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A 5' AMP-degrading activity appears during the course of development in Dictyostelium discoideum between the prestalk and prespore zones. This enzyme is referred as 5'-Nucleotidase (5NT). Given the critical role of cyclic AMP in cell differentiation in this organism, 5NT is thought to be involved in cell positioning during development. Southern blot analysis showed a single form of the gene. The expression of the 5nt gene is known to be developmentally regulated. The message appears first at about 5 hr of the Dictyostelium development and remains constant throughout the rest of the development. Primer extension indicated two potential transcriptional start sites (118 bp and 148 bp upstream of the ATG initiation codon) for the 5nt expression. The 5nt promoter region was cloned and analyzed to investigate the expression of 5nt. Analysis of the cloned 5nt promoter fused to lacZ enabled the localization of the 5nt expression in pstAB cells during development. To identify cis-acting regulatory sequences, a series of 5' and internal promoter deletions were generated and fused to a luciferase reporter gene. The reporter activity driven by the 1,212 bp promoter started at the early aggregation stage, in agreement with temporal expression of the 5nt gene. Also, the expression was induced by exogenous cAMP. The reporter activity was high and relatively equivalent for all deletion constructs that contained 547 bp or more of the promoter region. No luciferase activity was detected using 365 bp or less of the promoter. A gradual decrease in activity was observed when three deletion constructs between -547 and -365 bp were tested suggesting the presence of at least two cis-regulatory elements within this region. Internal deletion analysis indicated another potential regulatory region located between -307 and -226 bp. To identify protein factor(s) that bind specifically to these regulatory sequences, gel shift assays were performed. Two bands, 0.33 Rf and 0.13 Rf, were detected in both cytoplasmic and nuclear extracts using radiolabeled DNA fragments located between -227 and -198 bp and -252 and -203 bp of the promoter region, respectively. Competition experiments confirmed the specificity of binding. The protein factors in these DNA binding activities were purified using various chromatography techniques. Mass spectrometry analysis of the purified 70 kDa protein corresponding to the 0.33 Rf band activity and a subsequent search in the Dictyostelium genomic database revealed that the purified protein was a putative formyltetrahydrofolate synthase. / Ph. D.

Gene regulation is a critical aspect of normal development, energy conservation, metabolic control, and responses to environmental cues, diseases and pathogens in eukaryotic organisms. In order to appropriately respond to environmental changes and advance through the life cycle, an organism must manage the expression levels of a large number of genes by utilizing available gene regulation mechanisms. The developmental control of 5â -nucleotidase (5nt) expression in the model system Dictyostelium discoideum has provided a focal point for studies of gene regulation at the level of transcription. In order to identify temporally-regulated control elements within the promoter of the 5nt gene, 5â and internal promoter deletions were designed and fused to the luciferase and lacZ reporter genes, and reporter enzyme activity was measured in cells from the slug stage of development. The results from these experiments enabled the identification of a 250 bp region of the promoter, which was used as a template for subsequent site-directed mutagenesis experiments. These experiments involved altering 6-12 bp regions of the promoter by substitution. Twelve mutagenized promoters were fused to the luciferase and lacZ reporter genes, and activity was measured at the slug stage of development to more precisely locate cis-acting temporally-regulated control elements. In addition, cAMP induction experiments were performed on amoebae transformed with the mutagenized promoters to identify control elements within the promoter influenced by the presence of cAMP. The regions between -530 and -560 bp and -440 and -460 bp from the ATG translation start site. In order to evaluate the functions of the cis-acting promoter control elements, electromobility gel shift assays were performed to identify specific DNA-protein interactions on the 5nt promoter. These assays enabled the detection of a 0.13 Rf and 0.33 Rf binding activity to specific sites of the promoter. After characterization of these binding activities, both proteins were purified by a series of column chromatography techniques and characterized after mass spectrometry. The proteins purified were identified as formyltetrahydrofolate synthase and hydroxymethylpterin pyrophosphokinase. These enzymes function in the biosynthetic pathway of tetrahydrofolate and the production of folate coenzymes. The specific interactions of these enzymes with the 5nt promoter suggest these proteins may also function in regulating 5nt expression. / Ph. D.

