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Item Developmental gene expression in Eimeria bovis : characterization of stage specific genes of sporozoites and merozoites(Montana State University - Bozeman, College of Agriculture, 1995) Clark, Timothy GriffinItem The role of NSP1 in the regulation of rotavirus gene expression(Montana State University - Bozeman, College of Agriculture, 2003) Mitzel, Dana Nicole; Chairperson, Graduate Committee: Michele Hardy.Item Rotavirus NSP1 is an interferon system antagonist(Montana State University - Bozeman, College of Agriculture, 2008) Graff, Joel Wallace; Chairperson, Graduate Committee: Michele Hardy; Mark Jutila (co-chair)Rotaviruses cause severe gastroenteritis in mammals, including humans and livestock. Most rotavirus proteins have known functions, either as a mediator of virus replication or as a component of the infectious virus particle. The function of nonstructural protein 1 (NSP1) was unknown. However, it has been suggested that the function of NSP1 involved interactions with cellular proteins. Using the NSP1 encoded by a bovine rotavirus as the bait protein of a yeast-two hybrid interaction trap, interferon regulatory factor 3 (IRF3) was identified as an NSP1-interacting protein. Due to the importance of IRF3 in initiating an interferon response, we hypothesized that NSP1 acts to antagonize the interferon system. A comprehensive set of experiments yielded the following observations. Interferon-beta (IFNbeta) induction was blocked in wild-type, but not NSP1 null, infected cells. Expression of NSP1 in the absence of infection resulted in proteasome-mediated degradation of IRF3. A cysteine-rich zinc-binding region near the amino-terminus of all known NSP1 sequences resembles a domain found in hundreds of E3 ubiquitin ligases. Mutational analysis of the zinc-binding domain was consistent with NSP1 acting as an E3 enzyme. The NSP1 of a murine strain of rotavirus also interacted with IRF3. The NSP1 proteins encoded by the bovine and murine rotavirus strains showed low sequence homology (37% identity), which indicated that directing degradation of IRF3 may be a common property of NSP1 proteins from many different rotavirus strains. Screening of simian, bovine, and porcine rotavirus strains indicated that the porcine strain OSU was the only wild-type strain that was unable to direct IRF3 degradation. Characterization of OSU showed that this strain was able to block IFNbeta induction by antagonizing NFKB signaling, rather than IRF3 signaling. While analyzing NFKB signaling in rotavirus infected cells, we found that the F box protein, betaTrCP, of the Skp1/Cul1/F box multi-subunit E3 enzyme was targeted for degradation by OSU NSP1 and the NSP1 of a bovine rotavirus strain. Together, the results presented in this dissertation have determined that rotavirus NSP1 functions as an interferon antagonist by directing the proteasome-mediated degradation of IFNbeta induction signaling components, most likely by acting as an E3 ubiquitin ligase.Item Studies of gene expression control in the apicomplexan parasite Toxoplasma gondii(Montana State University - Bozeman, College of Agriculture, 2008) Behnke, Michael Sean; Chairperson, Graduate Committee: Michael W. White.Our understanding of global gene expression patterns and control of both developmental and strain specific aspects of Toxoplasma gondii has broadened in the past few years. A global approach was initially undertaken to construct the "transcriptome" for the Toxoplasma intermediate life cycle using serial-analysis-of-gene expression (SAGE). From this analysis, we confirmed the increased expression of known as well as novel mRNAs associated with the tachyzoite-to-bradyzoite transition. Accumulation of bradyzoite specific mRNAs in the bradyzoite SAGE libraries raises the possibility that transcriptional mechanisms play a key role in the developmental switch. To investigate this question, we adapted the dual luciferase model to recombinational cloning in order to construct a high throughput model for testing and mapping multiple bradyzoite promoters. Expression of luciferase from constructs matched previously published results indicating that developmental gene expression in Toxoplasma is controlled by elements contained in the 5'-flanking regions upstream of the protein coding regions. Promoter cis-elements from two genes, BAG1 and B-NTPase, have been fine mapped to 6-8 bp. EMSA assays confirm that these elements form sequence specific protein/DNA complexes. These data suggest that gene proximal cis-elements are required to initiate developmental gene expression, most likely by the binding of gene-specific trans-acting factors. Although strategies to identify these putative trans factors such as yeast one-hybrid are ongoing, we describe here a more global approach to identifying controllers of gene expression. We conducted an expression Quantitative Trait Locus (eQTL) mapping project that combines the power of Affymetrix microarray technology and QTL mapping to explore the genetic basis of differences in stage-specific gene expression in Type I versus III parasite strains and in F1 progeny from the I X III cross. Gene expression QTLs discovered in this analysis were either local or non-local to the associated transcriptional change and reflect proximal cis-mutations, transcription factors or central mechanisms that co-regulate gene expression during tachyzoite differentiation. Additionally, we identified segmental duplication events in various parents and progeny of the Type I X III cross that have a gene dosage effect on the level of gene expression in those parasites.