Corn is a valuable agricultural commodity in the United States and in the world. The causal agent of Stewart's wilt disease in corn, Pantoea stewartii subsp. stewartii, is a bacterial phytopathogen that is vectored into the plant by the corn flea beetle, Chaetocnema pulicaria. After entering the apoplast of the leaf, the bacteria cause water soaking symptoms before traveling to the plant xylem to form a dense biofilm, thereby blocking water transport and inducing necrosis and wilt. This results in reduced crop yield and may even lead to death of the corn plant. To better understand the in planta requirements of this pathogen, a whole transcriptome study was performed via RNA-Seq to determine genes differentially expressed in the bacteria while inside the corn. It was found that nutrient transporters and stress response genes were upregulated specifically when the bacteria are in their host plant, suggesting a response to nutrient availability and host defense in the xylem. Further elucidation of the genes required for the P. stewartii in planta lifestyle was performed via a reverse genetics approach where in-frame gene deletions and the corresponding complementation strains were constructed for genes that had shown a fitness defect in corn based on a previously published Tn-Seq study: genes encoding seven transcription factors, nsrR, iscR, lrp, nac, DSJ_00125, DSJ_03645, and DSJ_18135, as well as a hypothetical protein DSJ_21690. Investigation of the physiological role of these genes was performed using in planta virulence and competition assays for all strains. An in planta qRT-PCR analysis of bacterial gene transcription was also completed for the strains with deletions in nsrR and iscR. In vitro assays were performed on all strains to determine their capsule production and motility phenotypes. Taken together, it was seen that iscR is important for colonization capabilities in planta, both NsrR and IscR act as regulators, and lrp is important for full disease capabilities, perhaps due to reduced capsule and motility phenotypes. These findings lay the groundwork for finding potential disease intervention strategies not only against P. stewartii, but also other xylem-dwelling bacterial phytopathogens. In addition to exploring ways to enhance crop yield, an additional research area was on repurposing a byproduct of corn ethanol production, syrup. It was hypothesized that this corn-based syrup could be utilized as a carbon source to grown bacteria. In turn, the resulting bacterial biomass could then be added as a fish feed supplement in aquaculture. Syrup was tested as a growth medium for individual soil bacterial isolates as well as a full mixed bacterial community consortium to determine which bacteria could grow most efficiently, both in rate and yield. It was found that the highest growth rate and yield was from Bacillus species, some of which may have probiotic benefits to fish. Ultimately, the collective outcomes from these projects in basic research about a bacterial corn pathogen and applied research about beneficial microbes grown on a corn-based substrate are expected to improve scientific endeavors as well as agricultural practices. / Doctor of Philosophy / Corn is a top agricultural commodity in the United States, as a food for human consumption, a primary nutrient source used in animal feed, and a substrate consumed during biofuel production. These various corn-based industries are impacted by bacteria in multiple ways; in some cases, bacteria may cause disease that reduces crop yield, but other bacteria serve beneficial roles that enhance health. This dissertation research describes studies about the bacterium that causes Stewart's wilt disease in corn, Panteoa stewartii subsp. stewartii. In an initial experiment, the genes that P. stewartii expresses at the highest levels when it grows inside the corn plant were identified. These genes were deduced to be important for the ability of the bacterium to live successfully in this environment. This work was followed up with a more specific approach that examined the role of certain genes that were predicted to be master regulators of the expression of other genes in the ability of the P. stewartii to colonize the plant and/or cause disease. By identifying key bacterial genes, disease intervention strategies to combat Stewart's wilt and other similar bacterial plant pathogen diseases might become possible. Protecting corn yields is important for ethanol production. The final study of this dissertation examined the ability of bacteria to grow on a byproduct of ethanol production called syrup. The goal was to then use the biomass of these beneficial microbes as a food source for animals being produced in aquaculture facilities. Among the species tested, the highest growth rate and yield was from Bacillus subtilis, a safe-to-eat bacterium that has known beneficial health properties when consumed by fish. Overall, the research studies that were completed for this dissertation have the potential to improve agricultural practices by decreasing corn disease leading to increased corn yield and developing new downstream corn-based animal feed products.

