Fluorescence correlation spectroscopy (FCS) analyzes the fluctuations in the fluorescence intensity, which is emitted from a tiny excition volume, to obtain information about the concentration, the mobility, and the molecular interactions of labeled molecules. The more advanced fluorescence cross-correlation spectroscopy (FCCS) increases the precision in the determination of fl ow velocities and binding constants compared to standard FCS. The miniaturization in biomedical and chemical engineering has been developing rapidly, propelled by the vision of a fully functional laboratory on a single chip and its use in human therapeutics, for example, as implanted drug delivery system. A key requirement to fulfill this vision is the ability to handle small fl uid volumes. Handling liquids using the electrohydrodynamical principle circumvents many of the disadvantages of other systems. The complex flow pattern in the active region of such a pump could not be resolved by common tracking techniques. In this thesis, two-focus FCCS (2f-FCCS) was used to map the flow pro file inside a micropump. The high precision of 2f-FCCS in the determination of fl ow measurements even with small fluorescent particles allowed the measurement of the flow velocities induced by electrohydrodynamic forces acting on the solvent, while excluding the effects of dielectrophoretic forces acting on larger particles. Analysis of the fl ow data indicates a fl ow pattern that consists of two vortices of different size and opposite direction of rotation. The flow pattern derived by 2f-FCCS explains the observed complex particle trajectories in the force field and the accumulation of particles in well-de fined regions above the microelectrode array. In the second part of this thesis, the mechanism of RNA interference (RNAi) was studied by dual-color FCCS in vivo. RNAi is an evolutionary conserved gene silencing mechanism, which uses short double-stranded RNA molecules, called short interfering RNAs (siRNAs), as effector molecules. Due to its speci city and simplicity, RNAi yields a great potential for a widespread therapeutic use. To broaden the therapeutic applications, the in vivo stability of siRNAs has to be improved by chemical modi cations, but some of these modi fications inhibit the gene silencing mechanism. The presented FCCS assays are very well suited to investigate the individual assembly steps of RNAi machinery with very high specifi city and sensitivity in real time and to study the cleavage activity of the activated RNAi machinery. A direct correlation between activity of the RNAi machinery and the results from the FCCS measurements could be shown. The in fluence of several chemical modi cations on the assembly and activity of the RNAi machinery was investigated with these assays. / Fluoreszenz-Korrelations-Spektroskopie (FCS) analysiert die Fluktuationen im Fluoreszenzsignal eines kleinen angeregten Volumens, um Informationen über die Konzentration, die Bewegung und die Interaktionen der markierten Moleküle zu erhalten. Die Fluoreszenz-Kreuzkorrelations-Spektroskopie (FCCS) erhöht die Genauigkeit bei der Messung von Fließgeschwindigkeiten und Bindungskonstanten im Vergleich zur Standard-FCS. Die Miniaturisierung der Biomedizin und Chemie hat sich rapide entwickelt, angetrieben von der Vision eines kompletten Labors auf einem Chip und dem Einsatz dieses in der medizinischen Therapie, zum Beispiel als implantierter Medikamentenspender. Ein Schlüsselelement zur Erfüllung dieser Vision ist der Transport von kleinsten Flüssigkeitsmengen in diesen miniaturisierten Systemen. Der Transport von Flüssigkeiten mittels des elektrohydrodynamischen Prinzips umgeht viele Nachteile von anderen Systemen, allerdings zeigt eine solche Pumpe ein kompliziertes Strömungsbild in der aktiven Region, welches sich mit herkömmlichen Methoden wie Teilchenverfolgung nicht vermessen ließ. Hier wurde Zwei-Fokus-FCCS (2f-FCCS) genutzt, um das Strömungsbild in der Pumpe zu vermessen. Die hohe Genauigkeit der 2f-FCCS bei der Bestimmung von Fließgeschwindigkeiten auch mit kleinen fluoreszierenden Teilchen ermöglichte die Messung der Fließgeschwindigkeiten, aufgrund der auf das Lösungsmittel wirkenden elektrohydrodynamischen Kräfte, unter Ausschluss der auf größere Teilchen wirkenden dielektrophoretischen Kräfte. Die Analyse der Daten ergab, dass das Strömungsbild aus zwei entgegengesetzt rotierenden unterschiedlich großen Wirbeln besteht. Dieses Strömungsbild erklärt die komplizierten Teilchenbewegungsbahnen und die Anreicherung der Teilchen in klar abgegrenzten Bereichen über den Mikroelektroden. Im zweiten Teil dieser Arbeit wurde der RNAi-Mechanismus in lebenden Zellen mittels Zwei-Farben-FCCS untersucht. RNA Interferenz (RNAi) ist ein evolutionär erhaltener Geninaktivierungsmechanismus, der kurze doppelsträngige RNA Moleküle, so genannte kurze interferierende RNAs (siRNAs), als Effektormoleküle nutzt. Die Spezifi