UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a given thesis/dissertation in DRUM.
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Item Analysis of Genetic Regulatory Mechanisms that Control Ethanolamine Utilization in Enterococcus faecalis(2017) Gebbie, Margo Page; Winkler, Wade C; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this project, we studied the genetic regulatory mechanisms that affect utilization of ethanolamine, an abundant compound in the gastrointestinal environment. In Enterococcus faecalis, the ethanolamine utilization (eut) gene cluster encodes for a two-component regulatory system (TCS), comprised of a histidine kinase, EutW, which autophosphorylates upon sensing EA, and a cognate response regulator, EutV, which dimerizes upon receiving the phosphoryl group from EutW and binds the nascent transcript to prevent premature transcription termination. This TCS is responsible for coupling sensing of ethanolamine to production of eut transcripts. However, clues from other organisms had previously suggested that adenosylcobalamin (AdoCbl) might also be an important genetic regulatory signal for the E. faecalis eut genes. Indeed, we discovered a novel trans-acting noncoding RNA (EutX) that contained an AdoCbl-responsive riboswitch. Our data demonstrated that the riboswitch promotes a shortened form of EutX when cellular AdoCbl levels are replete. In contrast, a longer form is synthesized when AdoCbl levels are depleted. We demonstrated that structural motifs contained in the longer form of EutX act to sequester the EutV protein, preventing it from promoting transcription elongation of eut transcripts. These unexpected data revealed an important new type of regulatory mechanism for riboswitch RNAs. In support of this overall genetic regulatory model, we recapitulated the full genetic circuitry in a heterologous host. Using this system, we employed extensive site-directed mutagenesis to examine the functional importance of highly conserved EutV residues. This led to the identification of a cluster of positively charged residues, which we speculated are important determinants for RNA-binding activity. Consistent with this hypothesis, mutations of these residues resulted in loss of RNA-binding activity. Furthermore, we also explored whether the eut gene cluster was affected by additional genetic regulatory mechanisms. From these efforts, we concluded that oxygen is not a genetic regulatory feature of eut genes, in contrast to previously published speculation. However, we did find that it is likely to be repressed under conditions of high glucose. Therefore, these aggregate studies revealed new mechanisms of post-initiation genetic regulation, and showed how E. faecalis specifically controls expression of ethanolamine catabolism genes.Item Post-transcriptional regulation of spermatogenesis through intron retention in the fern Marsilea vestita(2013) Boothby, Thomas Christopher Clark; Wolniak, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Many rapidly developing systems rely on the use of stored transcripts to carry out their developmental program. The microspore of M. vestita transcribes and stores RNA during a requisite period of desiccation. Rehydration of the microspore triggers spermatogenesis to begin, a process that is mediated by the utilization of these stored RNAs. Here I investigate mechanisms controlling the spatial and temporal utilization of these stored transcripts. Next generation Solexa based RNAseq was conducted using poly(A)+ RNA isolates from specific time ranges during spermatogenesis. A reference transcriptome as well as temporally specific transcriptomes were assembled de novo and analyzed for gene ontology enrichments. This analysis revealed an overrepresentation of catalytic splicing and nuclear speckle factors early in development suggesting that some transcripts are not fully mature. An in house Visual Basic for Applications program was used to identify potential intron retaining transcripts (IRTs) within our transcriptomes. A large subset of IRTs was identified and in silico and molecular biological approaches demonstrated that these IRTs are matured in a spliceosome dependent fashion at different times during development. Intron retention appears to confer a translational block to IRTs and splicing of retained introns alleviates this block. IRTs appear to be associated with splicing machinery organized in nuclear speckles. These subnuclear domains aggregate during desiccation and upon rehydration are proportioned asymmetrically to spermatogeneous cells. It appears that intron retention mediates both the association and asymmetric distribution of IRTs with nuclear speckles as well as their temporal utilization through post-transcriptional splicing.