Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
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Item Structure and function of a prokaryotic argonaute from Pseudomonas aeruginosa(Montana State University - Bozeman, College of Agriculture, 2020) Erickson, Reece Sheridan; Chairperson, Graduate Committee: Blake WiedenheftArgonautes (Ago) are structurally and functionally diverse proteins present in all domains of life. A common feature of these ancient proteins is their ability to bind nucleic acid guides that target the protein to complementary sequences. Although eukaryotic argonautes (eAgo) have been well-studied, we still know very little about the function of prokaryotic argonautes (pAgo) in bacterial and archaeal species. To address this gap in our knowledge, my thesis focused on determining the biochemical properties as well as the cellular functions of a pAgo from the organism Pseudomonas aeruginosa PACS2 (PaAgo). Here, we show that PaAgo plays a role in regulating the expression of transposons within PACS2. I also present results indicating that deletion of the PaAgo gene and its neighboring genes causes toxicity to P. aeruginosa. Finally, I provide evidence that PaAgo and a neighboring protein are binding partners and form a multi-protein complex. Future work will focus on copurifying and sequencing PaAgo nucleic acid guides as well as clarifying the mechanisms guide acquisition and biological function.Item Analysis of the interaction between bovine neutrophils and rotavirus(Montana State University - Bozeman, College of Agriculture, 2002) Hanson, Angela JeanItem Biologically significant metabolites of several marine invertebrates(Montana State University - Bozeman, College of Letters & Science, 1986) Kinzer, Ken F.Item Investigating the efficacy of a flipped science classroom model(Montana State University - Bozeman, Graduate School, 2013) Tucker, Dina M.; Chairperson, Graduate Committee: Peggy Taylor; John Graves was a co-author of the article, 'Investigating the efficacy of a flipped science classroom model' submitted to the journal 'American biology teacher' which is contained within this thesis.An introductory honors level high school biology course was used to implement a flipped classroom model in the topic of biochemistry. Ten-minute lecture videos were recorded and collaborative work was completed during class. Surveys and questionnaires were used to collect data about student preference. The results indicate that students enjoyed their class more and learning biochemistry in this way was preferable to the traditional in-class lecture format.Item Molecular and biochemical characterization of wheat (Triticum aestivum. L) polyphenol oxidases (PPOs)(Montana State University - Bozeman, College of Agriculture, 2005) Jukanti, Aravind Kumar; Chairperson, Graduate Committee: Andreas Fischer.Polyphenol oxidases (PPOs) from several plant species, including wheat, have been implicated in the undesirable brown discoloration of food products. Wheat (Triticum aestivum L.) represents an interesting system to advance our understanding of plant PPO function for two important reasons, namely (1) the large size an complexity of its (allohexaploid) genome, and (2) its economic importance. Prior to this study, the molecular and biochemical properties of wheat PPOs were largely unknown. To remedy this situation, we have performed several BLAST searches of expressed sequence tag (EST) databases, using a known wheat PPO sequence as a search tool. This study suggested the presence of at least six PPO genes in hexaploid wheat, falling into two different phylogenetic clusters of three genes each. Presence of a wheat PPO multigene family was confirmed by Southern blotting. A combination of biochemical (enzyme purification and mass spectrometric analysis) and molecular (Northern) approaches confirmed that members of one cluster are not expressed in the developing kernels and senescing flag leaves, while regulation of one or several members of the other gene cluster controls PPO activity in these tissues. Our data, including immunoblotting and enzyme activity studies, further indicated that wheat PPOs are synthesized as inactive precursor (early kernel development) which are proteolytically processed and activated as the kernels mature. Activation of PPO precursor proteins was also demonstrated in vitro, in presence of purified trypsin. In these experiments, PPO activity increased during the first four hours and remained stable thereafter, indicating that the protein domains responsible for catalytic activity are quite stable. Research performed as a part of this dissertation has also demonstrated that wheat PPO activity is influenced by strong anionic detergents such as SDS and N-lauroylsarcosine. The corresponding experiments indicated that these detergents influenced both enzyme extraction and activity, at least in high-PPO wheat varieties. This work also has practical aspects, making PPO assays (as used by breeders for germplasm screening) more robust. In conclusion, as a result of this dissertation, wheat PPOs have emerged as a fascinating example of a plant multigene family with complex transcriptional and posttranscriptional regulation.Item Biochemical characterization of the [FeFe]-hydrogenase maturation protein HydE and identification of the substrate(Montana State University - Bozeman, College of Letters & Science, 2011) Boswell, Nicholas William Bradford; Chairperson, Graduate Committee: Joan B. BroderickHydrogenases catalyze the reversible reduction of protons using complex metal clusters with unusual ligands. The catalytic center of the [FeFe]-hydrogenases is called the H-cluster, and is characterized by a [4Fe-4S] cluster connected via a cysteine thiolate to a 2Fe subcluster coordinated by carbon monoxide and cyanide ligands as well as a bridging dithiolate. Assembly of the H-cluster is carried out by three hydrogenase maturation proteins: HydE, HydF, and HydG. HydF is a GTPase and has been implicated to serve as a scaffold for assembly of the 2Fe subcluster of the H-cluster. HydE and HydG are radical S-adenosylmethionine (SAM) enzymes and thus are thought to utilize reductive cleavage of SAM to initiate radical chemistry. HydG has been shown to catalyze the formation of the carbon monoxide and cyanide ligands of the H-cluster utilizing tyrosine as a substrate. HydE, therefore, has been proposed to be responsible for biosynthesis of the dithiolate ligand of the H-cluster. The aim of this study was to biochemically characterize active, Fe-S reconstituted HydE and to identify the substrate of this radical SAM enzyme. Questions to be studied also included studying the role of HydE in H-cluster maturation. This study used protein purified from recombinant E. coli. The purified protein was chemically reconstituted with iron and sulfide, and used for spectroscopic characterization and HPLC based activity assays. Colorimetric assays were also used for protein characterization and to test for the consumption of substrate. The results indicate that cysteine is likely the substrate of HydE. Activity assays show that HydE- catalyzed SAM cleavage is stimulated in the presence of cysteine, and HydF purified from different genetic backgrounds shows a spectroscopic shift in the lambda max when both HydE and cysteine are present during growth. Spectroscopic characterization confirms that HydE is an Fe-S containing radical SAM enzyme and that cysteine may be a substrate during [FeFe]-hydrogenase H-cluster maturation.