tät und Einfachheit der RNAi hat ihr ein weites Feld in der medikamentösen Therapie geöffnet. Zur Erweiterung dieses Feldes ist es nötig die Stabilität der siRNAs im Körper mittels chemischer Modi fikationen zu erhöhen. Einige dieser Modifikationen hemmen aber den RNAi-Mechanismus. Die hier vorgestellten FCCS Experimente sind sehr gut geeignet, um die einzelnen Schritte des Zusammenbaus der RNAi Maschinerie mit hoher Empfi ndlichkeit und Spezi fität in Echtzeit zu untersuchen und die Aktivität der RNAi Maschinerie zu studieren. Es konnte ein Zusammenhang zwischen der Aktivität der RNAi Maschinerie und den Ergebnissen der FCCS Messungen hergestellt werden. Der Einfluss von verschiedenen chemischen Modikationen auf den Zusammenbau und die Aktivität der RNAi Maschinerie wurde mit diesen neuartigen Methoden untersucht.

Motivation: Although various high-throughput technologies provide a lot of valuable information, each of them is giving an insight into different aspects of cellular activity and each has its own limitations. Thus, a complete and systematic understanding of the cellular machinery can be achieved only by a combined analysis of results coming from different approaches. However, methods and tools for integration and analysis of heterogenous biological data still have to be developed. Results: This work presents systemic analysis of basic cellular processes, i.e. cell viability and cell cycle, as well as embryonic stem cell pluripotency and differentiation. These phenomena were studied using several high-throughput technologies, whose combined results were analysed with existing and novel clustering and hit selection algorithms. This thesis also introduces two novel data management and data analysis tools. The first, called DSViewer, is a database application designed for integrating and querying results coming from various genome-wide experiments. The second, named PhenoFam, is an application performing gene set enrichment analysis by employing structural and functional information on families of protein domains as annotation terms. Both programs are accessible through a web interface. Conclusions: Eventually, investigations presented in this work provide the research community with novel and markedly improved repertoire of computational tools and methods that facilitate the systematic analysis of accumulated information obtained from high-throughput studies into novel biological insights.

No description available.

In 1990, Richard Jorgensen’s lab initiated a study to test if they could create a more vivid color petunia (Napoli et al. 1990). Their plan was to transform plants with the chalcone synthase transgene––the predicted rate limiting factor in the production of purple pigmentation. Much to their surprise, the transgenic plants, as well as their progeny, displayed a great reduction in pigmentation. This loss of endogenous function was termed “cosuppression” and it was thought that sequence-specific repression resulted from over-expression of the homologous transgene sequence. In 1998, Andrew Fire and Craig Mello described a phenomenon in which double stranded RNA (dsRNA) can trigger silencing of cognate sequences when injected into the nematode, Caenorhabditis elegans (Fire et al. 1998). This data explained observations seen years earlier by other worm researchers, and suggested that repression of pigmentation in plants was caused by a dsRNA-intermediate (Guo and Kemphues 1995; Napoli et al. 1990). The phenomenon––which soon after was coined RNA interference (RNAi)––was soon discovered to be a post-transcriptional surveillance system in plants and animals to remove foreign nucleic acids.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA tumor virus that has been associated with all epidemiological forms of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). Like other herpesviruses, KSHV undergoes two phases of life cycle (latent and lytic replication). During latency, the viral genome persists as a circular episome in the nucleus of the host cell and only a few viral genes are expressed, namely LANA (latency- associated nuclear antigen), Kaposin, vFLIP (viral FLICE inhibitory protein), vCyclin, and vIRF3/LANA2 (viral interferon regulatory factor 3). These viral genes are responsible for regulation of host cell proliferation, prevention of apoptosis, facilitation of immune evasion, and maintenance of the extrachromosomal viral genome during cell divisions. vIRF3 is a multifunctional nuclear protein that is constitutively expressed in KSHV positive PEL cells and Castleman's disease tumors, which expression causes dramatic changes of critical host pathways that are involved in the regulation of apoptosis, cell cycle, antiviral immunity, and tumorigenesis. In our study, we have demonstrated and elucidated predicted mechanism, by which vIRF3 enhances transcription activity of c-Myc. Moreover, we have clarified the previously unappreciated...

