyzed using quantitative real-time-PCR in 7-day-old seedlings as well as in 3- and 9-week-old rosette leaves. To address the question whether RpoT transcript accumulation is regulated differentially during plant development transcript abundance was measured during leaf development. Additionally, effects of the plants circadian rhythm on RpoT transcript accumulation were analyzed. Transcripts of all three RpoT genes were found to be strongly light-induced even in senescent leaves and only marginally influenced by the circadian clock. Further analyses employing different photoreceptor mutants and light qualities revealed the involvement of multiple receptors in the light-induction process. The biogenesis of mitochondria and chloroplasts as well as processes like respiration and photosynthesis require the activity of genes residing in at least two distinct genomes. There have to be ways of intracellular communication between different genomes to control gene activities in response to developmental and metabolic needs of the plant. In this study, it was shown that gene copy numbers drastically increased in photosynthetically inactive Arabidopsis seedlings. Mitochondrial DNA contents in cotyledons and leaves ranging in age from 2-day-old cotyledons to 37-day-old senescent rosette leaves were examined. A common increase in senescing rosette leaves and drastic differences between individual genes were found, revealing the importance of an integrative chondriome in higher plant cells.

Licht und Pflanzenhormone sind essentiell für das Wachstum und die Entwicklung von Pflanzen. Es ist nur wenig darüber bekannt, wie sie die Transkription organellärer Gene beeinflussen. In Arabidopsis thaliana gibt es drei kernkodierte Phagentyp-RNA-Polymerasen (RpoT), welche für die organelläre Transkription verantwortlich sind. Diese werden in die Plastiden (RpoTp), die Mitochondrien (RpoTm) oder zu beiden Organellen (RpoTmp) transportiert. Neben den beiden kernkodierten RNA-Polymerasen (NEP) existiert in den Plastiden eine plastidärkodierte RNA-Polymerase (PEP), welche zusätzliche Sigmafaktoren zur Promotererkennung benötigt. Um die Lichtabhängigkeit der Expression der RpoT Gene sowie NEP-transkribierter Chloroplastengene zu analysieren, wurde die Akkumulation von RpoT- und rpoB-Transkripten in 7-Tage alten Keimlingen unter verschiedenen Lichtbedingungen mittels quantitativer real-time PCR untersucht. Die Änderungen in der Transkriptakkumulation deuten darauf hin, dass rote, blaue und grüne Wellenlängen die Expression der drei RpoT Gene unterschiedlich stark stimulieren. Untersuchungen an verschiedenen Lichtrezeptor-Mutanten zeigten, dass die Lichtinduktion der RpoT Genexpression überaus komplex ist und ein interagierendes Netzwerk aus multiplen Rezeptoren und Transkriptionsfaktoren an der Signalweiterleitung beteiligt ist. Das Phytohormon Cytokinin wird durch Histidin Kinase Rezeptoren (AHK) detektiert. Es gibt drei unterschiedliche Rezeptoren: AHK2, AHK3 und AHK4. Diese sind Teil eines Zwei-Komponenten-Systems, welches Signale mit Hilfe einer Phosphorylierungskette überträgt. Der Einfluss von Cytokinin auf die plastidäre Transkription wurde mit Hilfe von Cytokininrezeptor-Mutanten untersucht, um die Funktion von AHK2, AHK3 und AHK4 zu analysieren. Um weitere Informationen darüber zu erhalten, wie die plastidäre Transkription durch PEP mittels Cytokinin reguliert wird, wurden die Hormoneffekte auf die plastidäre Transkription in Sigmafaktor-Mutanten untersucht. / Light and plant hormones are essential for plant growth and development. Only little information is available about how these signals influence the transcription of organellar genes. Arabidopsis thaliana possesses three nuclear-encoded phage-type RNA polymerases (RpoT) for organellar transcription. They are imported into plastids (RpoTp), mitochondria (RpoTm), or into both organelles (RpoTmp). Besides the two nuclear-encoded plastid polymerases (NEP), plastids contain an additional plastid-encoded RNA polymerase (PEP), which needs additional sigma factors for promoter recognition. Interested in the expression of RpoT genes and NEP-transcribed plastid genes in response to light we analyzed transcript levels of RpoT and rpoB genes in 7-day-old wild-type plants under different light conditions by quantitative real-time-PCR. The observed changes in transcript accumulation indicated that red, blue, and green light differentially stimulated the expression of all three RpoT genes. Further analyses using different photoreceptor mutants showed that light induction of RpoT gene expression is surprisingly complex based on a network of multiple photoreceptors an d downstream pathways. Cytokinin signals are perceived by the histidine kinase (AHK) receptor family. There exist three different membrane-bound receptors: AHK2, AHK3 and AHK4/CRE1. These receptors are part of a two-component signaling system which transfers signals via phosphorelay mechanisms. Interested in the potential role of AHK2, AHK3 and AHK4/CRE1 in the transduction of cytokinin signals into the chloroplast, we analyzed the influence of cytokinin on plastidial transcription in receptor mutants. To gain more information on how plastid transcription by PEP is regulated by cytokinin, the influence of cytokinin in sigma factor mutants was also studied.