Mammalian genomes are characterised by global and pervasive DNA methylation and this modification is generally thought to be inhibitory to transcription. An exception to this widespread DNA modification are genomic elements called CpG islands (CGI), contiguous regions of non-methylated DNA which encompass the transcription start site of two thirds of mammalian genes. Although CGIs represent the most prominent feature of mammalian promoters, the contribution of these elements to promoter function remains unclear. Work in this study shows that the histone lysine demethylase KDM2B (FBXL10/ JHDM1B) is a nuclear protein which binds specifically to non-methylated CpG dinucleotides and associates with CGI elements genome-wide through its zinc-finger CxxC (ZF-CxxC) DNA binding domain. Furthermore, in mouse embryonic stem cells, biochemical investigation revealed that KDM2B associates with Polycomb group E3 ubiquitin ligase RING1B to form a variant Polycomb repressive complex 1 (PRC1) characterized by the PCGF1 subunit. Considering that KDM2B has clear DNA-binding activity and that CGIs were reported to function as nucleation sites for polycomb repressive complexes, a potential role for KDM2B in mediating PRC1 recruitment to target genes was investigated. Stable depletion studies indicated that KDM2B is required for the normal targeting of RING1B to CGIs and the regulation of expression of a subset of Polycomb-occupied genes. By taking advantage of a genetic ablation system in which the DNA binding domain of KDM2B can be conditionally deleted, results in this thesis reveal that the ability of KDM2B to recognize non-methylated DNA is essential for polycomb domain formation and normal embryonic development. Finally, through the use of a de novo targeting assay, an unexpected PRC2 recruitment pathway was discovered which is dependent on PRC1-mediated H2AK119ub1 deposition. Together this work uncovers a novel mechanism linking KDM2B-dependent recognition of non-methylated DNA with recruitment of Polycomb proteins and provides the framework on which to further investigate the contribution of CGIs to formation of polycomb domains.

The zinc finger associated domain (ZAD) family of transcription factors from Drosophila melanogaster is not well described in the literature, in part because it is very difficult to study by traditional mutagenesis screens. Bioinformatic studies indicate this is due to overlapping functions remaining after a recent evolutionary divergence. I set out to use in vitro-binding techniques to identify the characteristics of the ZAD family and test this theory. I have constructed glutathione S-transferase (GST)-ZAD domain chimeric proteins for use in pull down protein binding assays,and GST-Zinc finger (ZnF) array domain chimera for electrophoretic mobility shift assays (EMSA). Protein binding assays indicated two putative conserved interactors, similar to the analogous KRAB system in mammals. ... Competitive bindings were carried out to show a specificity of binding conferred by the identified conserved positions. While the consensus binding sites show relatively few similarities, the predicted target genes identified by the consensus binding sites show significant overlap. The nature of this overlap conforms to the known characteristics of the ZAD family but points to a more positive selection to maintain conservation of function. / by Joseph Krystel. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Mitochondrial disorders resulting from defects in oxidative phosphorylation are the most common form of inherited metabolic disease. Mutations in the human mitochondrial translation elongation factor GFM1 have recently been shown to cause the lethal pediatric disorder Combined Oxidative Phosphorylation Deficiency Syndrome (COXPD1). Children harboring mutations in GFM1 exhibit severe developmental, metabolic and neurological abnormalities. This work describes the identification and extensive characterization of the first known mutations in iconoclast (ico), the Drosophila orthologue of GFM1. Expression of human GFM1 can rescue ico null mutants, demonstrating functional conservation between the human and fly proteins. While point mutations in ico result in developmental defects and death during embryogenesis, animals null for ico survive until the second or third instar larval stage. These results indicate that in addition to loss-of-function consequences, point mutations in ico appear to produce toxic proteins with antimorphic or neomorphic effects. Consistent with this hypothesis, transgenic expression of a mutant ICO protein is lethal when expressed during development and inhibits growth when expressed in wing discs. In addition, animals with a single copy of an ico point mutation are more sensitive to acute hyperthermic or hypoxic stress. Removal of the positively-charged tail of the protein abolishes the toxic effects of mutant ICO, demonstrating that this domain is necessary for the harmful gain-of-function phenotypes observed in ico point mutants. / Further, expression of GFP-tagged constructs indicates that the C-terminal tail enhances ectopic nuclear localization of mutant ICO, suggesting that mislocalization of the protein may play a role in the antimorphic effects of mutant ICO. Taken together, these results illustrate that in addition to loss-of-function effects, gain-of-function effects can contribute significantly to the pathology caused by mutation in mitochondrial translation elongation factors. / by Catherine F. Trivigno. / Thesis (Ph.D.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Longitudinals lacking gene (LOLA) is a transcription factor that is involved in a variety of axon guidance decisions in Drosophila melanogaster nervous system. Besides having a role as an epigenetic silencer and in the programmed cell death in Drosophila's ovary, this gene is also an example of complex transcription unit. LOLA is a transcription repressor and can generate 17 DNA - binding isoforms, through alternative splicing, each containing distinct zinc-finger proteins. This unique DNAbinding binding sequence to which LOLA-ZFP binds has been determined for four of the lola isoforms F, J, P and K. Also, bioinformatics' tool approach has been taken to identify the target genes that are regulated by these four LOLA splice variants. Future work will be done for the five other LOLA isoforms to categorize their putative DNA-binding sequences and subsequently their protein interactions. / by Bazila Qureshi. / Thesis (M.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