Neste estudo, investigamos o envolvimento de galectina-3 na fisiologia e no câncer de tiróide usando vários modelos biológicos e metodologias. Observamos que o gene LGALS3 apresenta um SNP no códon 98, mas não observamos correlação entre os genótipos deste SNP e fenótipo de câncer de tiróide. Na linhagem de tiróide de rato PCCl3, mostramos que a indução da expressão do oncogene RET/PTC promove o aumento da expressão de galectina-3, no entanto, a expressão de galectina-3, por si só, não confere vantagem de proliferação à célula. Por outro lado, na linhagem de carcinoma papilífero de tiróide TPC-1, a galectina-3 contribui para a sobrevivência da célula tumoral e progressão do ciclo celular, aumentando a expressão de c-Myc, diminuindo a expressão de p21 e caspase-3, e favorecendo a ativação de importantes vias envolvidas no controle do ciclo celular. Além disto, em modelos in vivo e in vitro, a galectina-3 interferiu na função e diferenciação da célula folicular tiroidiana, exercendo um papel indireto na regulação da expressão da tireoglobulina e atividade de TTF-1. / In this study, we investigate the involvement of galectin-3 in thyroid physiology and cancer using several biological models and methodologies. We observed that LGALS3 gene presents a SNP in codon 98, but no correlation between the genotype and the phenotype of benign or malignant thyroid tumor was observed. In the rat thyroid cell line PCCl3, we showed that the conditional induction of RET/PTC oncogene expression promotes the increase of galectin-3 expression, however, galectin-3 expression itself did not confer a proliferative advantage to cell. On the other hand, in papillary thyroid carcinoma cell line TPC-1 the galectin-3 contributes to tumor cell survival and cell cycle progression, increasing c-Myc expression, decreasing p21 and caspase-3 expression and cooperating to activation of important signaling pathways which are involved in the cell cycle control. In addition, in vitro and in vivo models the galectin-3 interferes in the differentiation and function of thyroid follicular cell, playing an indirect role in the regulation of thyroglobulin expression and TTF-1 activity.

