No description available.

Polyphenol oxidases (PPOs) are ubiquitous enzymes that oxidize phenols to quinones in the presence of molecular oxygen, often leading to tissue discolouration. They are sometimes considered as defense proteins but other functions, for example in phenolic compound biosynthesis, have also been found. In this thesis, bioinformatic searches were conducted to identify putative PPO genes from available genomes representing five Viridiplantae lineages: chlorophytes, bryophytes, lycophytes, monocotyledonous anthophytes and eudicotyledonous anthophytes. Duplicated PPO genes were found in most land plant genomes. A detailed investigation of the poplar (Populus trichocarpa) PPO gene family found nine genes that exhibit differential expression profiles during development and following stress, of which PtrPPO1 was the only significant wound-inducible PPO gene. A phylogenetic reconstruction of the poplar PPOs identified PtrPPO13 to be an unusual PPO homolog and it was studied in detail. Experimental evidence indicated that PtrPPO13 is expressed in most organs, and unlike most PPOs, is localized to the vacuole. Together, the phylogeny, gene expression and subcellular localization studies suggest that PPOs are likely to have variable physiological functions in plants and that PtrPPO13 is distinct from most typical PPOs. / Graduate / 0309

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "July 1998." Typescript. Vita. Includes bibliographical references (leaves 111-119). Also available on the Internet.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Not embargoed. Vita. Bibliography: 108-119.

A new disease of sugar beet, named Fusarium yellowing decline, was recently found in in the Red River Valley of MN and ND. This disease is caused by a novel pathogen named Fusarium secorum. Pathogens such as F. secorum secrete proteins during infection called ‘effectors’ that help establish disease. Since pathogenicity and disease development may depend on effector proteins produced by F. secorum during infection, effector protein identification furthers our understanding of the biology of this important pathogen. A list of 11 candidate effectors was generated previously. In this study, to characterize putative effectors, we developed a transformation system using polyethylene glycol–mediated transformation. Several mutant lines were created with an effector deleted from the genome using a split-marker knock-out strategy. To explore their role in pathogenicity, mutant strains have been inoculated to sugarbeet and compared to WT F. secorum.

Intercellular communication is crucial during animal development and tissue maintenance to ensure that correct patterns of cell types are generated to meet the needs of the organism. During lateral specification, intercellular communication resolves cell fate decisions between equipotent cells, creating fate patterns that are biased by external factors in some contexts, but appear stochastic in others. The Notch signaling pathway mediates lateral specification; small differences in Notch activity are amplified by regulatory feedback loops to robustly differentiate cell fates based on relative levels of Notch activity. It is often unclear how noise in the environment is processed by cells to generate differences in Notch activity that can be translated into stochastic, but robust, cell fate outcomes. The nematode Caenorhabditis elegans contains a simple, Notch-mediated, stochastic lateral specification event; a small, random difference in Notch activity between two cells, the α cells, is amplified so that one α cell assumes Anchor Cell (AC) fate and the other assumes Ventral Uterine precursor cell (VU) fate. Two upstream factors bias the outcome of the AC/VU decision depending on the length of the time interval between the births of the α cells: the relative birth order of the α cells and the onset of expression of the transcription factor HLH-2. It is unknown how these factors create a difference in the relative Notch activity level between the two α cells, and limitations of existing Notch reporters have prevented the direct observation of Notch activity levels required for determining the relationships. In this thesis, I describe a genetically-encoded Sensor Able to detect Lateral Signaling Activity, or SALSA, which uses changes in nuclear Red:Green fluorescence to indicate Notch activity. I demonstrated that SALSA captures expected Notch activity patterns in four paradigms in C. elegans, encompassing both Notch homologs, and reports low levels of Notch activity that were predicted but undetectable with other Notch activity reporters. Using SALSA, I showed that the first-born α cell is able to develop an advantage in Notch activity prior to the birth of the other α cell when the time interval between α cell births is long, but the α cell that gains the Notch activity advantage is random with respect to birth order when the time interval between α cell births is short. These results agree with the current model of the AC/VU decision. I also describe Flexon, a method for the conditional activation of strong gene expression in specific cell lineages using a lox-stop-lox cassette encoded into an artificial exon flanked by two artificial introns. A flexon can be placed into the coding region of a gene to prevent translation of a functional gene product; gene expression is restored to specific lineages through expression of a tissue-specific Cre driver that excises the flexon. I show that flexon can be used to make bright, long-lasting, tissue-specific fluorescent lineage markers. I also showed that the flexon could be used for conditional activation of an endogenous gene by inserting a flexon into rde-1 to severely reduce RNAi activity and restore gene function in specific tissues using Cre drivers.

