Variability of transgene expression levels resulting from gene silencing is considered as ahindrance to the successful application of plant genetic engineering. Towards alleviatinggene silencing, I decided to screen for novel genes involved in transgene silencing and toinvestigate how these genes regulate plant development. Genes encoding putative chromatinremodeling factors, especially those including a SET domain, were selected as candidatetargets. A bioinformatic analysis of the Arabidopsis SET genes (AtSET) was performed andthese genes were classified into 6 groups based on the domain architecture. RNA interference (RNAi) vectors were constructed for ~ 20 AtSET genes and wereintroduced into both wild type lines and transgenic lines silenced for a GFP reporter gene.Surprisingly, altered developmental phenotypes were only observed for three constructs,raising questions as to the effectiveness of the RNAi approach for the chosen Arabidopsissystem. To assess this situation, I targeted a phytoene desaturase (PDS) gene using the sameRNAi approach. Inactivation of PDS renders plant a readily identifiable phenotype. Whereasthe RNAi penetrance in Arabidopsis can be very high, the expressivity of RNAi in varioustissues and among different plants can vary dramatically. Contradictory to previous reports,I found that there is correlation between transcript level and silencing phenotype. Possiblereasons for this discrepancy are discussed. No apparent correlation between transgene copynumber and RNAi phenotypes was observed. Among the three RNAi constructs that caused an abnormal development inArabidopsis, K-23 which targets SuvR3 has the highest expressivity and could reactivate asilenced GFP locus. SuvR3 RNAi lines were selfed for six generations and were screenedfor morphological phenotypes. Abnormal number of flower organs, loss of viability of malegametophytes, and decreased seedling germination percentage were found in SuvR3 RNAilines. A progressive increase in both severity and frequency of abnormal phenotypes wereseen in subsequent generations, suggesting an epigenetic regulatory mechanism involvedwith SuvR3. Alternative splicing of SuvR3 was also observed in most of Arabidopsis tissues.One of the protein isoforms, SuvR3, lacks 16 amino acids within the highly conserved SETdomain. Possible effects of isoform interaction are proposed.

Optical transceiver module plays an important role in the optical communication system. The packaging quality of the module decides the ability of the communication. Since the light signal is transferred from a laser diode to an optical fiber, the light transfer efficiency between these two components becomes a very important work to be done. The micrometer dimension and the ultra-high performance requirement of these components lead to many problems in module packaging process. Among all the problems, the packaging of the fiber components is the most complicated. In this research, many key technologies are proposed to solve or improve the problems in the packaging of the fiber components. Thus, the performance of the module can be ensured. Two main topics of the fiber component packaging will be introduced here, the fiber-solder-ferrule (FSF) packaging and the machining of the fiber. In the packaging of the FSF, a positioning and a soldering technology are proposed to improve the packaging yield. For the positioning, a novel control strategy is constructed to shorten the positioning time and improve the positioning accuracy. Thus, the position of the fiber can be positioned at the center of the ferrule fast and precisely. The controller successfully completes the positioning command in 0.25sec with 1µm accuracy. And finally, the coupling efficiency can be hold. For the soldering of the FSF, an active soldering mechanism is developed to replace the passive manual operation. The mechanism successfully proofs the stability of the soldering and raises the yield from the 25% to 83%. In machining of the fiber, a fiber end polishing issue and a fiber inspection topic are addressed. For the fiber end polishing, an online force sensing mechanism is implemented. The force sensing mechanism can control the polished fiber tip offset within 1.5µm. So the fiber coupling efficiency can be maintained. A control strategy is designed to solve the polishing problems and reach the polishing requirement. At last, an interference-based fiber inspection method is proposed to find the splicing plane between two spliced fibers. The accuracy of the fiber cleaving in a cascaded fiber fabrication improves from 10µm to 1µm by observing the fiber splicing plane precisely. All the improvements of the above packaging technologies are proposed to raise or keep the performance of the transceiver module. So, the error between theories and experiments can be minimized. Meanwhile, a high stability and repeatability of the packaging can be achieved due to the automation of the positioning, force sensing, and inspection.