Gene deletions are a powerful method to uncover the cellular functions of a given gene in living systems. A limitation to this methodology is that it is not applicable to essential genes. Even for non-essential genes, gene knockouts cause complete absence of gene product thereby limiting genetic analysis of the biological pathway. Alternatives to gene deletions are mutants that are conditional, for e.g, temperature sensitive (ts) mutants are robust tools to understand temporal and spatial functions of genes. By definition, products of such mutants have near normal activity at a lower temperature or near-optimal growth temperature which is called as the permissive temperature and reduced activity at a higher, non-optimal temperature called as the non-permissive temperature. Generation of ts alleles in genes of interest is often time consuming as it requires screening a large population of mutants to identify those that are conditional. Often many essential proteins do not yield ts such alleles even after saturation mutagenesis and extensive screening (Harris et al., 1992; Varadarajan et al., 1996). The limited availability of such mutants in many essential genes prompted us to adopt a biophysical approach to design temperature-sensitive missense mutants in an essential gene of fission yeast. Several studies report that mutations in buried or solvent-inaccessible amino acids cause extensive changes in the thermal stability of proteins and specific substitutions create temperature-sensitive mutants (Rennell et al., 1991; Sandberg et al., 1995). We used the above approach to generate conditional mutants in the fission yeast gene spprp18+encoding an essential predicted second splicing factor based on its homology with human and S. cerevisiae proteins. We have used a missense mutant coupled with a conditional expression system to elucidate the cellular functions of spprp18+. Further, we have employed the same biophysical principle to generate a missense mutant in spago1+ RNA silencing factor that is non-essential for viability but has critical functions in the RNAi pathway of fission yeast. Fission yeast pre-mRNA splicing: cellular functions for the protein factor SpPrp18 Pre-mRNA splicing is an evolutionarily conserved process that excises introns from nascent transcripts. Splicing reactions are catalyzed by the large ribonuclear protein machinery called the spliceosome and occur by two invariant trans-esterification reactions (reviewed in Ruby and Abelson, 1991; Moore et al., 1993). The RNA-RNA, RNA–protein and protein-protein interactions in an assembly of such a large protein complex are numerous and highly dynamic in nature. These interactions in in vitro splicing reactions show ordered recruitment of essential small nuclear ribonucleic particles snRNPs and non–snRNP components on pre-mRNA cis-elements. Further these trans acting factors recognize and poise the catalytic sites in proximity to identify and excise introns. The precision of the process is remarkable given the diversity in architecture for exons and introns in eukaryotic genes (reviewed in Burge et al., 1999; Will and Luhrmann, 2006). Many spliceosomal protein components are conserved across various organisms, yet introns have diverse features with large variations in primary sequence. We hypothesize that co-evolution of splicing factor functions occurs with changes in gene and intron architectures and argue for alternative spliceosomal interactions for spliceosomal proteins that thus enabling splicing of the divergent introns. In vitro biochemical and genetic studies in S. cerevisiae and biochemical studies with human cell lines have indicated that ScPRP18 and its human homolog hPRP18 function during the second catalytic reaction. In S. cerevisiae, ScPrp18 is non-essential for viability at growth temperatures <30°C (Vijayraghavan et al., 1989; Vijayraghavan and Abelson, 1990; Horowitz and Abelson, 1993b). The concerted action of ScSlu7 - ScPrp18 heteromeric complex is essential for proper 3’ss definition during the second catalytic reaction (Zhang and Schwer, 1997; James et al., 2002). These in vitro studies also hinted at a possible intron -specific requirement for ScPrp18 and ScSlu7 factors as they were dispensable for splicing of intron variants made in modified ACT1 intron containing transcripts (Brys and Schwer, 1996; Zhang and Schwer, 1997). A short spacing distance between branch point adenosine to 3’splice site rendered the substrate independent of Prp18 and Slu7 for the second step (Brys and Schwer, 1996; Zhang and Schwer, 1997). Extensive mutational analyses of budding yeast ScPrp18 identified two functional domains and suggested separate roles during splicing (Bacikova and Horowitz, 2002; James et al., 2002). Fission yeast with its genome harboring multiple introns and degenerate splice signals has recently emerged as a unique model to study relationships between splicing factors and their role in genomes with short introns. Previously, studies in our lab had initiated genetic and mutational analysis of S. pombe Prp18, the predicted homolog of budding yeast Prp18. Genetic analysis showed its essentiality, but a set of missense mutants based on studies of budding yeast ScPrp18 (Bacikova and Horowitz, 2002) gave either inactive null or entirely wild type phenotype for the fission yeast protein. In this study, we have extended our previous mutational analysis of fission yeast Prp18 by