Regulation of gene expression is known to be a critical factor involved in proper development, responses to environmental cues, metabolism, energy conservation, and disease. Gene expression is regulated at several levels including transcription, mRNA splicing, post translational modification, and the rate of protein degradation. The developmental control of <i>alkaline phosphatase</i> (alp) in <i>Dicytostelium</i> has provided a focal point for the study of gene regulation at the level of <i>de novo</i> synthesis. The localization of <i>alkaline phosphatase</i> (alp) expression during development was characterized by fusing the 5' flanking sequence to the <i>lacZ</i> reporter and using an <i>in situ</i> β-galactosidase staining method. The localization of </i>lacZ</i> expression corresponds with that of the endogenous ALP enzyme suggesting that <i>alp</i> is regulated at the level of transcription. In order to identify temporal regulatory elements within the <i>alp</i> promoter a series of 5' and internal promoter deletions were generated and fused to the <i>lacZ</i> reporter. The data from these promoter deletion constructs indicated a regulatory element within the -683 to -468 bp sequence that is required for normal expression of <i>alp</i> during development. A series of small internal and 5' promoter deletions were designed within the -683 to -468 bp regulatory sequence. The results from these promoter deletion-reporter gene fusions suggested a DNA regulatory element is located within a 26-bp sequence beginning at the -620 bp site. The function of <i>cis</i>-acting regulatory elements were evaluated using the electromobility shift assay (EMSA) to identify sequence specific DNA-protein interactions on the <i>alp promoter</i>. We report the characterization of three DNA-binding activities with the 20% ammonium sulfate (AS) slug nuclear fraction. These DNA-binding activities appear to be related as they all require magnesium or calcium for effective binding to the <i>alp</i> promoter. Interestingly, the DNA-binding proteins appeared to interact with a GT-rich sequence that contained a G-box binding factor (GBF) consensus element. Additionally, a DNA-binding activity observed in the 80% AS slug nuclear extract was characterized and sequentially purified using conventional and affinity chromatography techniques. The DNA-binding protein was identified as TFII, a protein that was previously identified during the investigation of <i>glycogen phosphorylase-2 (gp2)</i> regulation. A comparison of the <i>alp</i> and <i>gp2</i> probes used to identify TFII suggests a DNA-binding site, ACAATGN₈₋₁₂CACTA. The ability of TFII to bind specifically with the promoter of two functionally different genes suggests that it may regulate the temporal and/or spatial expression of several <i>Dictyostelium</i> genes. / Ph. D.

Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue hemorrhagic fever, and yellow fever. In depth genetic studies of vector species have been made possible due to the availability of genome sequences and techniques for producing stably transformed mosquitoes. These resources have also contributed to the establishment of new genetics-based approaches to the control of vector borne disease. Genetic studies of Ae. aegypti have benefited from the ability to drive targeted transgene expression, however a ubiquitous inducible promoter has not been identified in this mosquito. The Drosophila melanogaster heat shock 70 promoter has been shown to drive inducible expression in heterologous systems; however, DmHsp70 possesses significant basal activity in Aedes aegypti. This study characterized the sequence and expression of the heat shock 70 genes of Aedes aegypti. AaHsp70 genes were found to be organized in two clusters, each comprised of three divergent pairs. AaHsp70 genes exhibited robust expression upon heat shock in larvae, pupae, and adults as well as in heads, salivary glands, midguts and ovaries. Genomic regions upstream of AaHsp70 genes were found to drive heat-inducible expression of a reporter in both cell and embryo assays. Deletion analysis of AaHsp70-derived promoters yielded two ~1.5 kb genomic fragments that maintained robust heat inducibility in these systems. Aedes aegypti were transformed with AaHsp70-luciferase gene cassettes using the transposable element Mos1. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Heat-induced expression of luciferase was observed in transgenic larvae, pupae and adults as well as heads, midguts and ovaries but not salivary glands, with levels varying between transgenic strains. The effect of heat shock on the endogenous RNAi pathway as well as the effect of blood feeding on the expression of AaHsp70 genes was investigated, though reproducible results could not be obtained using the assays employed. In conclusion, the heat shock 70 gene family of Aedes aegypti was identified and characterized. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of anti-pathogen molecules. / Ph. D.

