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Item Allosteric activation of the CRISPR-associated transcription factor Csa 3 by cyclic tetra-adenylate (cA 4)(Montana State University - Bozeman, College of Letters & Science, 2020) Charbonneau, Alexander Anthony; Chairperson, Graduate Committee: C. Martin LawrenceThe CRISPR-Cas immune system provides adaptive and heritable immunity to archaea and bacteria to combat viral infection, and is a source of biochemical tools to researchers. This work combines structural biology and biochemical approaches to provide insight into mechanisms prokaryotes use to control the CRISPR-Cas immune system, linking subsystems into a coordinated response. The first structure of S. solfataricus Csa3 determined by Lintner et al. revealed a dimer with a C-terminal wHTH DNA-binding domain and an N-terminal CARF domain with a putative ligand binding site predicted to bind a two-fold symmetric molecule with both negatively charged and hydrophobic/aromatic moieties, such as dinucleoside polyphosphates or nucleic acid molecules. 1 Later, analysis by Topuzlu et al. of the A. fulgidis Csx3 structure containing a 4 base RNA molecule in a binding pocket revealed similarities between Csx3 and the Csa3 CARF domain, and suggested CARF proteins could bind cyclic or pseudosymmetric linear RNA tetranucleotides represented by the ring-shaped RNA density in the Csx3 binding pocket. 2 Functional studies with S. islandicus Csa3 identified that SiCsa3 regulates transcription of acquisition genes (cas1, cas2, and cas4) and several CRISPR loci. 3,4 Additionally, two groups simultaneously showed that the Type III surveillance complexes produce cyclic oligoadenylate messengers, including cyclic tetra-adenylate (cA 4), which allosterically regulate the RNase activity of Csm6 and Csx1, other CARF proteins. 5,6 These advances support the original predictions by Lintner et al. and suggest that Csa3 binds a cyclic RNA as proposed by Topuzlu et al. Binding of cA 4 likely allosterically causes conformation changes in the wHTH, and regulates the protein's transcriptional regulation. We present the crystal structure of S. solfataricus Csa3 complexed with cyclic tetraadenylate (cA 4). cA 4, as predicted, 1 is bound in the CARF domain 2-fold symmetric pocket, which stimulates conformational changes in the C-terminal domain. Additionally, we identify the presence of a palindromic predicted binding motif upstream of the Type I-A(2) acquisition cassette and CRISPR loci C and D, and reveal through EMSA analysis that Csa3 binds dsDNA nonspecifically with high affinity. Finally, ring nuclease activity is not detected in Csa3, suggesting longer term potentiation of the cA 4 in Csa3 than observed for Csx1/Csm6. 5,6Item The effects of acute and chronic prenatal alcohol exposure on lymphoid organ development and immune function in C57BL/6 mice(Montana State University - Bozeman, College of Agriculture, 1990) Huang, ChingItem Identification and characterization of a novel transcription factor that regulates NCF2 expression via the TNF-alpha responsive region(Montana State University - Bozeman, College of Agriculture, 2007) Anderson, Mary Cloud Bosworth Ammons; Chairperson, Graduate Committee: Mark T. Quinn.The multicomponent NADPH oxidase is an essential enzyme complex found in professional phagocytic cells that mediates innate immune defence against multiple pathogens through the production of reactive oxygen species. The vital and functionally limiting cytosolic component of the NADPH oxidase, p67 phox, is transcriptionally regulated by TNF-alpha at the TNF-alpha Responsive Region (TRR) in the intragenic region of Neutrophil Cytosolic Factor 2 (NCF2), the gene which codes for p67 phox. The aim of this dissertation is to identify and charaterize the factor(s) that binds the TRR and regulates NCF2 expression in response to TNF-alpha. Using the TRR as bait in a yeast one-hybrid screen, we identified Pleomorphic Adenoma Gene-Like 2 (PLAGL2) as a candidate regulator of NCF2 expression. In vitro and in vivo analysis confirmed that PLAGL2 binds to the TRR in NCF2 and that endogenous PLAGL2 binding is enhanced in the presence of TNF-alpha. Knock-down of endogenous PLAGL2 expression inhibited TNF-alpha-mediated NCF2 expression, p67 phox expression, and subsequent superoxide production. Characterization of PLAGL2 binding to the NCF2 TRR identified the essential core sequence which is recognized by zinc fingers six and seven of PLAGL2. Using PLAGL2 as bait in a yeast two-hybrid screen, we identified ubiquitin protein ligase (E2I), positive cofactor 2 (PC2,) four and a half