Leukocytes express a family of high molecular weight glycoproteins called leukocyte common antigens (CD45) which have tyrosine phosphatase activity and are involved in phosphotyrosine signal transduction. Antibodies to different CD45 isoforms distinguish functionally different CD4+ T cell subsets in humans, rats, and mice. Selected protein isoforms are expressed through a process of exon splicing which is cell-type and differentiation-state specific. Splicing of the three variable exons, A, B, and C, which encode amino acids located near the extracellular amino terminus of the protein, potentially results in generation of eight different mRNA transcripts. The purpose of this study was to determine the relative levels of all eight different CD45 transcripts present in a panel of murine CD4+ T cell lines and normal murine and human CD4+ T cell subsets separated with antibodies to CD45 variable exons. I show, as expected, that the broad features of CD45 surface isoform expression in these cells can be accounted for by the relative amounts of the eight differentially spliced transcripts. Unexpectedly, all the differences in CD45 isoform expression among the CD4+ T cell subpopulations that I measured could be accounted for by differences in the overall level of variable exon expression. I did not see differences among T cell populations in the relative expression of particular variable exons. Exon B was always found in greater abundance than exons C or A. Of the dual exon species, only AB and BC were found in CD4+ T cells. The AC species was undetectable. Human CD4+ T cells, especially those in the naive subset, express higher levels of CD45 variable exons than murine CD4+ T cells. In unrelated studies, I have generated a rat-mouse hybridoma which secretes a rat IgG antibody reactive with mouse CD45. I show that the monoclonal antibody, 25D10, defines a novel epitope consistent with a post-translational modification of CD45, similar but distinct from the epitope recognized by monoclonal antibody RA3.6B2 (anti-B220). This conclusion is based on evidence that it precipitates similar molecular weight bands from cells as does a framework monoclonal antibody to CD45, yet has a distinct cell surface expression as determined by flow cytometric analysis. It stains activated Th cell lines at a higher intensity than resting Th cells, stains 60-70% of splenocytes, and 25-30% of lymph node cells. It stains all class II positive cells but not freshly isolated CD4+, CD8+ T cells or CD45 transfected fibroblasts.

Over 90% of human genes produce precursor mRNA (pre-mRNA) that undergoes splicing, an RNA processing mechanism. Alternative splicing (AS) of pre-mRNA allows a gene to generate multiple coding and non-coding isoforms by removing introns and ligating distinct exonic combinations. It is a mechanism that plays a major role in driving molecular diversity in mammals. This process is tightly regulated to determine the types and levels of protein products expressed in specific cellular contexts. Cis-acting splicing regulatory elements (SREs) found within the pre-mRNA are recognized and bound by RNA-binding proteins that either assist or interfere with the recruitment of the spliceosome. In the field of splicing, a long-standing goal has been to develop a “splicing code”, or a set of rules to understand the splicing patterns of a gene in a predictable manner. It is essential to highlight the significance of sequence context for SREs and the potential impact that distal intronic elements can have on splice site selection to better understand splicing. Given the importance of sequence context and the involvement of distal intronic regions in splicing, future approaches aimed at identifying SREs should consider these factors.This thesis will describe the process of the identification and validation of two novel distal intronic SREs located in critical disease exons. Importantly, these findings were made by combining experimental and computational approaches and through the development of a high-throughput SRE screening methodology. Chapter 1 will provide a general context for splicing, in particular AS, as an important mechanism among a plethora of RNA regulatory functions. The significance of AS regulation will be explored, as it plays a key role in the occurrence of physiological events and incorrect regulation can trigger disease. I will also introduce several methods used to study SREs, with experimental efforts primarily focusing on exonic and proximal intronic sequences. Additionally, as mis-splicing is associated with disease, there is a high interest in modulating splicing with novel therapeutic interventions, the development of which benefits from an increased understanding of SREs. Specifically, I will provide a landscape view of the splicing research field for the genes spinal muscular atrophy 2 (SMN2) and microtubule-binding protein tau (MAPT), given their relevance to our discoveries. As there is currently a paucity of high-throughput methods for studying SREs, especially those that allow for analysis of SREs in a near-native sequence context, I will introduce the CRISPR-Cas system (dCas13d) as a potential splicing modulator. This system will form the foundation for developing a tool to help us understand splicing regulation. Chapter 2 will discuss the discovery of a distal SRE regulating MAPT exon 10 splicing divergence in the primate lineage. Our lineage-specific AS analysis found that MAPT exon 10 shows a two-step evolutionary shift in the Catarrhine and hominoid lineages. The previously identified splicing regulatory elements cannot explain this evolutionary shift. Instead, a key splicing factor, muscleblind-like (MBNL), was found to be a major contributor to the observed splicing pattern divergence. Further mechanistic dissection revealed divergent, distal regulatory sequences in intron 10 that are recognized by MBNL. Based on this finding, we also demonstrated the potential of developing a therapeutically compatible strategy to target the MBNL binding sites by a steric hindrance to modulate exon 10 splicing effectively. Chapter 3 will discuss the development of a method allowing for a more unbiased, high-throughput screening of SREs, including those in the distal intronic regions. This method relies on the nuclease-inactive dRfxCas13d to modulate splicing. We use a dual-color fluorescent splicing reporter to identify the impact of splicing in a high-throughput manner. For our proof of concept, we use SMN2 exon 7 to identify SREs that influence the splicing of this exon and corroborate our findings with known SMN2 SREs. We performed a screen on the SMN2 dual-color splicing reporter using a gRNA library and obtained highly reproducible results. The screen also correctly identified gRNAs targeting known SREs, including the well-studied exonic regions and the downstream ISS-N1 element, the target of the ASO therapeutic known as nusinersen. Importantly, this screen also discovered novel splicing inhibiting gRNAs in a more distal region of the downstream intron, suggesting that a robust splicing enhancer was targeted. Previous studies likely overlooked this region due to its distance from the exon. This novel approach allows for the simultaneous screening of sizeable genetic regions using a large-scale gRNA library.

No description available.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "July 1998." Typescript. Vita. Includes bibliographical references (leaves 215-225). Also available on the Internet.