adopting biophysical and computational approaches to generate temperature-sensitive mutants. A missense mutant was used to understand the splicing functions and interactions of SpPrp18 and the findings are summarized below. Fission yeast SpPrp18 is an essential splicing factor with transcript-specific functions and links efficient splicing with cell cycle progression We initiated our analysis of SpPrp18 by adopting a biophysical approach to generate ts mutants. We used the PREDBUR algorithm to predict a set of buried residues, which when mutated could result in a temperature-sensitive phenotype that complements the null allele at permissive temperature. These predictions are based upon two biophysical properties of amino acids: 1) Hydrophobicity, which is calculated in a window of seven amino acids 2) Hydrophobic moment, which is calculated in a sliding window of nine amino acids in a given protein sequence. Several studies correlate these properties to protein stability and function (Varadarajan et al., 1996). One of the buried residue mutants V194R, in helix 1 of SpPrp18 conferred weak temperature- sensitivity and strong cold-sensitivity even when the protein was over expressed from a plasmid. Through semi-quantitative RT-PCR we showed splicing-defects for tfIId+ intron1 in these cells even when grown at permissive temperature. The primary phenotype was the accumulation of pre-mRNA. Further, we showed this splicing arrest is co-related with reduced levels of SpPrp18 protein, linking protein stability and splicing function. Next we examined the effects of this mutation on function by further reduction of protein levels. This was done by integrating the expression cassette nmt81:spprp18+/spprp18V194R at the leu1 chromosomal locus and by metabolic depletion of the integrated allele. Through RT-PCRs we demonstrated that depletion of wild type or missense protein has intron specific splicing defects. These findings showed its non-global and possibly substrate-specific splicing function. In the affected introns, precursor accumulation is the major phenotype, confirming prior data from our lab that hinted at its likely early splicing role. This contrasts with the second step splicing role of the human or budding yeast Prp18 proteins. Previous data from our lab showed loss of physical interaction between SpPrp18 and SpSlu7 by co-immunoprecipitation studies. This again differs from the strong and functionally important ScPrp18 and ScSlu7 interaction seen in budding yeast. We show the absence of charged residues in SpSlu7 interaction region formed by SpPrp18 helix1 and helix2 which can explain the altered associations for SpPrp18 in fission yeast. Importantly, as the V194R mutation in helix 1 shows splicing defects even at permissive temperature, the data indicate a critical role for helix 1 for splicing interactions, possibly one that bridges or stabilizes the proposed weak association of SpPrp18-SpSlu7 with a yet unknown splicing factor. We also investigated the effects of mutations in other helices; surprisingly we recovered only mutations with very subtle growth phenotypes and very mild splicing defects. Not surprisingly, stop codon at L239 residue predicted to form a truncated protein lacking helices 3, 4 and 5 conferred recessive but null phenotype implicating essential functions for other helices. Other amino acid substitutions at L239 position had near wild type phenotype at 30°C and 37°C. Helix 3 buried residue mutant I259A conferred strong cold-sensitivity when over expressed from plasmid, but semi quantitative analysis indicated no splicing defects for intron1 in the constitutively expressed transcript tfIId+. These findings indicate cold sensitivity either arises due to compromised splicing of yet unknown transcripts or that over-expressed protein has near wild type activity. We find mutations in the helix 5 buried residues L324 also conferred near WT phenotype. Earlier studies in the lab found that substitution of surface residues KR that are in helix 5 with alanine lead to null phenotypes (Piyush Khandelia and Usha Vijayraghavan unpublished data). We report stable expression of all of these mutant proteins; L239A, L239P, L239G, I259A, I259V, L324F, L324A as determined by our immunoblot analysis at 30°C and 37°C. The mild phenotypes of many buried residues can be attributed to orientation of their functional groups into a protein cavity between the helices. Lastly, our microscopic cellular and biochemical analysis of cellular phenotypes of spprp18 mutant provided a novel and direct role of this factor in G1-S transition of cell cycle. Our RT-PCR data suggest spprp18+ is required for efficient splicing of several intron containing transcripts involved in G1-S transition and subsequent activation of MBF complex (MluI cell cycle box-binding factor complex) during S-phase and shows a mechanistic link between cell cycle progression and splicing. A tool to study links between RNA interference, centromeric non-coding RNA transcription and heterochromatin formation S.pombe possesses fully functional RNA interference machinery with a single copy for essential RNAi genes ago1+, dcr1+ and rdp1+. Deletion of any of these genes causes loss of heterochromatinzation with abnormal cytokinesis, cell-cycle deregulation and mating defects (Volpe et al., 2002). In S.pombe, exogenous or endogenously generated dsRNA’s from transcription of centromeric repeats are processed by the