Chloroplasten höherer Pflanzen haben kleine Genome. Trotzdem ist ihre Transkriptionsmaschinerie sehr komplex. Plastidäre Gene werden von plastidenkodierten (PEP) und kernkodierten RNA-Polymerasen (NEP) transkribiert. In der vorliegenden Arbeit wurden Promotoren plastidärer Gene und Operons von Arabidopsis thaliana charakterisiert. Zur Unterscheidung zwischen NEP- und PEP-Promotoren wurden erstmals spectinomycinbehandelte, chlorophylldefiziente Arabidopsis-Pflanzen mit fehlender PEP-Aktivität verwendet. Obwohl für einige Gene auch einzelne Promotoren lokalisiert wurden, wird die Transkription der meisten plastidären Gene und Operons an multiplen Promotoren initiiert. Der Vergleich plastidärer Promotoren von Tabak und Arabidopsis zeigte eine hohe Vielfältigkeit der Promotornutzung, die möglicherweise auch in anderen höheren Pflanzen vorkommt. Dabei stellt die individuelle Promotornutzung eine speziesspezifische Kontrollmöglichkeit der plastidären Genexpression dar. Das Kerngenom von Arabidopsis beinhaltet zwei Kandidatengene der NEP, RpoTp und RpoTmp, welche Phagentyp-RNA-Polymerasen kodieren. In der vorliegenden Arbeit wurde die Wirkung veränderter RpoTp-Aktivität auf die Nutzung von NEP- und PEP-Promotoren in transgenen Arabidopsis-Pflanzen mit verminderter und fehlender RpoTp-Aktivität untersucht. Im Keimlingsstadium konnten Unterschiede in der Promotornutzung zwischen Wildtyp und Mutanten beobachtet werden. Fast alle NEP-Promotoren wurden in Pflanzen mit verringerter oder fehlender RpoTp-Aktivität genutzt. Dabei zeigten nur einige von ihnen eine geringere Aktivität, andere wiederum waren sogar verstärkt aktiv. Der starke NEP-Promotor des essentiellen ycf1 Gens wurde in jungen Keimlingen ohne funktionelle RpoTp nicht genutzt. Die Ergebnisse zeigen, dass NEP gemeinsam von beiden Phagentyp-RNA-Polymerasen RpoTp und RpoTmp repräsentiert wird und dass beide sowohl eine überlappende, als auch eine spezifische Rolle in der Transkription plastidärer Gene innehaben. / Although chloroplasts of higher plants have small genomes, their transcription machinery is very complex. Plastid genes of higher plants are transcribed by the plastid-encoded plastid RNA polymerase PEP and the nuclear-encoded plastid RNA polymerases NEP. Here, promoters of plastid genes and operons have been characterized in Arabidopsis thaliana. For the first time spectinomycin-treated, chlorophyll-deficient Arabidopsis plants lacking PEP activity have been used to discriminate between NEP and PEP promoters. Although there are plastid genes that are transcribed from a single promoter, the transcription of plastid genes and operons by multiple promoters seems to be a common feature. Comparison of plastid promoters from tobacco and Arabidopsis revealed a high diversity, which my also apply to other plants. The diversity in individual promoter usage in different plants suggests that there are species-specific solutions for attaining control over gene expression in plastids. The nuclear genome of Arabidopsis contains two candidate genes for NEP transcription activity, RpoTp and RpoTmp, both coding for phage-type RNA polymerases. In this study the usage of NEP and PEP promoters has been analysed in transgenic Arabidopsis plants with reduced and lacking RpoTp activity. Differences in promoter usage between wild type and mutant plants were most obvious early in development. Nearly all NEP promoters were active in plants with low or lacking RpoTp activity, though certain promoters showed reduced or even increased usage. The strong NEP promoter of the essential ycf1 gene was not transcribed in young seedlings without functional RpoTp. These results provide evidence for NEP being represented by two phage-type RNA polymerases RpoTp and RpoTmp that have overlapping as well as specific functions in the transcription of plastid genes.

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.

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.