The zinc finger associated domain (ZAD) containing family of transcription factors is not well described in the literature, in part because it is very difficult to study by mutagenesis. We used in vitro-binding techniques to identify characteristics of the ZAD family, by constructing glutathione Stransferase (GST)-ZAD domain chimeric proteins for use in protein binding assays, and GST-Zinc finger array domain chimera for binding site selections. Protein binding assays indicated a possible shared cofactor, as seen in the analogous KRAB system in mammals. DNA binding assays have provided a consensus binding sequence for five of the ZAD proteins, consistent with previously reported work on ZAD and unpublished work on mammalian transcription factors. Research is ongoing with an additional ~50 ZAD proteins to more fully map the binding characters of ZAD proteins. / by Joseph Krystel. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

L’ARN non-codant 7SK forme la charpente d’un complexe, 7SK snRNP, qui régule l’activité du facteur d’élongation de la transcription P-TEFb, intervenant dans la levée des pauses transcriptionelles chez les métazoaires. Le 7SK snRNP comprend les protéines LARP7, essentielle pour la stabilité de l’ARN 7SK et MePCE, participant à sa coiffe. Dans le cadre d’une investigation du rapport entre structure et fonction de l’ARN7SK, le projet était de comprendre commen la protéine LARP7 reconnait et assemble l’ARN dans le 7SK snRNP. La protéine LARP7, membre d’une famille reliée à laprotéine La, est spécifique de 7SK. Les éléments responsables de l’interaction ont été analysés par des méthodes biochimiques dans des complexes reconstitués à partir d’ARN synthétique et de protéines recombinantes. Le module La, dans la région N-terminale, reconnaît et lie les trois uridines à l’extrémité 3’ de l’ARN et, additionellement,une séquence conservée au pied de la tigeboucle en 3’, induisant une conformation fermée de l’ARN. L’autre extrémité de la protéine comprend un domain RRM de reconnaissance de l’ARN, qui se lie à la boucle apicale de la tige-boucle 3’. La protéine LARP7 reconnaît également une région conservée au centre de l’ARN. Dans l’ensemble, LARP7 utiliserait ses domaines terminaux et central pour envelopper l’ARN et le stabiliser. Au cours de ces travaux, une interaction directe du domaine C-terminal avec la tige-boucle 5’ a également été mise en évidence. Celle-ci comprend le site de liaison à la HEXIM, la protéine qui déclenche l’interaction avec P-TEFb et un rôle fonctionnel de LARP7 est envisagé. / The non-coding RNA 7SK is the scaffold for the 7SK snRNP complex that regulates PTEFb, the positive transcription elongation factor, which relieves transcription pauses in metazoans. The 7SK snRNP comprises the proteins LARP7, essential for 7SK stability and MePCE, involved in capping. In the frame of an investigation of how the structure of the7SK RNA sustains its function, the project was to understand how is the RNA recognized and assembled in the 7SK snRNPby the associated protein LARP7. LARP7, a La-related protein is specific for 7SK. The elements responsible for the interaction were investigated by biochemical approaches in vitro with complexes reconstituted from purified recombinant proteins and transcribed RNA. The La-module of LARP7 recognizes and binds the triplet of uridines at the 3’-end of the 7SK RNA and additionally binds to a conserved region at the foot of the 3’-hairpin.This may stabilize a closed conformation of the 7SK. On the other end of the LARP7molecule, the C-terminal domain comprising a RRM (RNA Recognition Motif) binds to the apical loop of the 3’hairpin. Further investigations showed that a conserved region in the core of the RNA is also involved. On the whole, this strongly suggests thatLARP7 wraps around 7SK using its N terminal, C-terminal and linker domains to ensure the RNA stabilization into a functional core. In the course of the investigation, was revealed a direct interaction of the C-terminal domain of LARP7 with the 5’-hairpin of the RNA, which is responsible for 7SK function as it contains the binding site of HEXIM, the protein which bridges 7SK and P-TEFb. A possible functional role of LARP7 is envisioned.