Eukaryotic gene expression requires hundreds of proteins and several RNA factors to facilitate nuclear RNA processing. These RNA processing events include RNA transcription, pre-mRNA splicing, pre-ribosomal RNA (pre-rRNA) processing and trafficking of RNA to its proper location in the cell. As we learn more about the molecular details of the factors governing these highly coordinated processes it is becoming increasingly clear that a subset of factors participate in multiple RNA processing pathways to ensure faithful gene expression. Our work completes the characterization of the Abelson pre-mRNA splicing mutants. We have discovered that the prp27-1 splicing mutant is a severe loss of function allele of RAT1, an essential 5’→3’ exoribonuclease. Several alleles of RAT1 have been previously isolated with each conferring an array of phenotypes thus making the elucidation of its essential in vivo function difficult. We set out to determine how mutations within a specific region determines the RNA processing pathway in which Rat1p has been implicated to function within. In our analysis of Rat1p function we discovered the prp27-1 allele exhibits novel 3’ end processing defects never reported in previous rat1 mutants. We performed mutational analysis to examine the coupling of 5’ and 3’ exonucleolytic activities in nuclear RNA processing events. Through our study we have discovered a means by which the cell coordinately regulates the nuclear RNA degradation complexes to ensure efficient processing of pre-RNAs for the faithful execution of eukaryotic gene expression. Additionally, we offer evidence in support of role for Rat1p in promoting mitotic events in vivo. / text

Im Rahmen dieser Arbeit wurde Organisation, Expression und Funktion des humanen Peroxisomal-Testis-Spezifisch-1(PXT1)-Gens untersucht. Die mRNA des humanen PXT1-Gens enthält nicht wie bisher bekannt zwei Exons, sondern fünf Exons. Die Expression von drei putativen Exons stromaufwärts konnte in dieser Arbeit bestätigt werden. Die Ergebnisse qualitativer und quantitativer Real Time-PCR zeigen, dass sich das Exon 1 aus drei unterschiedlich gespleißten Einheiten (Exons 1a, 1b und 1c) zusammensetzt. Das humane PXT1-Gen unterliegt dem alternativen Spleißen, wovon die Exons 1b, 1c, 2 und 4 betroffen sind, was Sequenzanalysen zeigen. Sechs Transkripte konnten insgesamt identifiziert werden. Die zusätzlichen Exons haben Auswirkungen auf die Proteinstruktur aufgrund der Verlängerung des ORF, kodierend für einst 51 Aminosäuren, auf 134. Im längeren Protein wird die BH3 interacting domain (BID) nachgewiesen, von der eine proapoptotische Funktion bekannt ist. Aufgrund des alternativ gespleißten Exon 4 und der daraus resultierenden Leserasterverschiebung existiert ein verkürztes Protein, in dessen mRNA sich ein vorzeitiges Stopkodon befindet. Die proapoptotische Domäne ist nicht mehr nachweisbar. In silico-Analysen zeigen, dass die Sequenzen der Exons 1a und 1b von PXT1 sich mit dem KCTD20-Gen überlappen, das für einen Kaliumkanal kodiert.  Im Unterschied zum murinen, testisspezifischen Pxt1-Gen, ist das humane Homolog trotz Prädominanz im Testis auch schwächer in anderen Geweben nachweisbar.  Zur weiteren Klärung der proapoptotischen Funktion von Pxt1 in Keimzellen wurde am Mausmodell (Pxt1-Knockout-Maus) die Anzahl an DNA-Strangbrüchen untersucht. Im Vergleich zu den Kontrolltieren (C57BL/6J) zeigt die Pxt1-Knockout-Maus eine signifikant erhöhte Anzahl an Spermien mit DNA-Strangbrüchen. Dieses Ergebnis bestätigt die Annahme, dass das PXT1/Pxt1-Gen eine Art Entsorgungsfunktion für beschädigte Spermien ausübt. Im zeitlichen Verlauf zeigte sich aber, dass die Spermien der Knockout-Tiere nicht sensibler als die Wildtyp-Tiere auf DNaseI reagieren.  Als mögliches Kandidatengen für Mutationsanalysen bei Männern mit Fertilitätsstörungen wurden 55 Patienten mit Fertilitätsstörungen (Azoo- oder Oligozoospermie) auf Punktmutationen im PXT1 untersucht. Eine Mutation konnte nicht identifiziert werden. Des Weiteren wurde die DNA der Patienten auf Copy Number Variations analysiert. Sowohl heterozygote als auch homozygote Duplikationen konnten im Exon1, bestätigt mithilfe der arraybasierten Comparativen Genomischen Hybridisierung (aCGH), vereinzelt auch in Exon2 und Exon3 nachgewiesen werden. Zusätzlich konnte bei einem Patienten eine Deletion nachgewiesen werden. Die bestätigte Duplikation im Exon 1 besitzt aber keinen Krankheitswert, da sie in einem Kontrollkollektiv eine Prävalenz von 41% in heterozygoter und 10% in homozygoter Form besitzt.