RNaseIII enzyme dicer to form siRNA. These siRNA’s are loaded in Ago1 to form minimal RNA induced silencing complex (RISC) complex or specialized transcription machinery complex RNA induced transcriptional silencing (RITS) complex and target chromatin or complementary mRNAs for silencing. Thus as in other eukaryotes, fission yeast cells deploy RNAi mediated silencing machinery to regulate gene-expression and influence chromatin status. Several recent studies point to emerging new roles of RNAi and its association with other RNA processes (Woolcock et al., 2011; Bayane et al., 2008; Kallgren et al., 2014). Many recent reports suggest physical interactions of RISC or RITS and RNA dependent RNA polymerase complex (RDRC) with either some factors of the spliceosomal machinery, heterochromatin machinery (CLRC complex) and the exosome mediated RNA degradation machinery (Bayne et al., 2008 and Chinen et al., 2010 ; Hiriart et al., 2012; Buhler et al., 2008; Bayne et al., 2010 ). Thus we presume conditional alleles in spago1+ will facilitate future studies to probe the genetic network between these complexes as most analyses thus far rely on ago1∆ allele or have been based on proteomic pull down analyses of RISC or RITS complexes. In this study, we employed biophysical and modeling approaches described earlier to generate temperature sensitive mutants in spago1+ and spdcr1+. We tested several mutants for their ability to repress two reporter genes in a conditional manner. Our modeling studies on SpAgo1 PAZ domain indicated structural similarities with human Ago1 PAZ domain. We created site-directed missense mutants at predicted buried residues or in catalytic residues. We also analyzed the effects of random amino acid replacements in specific predicted buried or catalytic residues of SpAgoI. These ago1 mutants were screened as pools for their effects on silencing of GFP or of ura4+ reporter genes. These assays assessed post transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) activity of these mutants. We obtained three temperature sensitive SpAgo1 mutants V324G, V324S and L215V while the V324E replacement was a null allele. Based upon our modeling, a likely explanation for the phenotype of these mutants is structural distortion or mis-orientation of the functional groups caused due to these mutations, which affect activity in a temperature dependent manner. This distortion in the PAZ domain may affect binding of siRNA and thereby lead to heterochromatin formation defects that we observed. Our data on the SpAgo1 V324 mutant shows conditional centromeric heterochromatin formation confirmed by semi quantitative RT-PCR for dh transcripts levels that shows temperature dependent increase in these transcripts. We find reduced H3K9Me2 levels at dh locus by chromatin immunoprecipitation (ChIP) assay, linking the association of siRNAs for establishment of heterochromatin at this loci. The data on PTGS of GFP transcripts show SpAgo1 V324G mutation has decreased slicing activity as semi-quantitative RT-PCR for GFP transcripts show increased levels at non permissive temperature. These studies point out the importance of siRNA binding to the PAZ domain and its effect on slicing activity of SpAgo1. The mutations in Y292 showed residue loss of centromeric heterochromatin formation phenotype. Thus, we ascribe critical siRNA binding and 3’ end recognition functions to this residue of SpAgo1. These studies point out functional and structural conservation across hAgo1 and SpAgo1. Adopting the aforementioned biophysical mutational approach, we generated mutants in spdcr1+ and screened for those with conditional activity. Our modeling studies on SpDcr1 helicase domain shows it adopts the conserved helicase domain structure seen for other DEAD Box helicases. Our data on mutational analysis of a conserved buried residue I143 in the walker motif B created inactive protein. The data confirm critical functions for dicer in generation of siRNAs and also in recognition of dsRNA ends. Mutants in buried residues L1130 and I1228 of RNase IIIb domain were inactive and the proximity of these residues to the catalytic core suggest that the critical structural alignment of catalytic residues is indispensable for carrying out dsRNA cleavage to generate siRNAs. We also attribute critical catalytic functions to SpDcr1 D1185 residue for generation of siRNA and heterochromatin formation as measured by our transcriptional gene silencing assay. Our studies employing biophysical and computational approaches to design temperature-sensitive mutants have been successfully applied to an essential splicing factor SpPrp18, which was refractory for ts mutants by other methods. Using a missense mutant, we showed its intron-specific splicing function for subsets of transcripts and deduced that its ubiquitous splicing role is arguable. We have uncovered a link between the splicing substrates of SpPrp18 and direct evidence of splicing based cell cycle regulation, thus providing a mechanistic link to the cell cycle arrest seen in some splicing factor mutants. The same methodology was applied to another important biological pathway, the RNAi machinery, where central factors SpAgoI and SpDcrI were examined We report the first instance of conditional gene silencing tool by designing Ago1 ts mutants which will be useful for future studies of the global interaction network between RNAi and other RNA processing events.