Abstract not available

<p>Nitrogen is an essential building block of proteins and nucleic acids and, therefore, crucial for the biosphere. Nearly 79 % of the air consists of nitrogen, but in the form of nitrogen gas (N₂), which cannot be utilized by most organisms. Nitrogen-fixing microorganisms such as cyanobacteria have a central role in supplying biologically useful nitrogen to the biosphere. Therefore, it is important to achieve further understanding of control mechanisms involved in nitrogen fixation and related processes. </p><p>This thesis concerns different molecular aspects of the transcription factor NtcA from the heterocystous cyanobacterium <i>Anabaena</i> PCC 7120. Apart from performing oxygenic photosynthesis, <i>Anabaena</i> PCC 7120 is also capable of fixing nitrogen. NtcA is a protein regulating transcription of a wide range of genes and in particular genes involved in cyanobacterial global nitrogen control. NtcA binds as a dimer to the promoter regions of target genes such as those involved in nitrogen fixation and heterocyst differentiation. </p><p>NtcA from <i>Anabaena</i> PCC 7120 was heterologously expressed in <i>E. coli</i> and a high yield of recombinant protein was achieved through purification by Ni-IMAC chromatography. The purified NtcA was used to examine DNA binding motifs preferred by NtcA <i>in vitro </i>using a semi-random library of DNA sequences. The preferred binding sequence for NtcA is TGTA – N₈ – TACA and at least five of the bases in the palindromic binding site are necessary for binding. Differences in the consensus sequence in vivo may reflect variations in the structural conformation of NtcA under various physiological conditions. </p><p>Since an earlier study suggested redox-regulated NtcA-DNA binding the role of the two cysteine residues of NtcA were investigated. Binding studies using three mutants, Cys157Ala, Cys164Ala, and Cys157Ala / Cys164Ala, demonstrated that all these NtcA variants bind to DNA with a slightly higher affinity in the presence of the reducing agent DTT. The studies indicate that the binding mechanism is not dependent on a conformational change of NtcA caused by breaking of intra-molecular disulfide bonds. </p><p>Crystallization followed by structural studies rendered a partial crystal structure of NtcA. The structure verifies that NtcA is a dimeric protein. Each subunit has three domains: the N-terminal domain, a dimerization helix connecting the N-terminal domain with the C-terminal domain, as well as making up the dimer interface, and a C-terminal domain including the DNA binding helix-turn-helix motif.</p><p>Furthermore, an NtcA binding site was found in the promoter region of the<i> hupSL</i> gene, encoding an uptake hydrogenase in <i>Nostoc punctiforme</i> (ATCC 29133), indicating that yet another gene is transcriptionally controlled by NtcA, thereby further emphasizing the multifaceted role of NtcA in cyanobacteria.</p>

<p>Adenovirus E1A is needed to activate early viral genes and induce cell cycle progression to optimise the conditions for viral replication. This is mostly achieved through interactions between the first exon of E1A and cellular transcriptional regulatory proteins. The carboxy terminus of E1A binds the cellular corepressor of transcription C-terminal Binding Protein (CtBP), resulting in derepression of CtBP target genes. </p><p>Inducible stable U2OS cell lines were established, expressing wild type E1A (E1Awt) and a mutant unable to bind CtBP (E1A∆CID). Low inducible levels and loss of protein expression after prolonged induction together with induction of apoptosis were consistent with the fact that wild type E1A is a cytotoxic protein and correlated with the ability of CtBP to repress proapoptotic genes. E1A∆CID did not induce apoptosis and could be expressed at high levels for prolonged time periods. Moreover, the binding of CtBP contributed to E1A-induced activation of viral E1B and E4 genes, through possible targeting of Sp1 and ATF transcription factors.</p><p>In a micorarray study on mRNA levels in E1A-expressing cells, several genes consistent with the tumour suppressive and apoptotic properties of E1Awt were identified as differentially expressed. Furthermore, the differences between the two cell lines correlated with the presence of binding sites for CtBP-interacting transcription factors in the promoters of regulated genes, enabling the possible identification of new CtBP target genes. </p><p>Finally, a molecular characterisation of the CtBP mechanism of repression revealed that positioning proximal to the basal promoter element was required for efficient repression, suggesting that CtBP interferes with the basal transcriptional machinery. Two separate domains were identified in CtBP, conferring transcriptional repression and activation when expressed alone, achieved through their interaction with HDACs and HATs, respectively. However, together they cooperate to ensure maximal repression through recruitment of histone deacetylase and inhibition of histone acetyl transferase activity.</p><p>Together, these data shows important modulation of E1A activities by the binding of CtBP and suggests the involvement of acetylation/deacetylation complexes for the regulation of E1A function.</p>