Therapeutische Erfolge von IVIG sind gut dokumentiert, aber die zu Grunde liegenden molekularen Mechanismen sind noch nicht vollständig erforscht. Molekulare Analysen unseres Labors über die Interaktion von IVIG mit Fabs von Patienten, die an einer autoimmunen Thrombozytopenie (ITP) leiden zeigten, dass die am häufigsten selektierten Fab von den V3-23 und V3-30 VH-Keimbahngenen abstammten. Eine weitere Studie mit IgG und IgM Phagen-Display Bibliotheken von einem gesunden Spender zeigten ebenfalls eine bevorzugte Reaktivierung von IVIG mit Fabs vom Ursprung der V3-23 und V3-30 Gene. Es konnte gefolgert werden, dass diese Interaktion von IVIG mit Fabs von diesen zwei VH-Genen weder alleine auf den Gesundheitsstatus des Spenders zurückzuführen war, noch auf eine zuvor erfolgte Behandlung mit IVIG. Diese Dissertation wurde unter Verwendung der Phagen-Display Technologie unternommen, um die molekulare Interaktion von IVIG mit Antikörpern zu erforschen, die von einem Patienten kloniert wurden, der an einem systemischen Lupus erythematodes und rheumatischem Fieber leidet. Die Resultate waren mit den früheren Studien zu vergleichen, insbesondere mit den Daten eines Patienten, der zu der ITP einen Lupus entwickelte. 23 Fabs, welche 7 unabhängige Klone repräsentierten, wurden isoliert. Im Gegensatz zu von Patienten mit ITP abstammenden Klonen reagierte keines von den in dieser Studie selektierten Fabs mit Thrombozyten. Die über IVIG gebundene Fab-Phagen stammten hierbei ausschließlich von den V3-23 und V3-30 VH-Genen ab. Darüber hinaus wurde beobachtet, dass von diesen Fabs verschiedene CDR3 Regionen einschließlich verschiedenen D- und JH-Gensegmenten benutzt wurden. Die Ergebnisse zeigten weiterhing, dass die Bindung von IVIG an die Fabs unabhängig von der Leichten Kette war. Ihrem Keimbahngen-Ursprung entsprechend hatten die Fabs Aminosäuren an Positionen in den FR1, FR3 und im 3'-Ende von CDR2, die dafür bekannt sind, dass sie für die Bindung des B-Zell-Superantigens Staphylococcus Protein A (SpA) essentiell sind. Es wurde gezeigt, dass sich zwar einige von den Fabs stark an SpA banden, aber keine Korrelation in der Intensität zur Bindung mit IVIG vorlag. Einige Fabs zeigten eine schwache Bindung an HIV gp120, einem anderen B-Zell-Superantigen. Zusammenfassend lässt sich aus der vorliegenden Studie und den vorherigen Ergebnissen schließen, dass ein Anteil von IVIG wie ein B-Zellen Superantigen funktionieren könnte, das für die Bildung und Regulation des normalen B-Zellen Repertoires wichtig ist. Der Bindungsmechanismus scheint ähnlich, aber nicht identisch mit dem der anderen getesteten B-Zellen-Superantigene zu sein. / The beneficial therapeutic effects of IVIG are well documented, but the underlying molecular mechanisms are not fully understood. Recent investigations from our laboratory into the molecular analysis of Fabs bound by IVIG from patients suffering from autoimmune thrombocytopenia revealed that the most frequently selected Fabs originated from the V3-23 and V3-30 VH germline genes. A subsequent study with IgG and IgM phage display libraries from a healthy donor also demonstrated a preferential reactivity of IVIG to Fabs of V3-23 and V3-30 origin. That study revealed that the unique reactivity of IVIG to Fabs of these two VH gene loci was not restricted to the autoimmune nature of the donors, neither to previous treatment with IVIG. One of the thrombocytopenia patients developed lupus. This study was undertaken to study the molecular interaction of IVIG with antibodies selected from a patient suffering from systemic lupus erythematosus and rheumatic fever using phage display technology, and to compare the results with the previous studies. Twenty-three Fabs representing seven independent clones were isolated. In contrast to ITP-derived clones, none of the Fabs selected in this study reacted with platelets. The Fab phages bound by IVIG were sequenced in order to determine their VH gene usage and clonal relatedness. V3-23 and V3-30 VH genes were found to be exclusively utilized by the Fab phages bound by IVIG. Moreover, different CDR3 regions including different D and JH gene segments were observed to be used by these Fabs. The results further showed that the binding of IVIG to the Fabs was independent of the light chain since different light chains were observed to be associated with the VH3 immunoglobulins. Detailed sequence analysis of the Fabs revealed the presence of amino acid residues at positions within FR1, FR3, and the 3' end of CDR2 that are known to be contacted by the B cell superantigen Staphylococcus protein A (SpA). Some of the Fabs were shown to bind strongly to SpA, but there was no correlation with the binding-intensity to IVIG. Some bound very weakly to HIV gp120, another B cell superantigen. This study, together with previous results, suggests that a subset of IVIG may function as a B cell superantigen that may significantly shape the B cell repertoire. The binding mechanism appears to be similar but not identical to the other tested B cell superantigens.