Uncontrolled spreading of genetically modified (GM) plants is one of the main concerns about their cultivation. Inducible RNA interference against an essential gene could be a tool for control of GM plants. After spraying with a chemical inducer, the essential gene will be silenced so the treated GM plant will die. For testing this strategy we chose two key enzymes of nitrogen metabolism, glutamate synthase (GOGAT) and glutamine synthetase (GS). GS processes ammonium ions into glutamine, then GOGAT transfers the amide group from glutamine to 2-oxoglutarate to form two glutamates. GS/GOGAT cycle is the main pathway for assimilation of ammonium ions, which could be toxic to plants in a higher concentration. Disruption of ammonium assimilation during photorespiration causes a strong inhibition of photosynthesis. The aim of this work was to describe the effects of silencing GOGAT and GS genes in Arabidopsis thaliana. To induce silencing, RNAi hairpin constructs under a control of constitutive or estradiol-inducible promoter were prepared. In selected independent transformants with the inducible hairpin against GOGAT, chlorosis and reduced growth were observed after the estradiol treatment in in vitro conditions. However, the spraying with estradiol was tricky, at the whole plant level, the induction of...

Background Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species. Methodology/Principal Findings We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster. Conclusions/Significance The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.

Huntington’s disease (HD) is an autosomal dominant, progressive neurodegenerative disorder. Invariably fatal, HD is caused by expansion of the CAG repeat region in exon 1 of the Huntingtin gene which creates a toxic protein with an extended polyglutamine tract 1. Silencing mutant Huntingtin messenger RNA (mRNA) is a promising therapeutic approach 2-6. The ideal silencing strategy would reduce mutant Huntingtin while leaving the wild-type mRNA intact. Unfortunately, targeting the disease causing CAG repeat expansion is difficult and risks targeting other CAG repeat containing genes. We examined an alternative strategy, targeting single nucleotide polymorphisms (SNPs) in the Huntingtin mRNA. The feasibility of this approach hinges on the presence of a few common highly heterozygous SNPs which are amenable to SNP-specific targeting. In a population of HD patients from Europe and the United states, forty-eight percent were heterozygous at a single SNP site; one isoform of this SNP is associated with HD. Seventy-five percent of patients are heterozygous at least one of three frequently heterozygous SNPs. Consequently, only five allele-specific siRNAs are required to treat three-quarters of the patients in the European and U.S. patient populations. We have designed and validated siRNAs targeting these SNPs. We also developed artificial microRNAs (miRNAs) targeting Huntingtin SNPs for delivery using recombinant adeno-associated viruses (rAAVs). Both U6 promoter driven and CMV promoter driven miRNAs can discriminate between matched and mismatched targets in cell culture but the U6 promoter driven miRNAs produce the mature miRNA at levels exceeding those of the vast majority of endogenous miRNAs. The U6 promoter driven miRNAs can produce a number of unwanted processing products, most likely due to a combination of overexpression and unintended export of the pri-miRNA from the nucleus. In contrast, CMV-promoter driven miRNAs produce predominantly a single species at levels comparable to endogenous miRNAs. Injection of recombinant self complementary AAV9 viruses carrying polymerase II driven Huntingtin SNP targeting miRNAs into the striatum results in expression of the mature miRNA sequence in the brain and has no significant effect on endogenous miRNAs. Matched, but not mismatched SNP-targeting miRNAs reduce inclusions in a knock-in mouse model of HD. These studies bring us closer to an allele-specific therapy for Huntington’s disease.