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Objetivo general Ampliar el conocimiento de la cascada de señalización de TGF-β en células humanas mediante el estudio de TβRII y sus variantes de splicing, que permita contribuir al conocimiento básico de dicha cascada así como al desarrollo de nuevas estrategias terapéuticas y de diagnóstico de enfermedades en las cuales la señalización de TGF-β se encuentra desregulada. Objetivos específicos - Caracterizar una nueva variante de splicing de TβRII en células humanas. - Evaluar los niveles de ARNm de las distintas variantes de splicing de TβRII en células humanas tumorales y no tumorales, que permita detectar patrones diferenciales entre ellas o patrones específicos de tipo celular. - Evaluar si los niveles de los ARNm de las variantes de splicing de TβRII en distintas poblaciones leucocitarias de pacientes con AR pueden ser utilizados como biomarcadores de diagnóstico y/o de determinación de la actividad y/o de pronóstico de la enfermedad.

RNA Binding Motif (RBM) domain proteins RBM5 and RBM10 have been shown to influence apoptosis, cell cycle arrest and splicing in transformed cells. In this study, RBM5 and RBM10 were examined in non-transformed cells in order to gain a wider range of knowledge regarding their function. Expression of Rbm5 and Rbm10, as well as select splice variants, was examined at the mRNA and protein level throughout H9c2 skeletal and cardiac myoblast differentiation. Results suggest that Rbm5 and Rbm10 may (a) be involved in regulating cell cycle arrest and apoptosis during skeletal myoblast differentiation and (b) undergo post-transcriptional or translational regulation throughout myoblast differentiation. All in all, the expression profiles obtained in the course of this study will help to suggest a role for Rbm5 and Rbm10 in differentiation, as well as possible differentiation-specific target genes with which they may interact.

Histone deacetylases (HDAC) 1 and 2 play crucial role in chromatin remodeling and gene expression regimes, as part of multiprotein corepressor complexes. Protein kinase CK2-driven phosphorylation of HDAC1 and 2 regulates their catalytic activities and is required to form the corepressor complexes. Phosphorylation-mediated differential distributions of HDAC1 and 2 complexes in regulatory and coding regions of transcribed genes catalyze the dynamic protein acetylation of histones and other proteins, thereby influence gene expression. During mitosis, highly phosphorylated HDAC1 and 2 heterodimers dissociate and displace from mitotic chromosomes. Our goal was to identify the kinase involved in mitotic phosphorylation of HDAC1 and 2. We postulated that CK2-mediated increased phosphorylation of HDAC1 and 2 leads to dissociation of the heterodimers, and, the mitotic chromosomal exclusions of HDAC1 and 2 are largely due to the displacement of HDAC-associated proteins and transcription factors, which recruit HDACs, from chromosomes during mitosis. We further explored the role of un- or monomodified HDAC1 and 2 complexes in immediate-early genes (IEGs), FOSL1 (FOS-like antigen-1) and MCL1 (Myeloid cell leukemia-1), regulation. Dynamic histone acetylation is an important regulator of these genes that are overexpressed in a number of diseases and cancers. We hypothesized that transcription dependent recruitment of HDAC1 and 2 complexes over the gene body regions plays a regulatory role in transcription and splicing regulation of these genes. We present evidence that CK2-catalyzed increased phosphorylation of HDAC1 and 2 regulates the formation of distinct corepressor complexes containing either HDAC1 or HDAC2 homodimers during mitosis, which might target cellular factors. Furthermore, the exclusion of HDAC-recruiting proteins is the major factor for their displacement from mitotic chromosomes. We further demonstrated that un- or monophosphorylated HDAC1 and 2 are associated with gene body of FOSL1 in a transcription dependent manner. However, HDAC inhibitors prevented FOSL1 activation independently of the nucleosome response pathway, which is required for IEG induction. Interestingly, our mass spectrometry results revealed that HDAC1 and 2 interact with a number of splicing proteins, in particular, with serine/arginine-rich splicing factor 1 (SRSF1). HDAC1 and 2 are co-occupied with SRSF1 over gene body regions of FOSL1 and MCL1, regardless of underlying splicing mechanisms. Using siRNA-mediated knockdown approaches and HDAC inhibitors, we demonstrated that alternative splicing of MCL1 is regulated by RNA-directed localized changes in the histone acetylation levels at the alternative exon. The change in histone acetylation levels correlates with the increased transcription elongation and results in change in MCL1 splicing by exon skipping mechanism. Taken together, our results contribute to further understanding of how the multi-faceted HDAC1 and 2 complexes can be regulated and function in various processes, including, but not limited to, transcription regulation and alternative splicing. This can be an exciting area of future research for therapeutic interventions.