LIM domain 3 (FHL3), and chromodomain helicase DNA binding protein 3 (CHD3) as putative PLAGL2 binding partners. In vitro and in vivo analysis confirmed that PLAGL2 binds PC2 434-748, FHL3, and CHD3 via interactions mediated through unique and overlapping domains of PLAGL2. In conclusion, we have identified a novel regulator of NCF2 transcription and have further characterized the features of PLAGL2 binding and recognition of the TRR sequence in the intragenic region of NCF2. Finally, through identification of PLAGL2 binding partners, we have begun to determine a model by which PLAGL2 regulates transcription in a context dependent manner.Item Molecular and functional characterization of bovine C5a receptor(Montana State University - Bozeman, College of Agriculture, 2006) Nemali, Sailasree; Chairperson, Graduate Committee: Mark T. Quinn.Anaphylatoxin C5a is an important chemotactic factor for bovine neutrophils, and is the earliest inflammatory agent formed during bovine mastitis. Bovine neutrophils respond to C5a and its truncated form C5a des arg with similar affinities unlike human neutrophils. Therefore, to test the hypothesis that the bovine C5a receptor structure and signaling differ from that of the human C5a receptor, we cloned and analyzed the bovine C5a receptor. In the present investigation, the bovine C5a receptor encoding cDNA from bovine bone marrow was cloned and the recombinant C5a receptor protein was expressed in CHO-K1 cells. Analysis of predicted C5a receptor amino acid sequence demonstrated 69.1%, 71.3%, 59.4%, 61.6%, and 33.2% identity with that of human, dog, mouse, rat, and trout respectively. The bovine C5a receptor mediated signaling was via pertussis toxin insensitive Gá-protein, and mediated L-selectin shedding on the activated neutrophils. The homologous-, and heterologous desensitization experiments provided further evidence that C5a and C5a des arg could desensitize C5a receptor signaling as well as IL-8 receptor mediated signaling. We further analyzed the expression profiles of the C5a receptor on bovine peripheral blood leukocytes with the receptor-specific antibody and FITC labeled C5a des arg. The monoclonal antibody as well as C5a des arg FITC failed to detect C5a receptor on peripheral blood mononuclear cells. Furthermore, we probed for the expression of C5a receptor gene and protein expression in Mac-T cell line--an immortalized bovine mammary epithelial cell line--, to test the hypothesis that bovine epithelial cells express C5a receptor protein. The results of RT-PCR analysis and FACS analysis for the expression of C5a receptor demonstrated the presence of the C5a receptor in these cells. This investigation led to the elucidation of the structure and function of bovine C5a receptor in neutrophils. The analysis of the C5a receptor expression in peripheral blood leukocytes demonstrated that it was constitutively expressed in neutrophils only. Mac-T, a mammary epithelial cell line expresses C5a receptor. Future studies understanding the function of C5a receptor in these cells will contribute to the knowledge of bovine inflammation.Item Mid-and late-gestation lethality in mice lacking the N terminus of TATA-binding protein(Montana State University - Bozeman, College of Agriculture, 2004) Hobbs, Nicole Kay; Chairperson, Graduate Committee: Edward E. Schmidt.TATA-binding protein (TBP) is a transcription factor comprised of a 180 amino acid core that is shared by all eukaryotes. TBP also has an N-terminal region that, in vertebrates, is highly conserved. We have generated mice bearing a mutant tbp allele, tbp deltaN, that lacks 111 of the 135 amino acids of the vertebrate-specific N terminus. Most homozygous mutants, tbp deltaN/deltaN, die at midgestation from an apparent defect in their placentas. tbp deltaN/deltaN fetuses were rescued at this midgestational crisis if supplied with a wild-type tetraploid placenta. tbp deltaN/deltaN fetuses also survived in immune-normal mothers when fetal/placental beta 2m expression was genetically disrupted. When reared in immunocompromised mothers, tbp deltaN/deltaN fetuses also survived midgestation. These results suggest the N terminus of TBP functions in beta 2M-dependent processes and within the placenta to favor immunotolerance during pregnancy at midgestion. Beyond midgestation, tbp deltaN/deltaN fetuses that survive in immunocompromised mothers were found to be runted at the perinatal period and died shortly after birth. These latter results suggest that the N terminus of TBP also functions in non-immune processes required for normal birth weight and successful pregnancy.