This dissertation details three studies dealing with the manipulation of nucleicacids. In the first investigation, each of the four natural nucleobases were analyzed for theability to serve as a universal template at the ligation junction of a T4 DNA ligasereaction. This resulted in the first instance of sequence-independent ligation catalyzed byany DNA ligase. Although all of the nucleobases display universal templatingcapabilities, thymidine and guanosine provided the most effective results. In addition,lowered MgCl2 and ATP concentrations, as well as the inclusion of DMSO, also aided inthe sequence-independent ligation reported here. In the course of these studies, currentmethods of removing urea from denaturing-gel purified nucleic acids provedcumbersome. Therefore, in the second study simple butanol extraction was examined as ameans to eliminate urea from nucleic acid solutions. Stepwise butanol extraction was themost effective approach to solving this problem and provided a much needed techniquefor nucleic acid purification. This type of extraction also does not result in significantlosses of nucleic acid sample. The third study exploits the molecular recognition andcatalytic properties inherent in an autocatalytic group I intron to develop a ribozyme thatcan replace the 5' end of an RNA substrate with a different RNA. This 5' replacementsplicing reaction can potentially repair mutations on the 5' ends of RNA transcripts thatlead to a variety of genetic mutations. The model system was a common mutation in asmall model mimic of the k-ras gene in vitro, which predisposes individuals to lungcancer. This 5' replacement splicing reaction occurred in vitro using this small modelsystem; the reaction was also enhanced by the alteration of the molecular interactionsinvolved. The results and implications of each of these studies are detailed in thisdissertation.

Group I introns belong to a class of large RNAs that catalyze their own excision from precursor RNA through a two-step process called self-splicing reaction. These self-splicing introns have often been converted into ribozymes with the ability site specifically cleave RNA molecules. One such ribozyme, derived from a self-splicing Pneumocystis carinii group I intron, has subsequently been shown to sequence specifically excise a segment from an exogenous RNA transcript through trans excision-splicing reaction.The trans excision-splicing reaction requires that the substrate be cleaved at two positions called the 5' and 3' splice sites. The sequence requirements at these splice sites were studied. All sixteen possible base pair combinations at the 5' splice site and the four possible nucleotides at the 3' splice site were tested for reactivity. It was found that all base pair combinations at the 5' splice site allow the first reaction step and seven out of sixteen combinations allow the second step to occur. Moreover, it was also found that non-Watson-Crick base pairs are important for 5' splice site recognition and suppress cryptic splicing. In contrast to the 5' splice site, 3' splice site absolutely requires a guanosine.The pathway of the trans excision-splicing reaction is poorly understood. Therefore, as an initial approach, a kinetic framework for the first step (5' cleavage) was established. The framework revealed that substrate binds at a rate expected for RNA-RNA helix formation. The substrate dissociates with a rate constant (0.9 min-1), similar to that for substrate cleavage (3.9 min-1). Following cleavage, the product dissociation is slower than the cleavage, making this step rate limiting for multiple-turnover reactions. Furthermore, evidence suggests that P10 helix forms after the 5' cleavage step and a conformational change exists between the two reaction steps of trans excision-splicing reaction. Combining the data presented herein and the prior knowledge of RNA catalysis, provide a much more detailed view of the second step of the trans excision-splicing reaction.These studies further characterize trans excision-splicing reaction in vitro and provide an insight into its reaction pathway. In addition, the results describe the limits ofthe trans excision-splicing reaction and suggest how key steps can be targeted for improvement using rational ribozyme design approach.