Verticillium is a soil-borne fungus that is one of the world's foremost plant pathogens. Commercial plant grafting suggests that resistance occurs in the root; this conflicts with decades of research indicating that resistance occurs in the stem. The goal of this thesis work was to use an alternative approach to determine the location of resistance by expressing the Ve1 gene using organ-specific promoters. Promoter sequences for the stem-specific gene, Ribulose 1,5-bisphosphate carboxylase oxygenase small chain 2A (Rbsc2A), and root-specific gene, Tobacco Mosaic Virus Induced (TMVi) were taken from the Sol Genomics Network (SGN) database, cloned into constructs with the Ve1 gene and susceptible tomato germplasm was transformed using Agrobacterium tumefaciens. Preliminary results suggest that resistance may not be localized and expression of the Ve1 gene in either the root or the stem is sufficient to develop whole plant resistance to the Verticillium pathogen.

Magister Scientiae - MSc / Ischemic heart disease is a disease which is characterized by the reduced blood supply to the heart. According to WHO 2013, ischemic heart disease is one of the major causes of death globally. For this reason, it is imperative to search for methods whereby heart cells can be protected from cell death. The upregulation of heat shock proteins (Hsps) is one of the major techniques which can be used to protect the heart cells from Hsps cell death and improve the tolerance to ischemic stresses in various models. The increased expression of Hsps during heat shock pre-conditioning is regulated by heat shock transcription factors (HSFs). HSFs orchestrate the initiation of gene expression by binding to sequence motifs, known as cis-acting regulatory elements (CAREs). Since gene expression is regulated at a transcriptional level, it is expected that functionally related genes (e.g. heat shock response genes) might also be regulated by the same transcription factors (TFs). In this study an in silico approach was performed to identify the promoter sequences of 50 known heat shock responsive genes using Genomatix Software. This software was also used to identify transcription factor binding sites that are statistically over represented in the promoter sequences of these genes. The use of the Electrophoretic Mobility Shift Assay was included to confirm that protein cell lysates of stressed cells contain proteins (TFs) that bind to this sequence (SP1F_KLFS_01). Luciferase promoter reporter assay were also used to iii investigate the transcriptional activity of mutant promoter constructs in which the SP1F_KLFS_01 was mutated. SP1F_KLFS_01 is a ±25 base pair sequence that was identified in the promoter sequences of 19 heat shock responsive genes, including the well-known Hsp70 and Hsp90. This sequence is a potential binding site for two TFs, Specificity Protein-1 and Krueppel like TFs. Consequently, the aim of this study is to identify CAREs that are statistically over-represented in the promoter regions of heat shock response genes. In conclusion, in vitro experiments of this study did not support the findings of the in silico experiments, therefore additional methods should be implemented to expand the investigation for the involvement of cis-acting regulatory elements in the regulation of heat shock proteins in cardiomyocytes, prior to heat shock.

Encapsulated Escherichia coli are responsible for a number of life threatening infections of man. These range from urinary tract infections to septicemia and neonatal meningitis. A common property of these E. coli strains is the expression of a polysaccharide capsule or K antigen. The expression of a capsule is an essential virulence factor protecting the bacterium from host defenses. Like many virulence factors capsule gene expression is regulated by temperature, such that at 37 0C inside the host the capsule is expressed whereas at 20 0C it is not. The project used the K5 capsule gene cluster as a model system to study in detail the regulation of capsule gene expression. Expression of E. coli K5 gene cluster is regulated at the transcriptional level by two convergent promoters PR1 and PR3. The temperature regulation-dependent expression is in part controlled at the level of transcription by complex regulatory network involving the regulators SlyA, H-NS and IHF acting at PR1 and PR3. A large 5’ untranslated region (5’ UTR) is involved in transcriptional regulation by interacting with global regulator proteins. In this study, a combination of lacZ reporter gene fusions, 5’ RACE analysis and site-direct mutagenesis at promoter functional elements were used to investigate the promoter. These studies identified that the PR1 promoter was more complex than initially thought and contains, in addition to previously characterized PR1-1 promoter at +1, three additional tandem promoters PR1-2, PR1-3 and PR1-4 transcribing in the same direction from the site +133, +142 and +182, respectively. In order to analyse the contribution for the transcription from PR1 among these multiple promoters, these multiple tandem promoters’ activities were measured by β-galactosidase assay and Real-time quantitative reverse PCR assay. We determined that PR1-2 and PR1-3 are two cryptic promoters with very low transcription activity while PR1-1 and PR1-4 are the major promoters that contributed evenly to the total transcripts into kps operon in the mid-exponential phase. Furthermore, we demonstrated that the promoter PR1-1 and PR1-4 are tightly coupled and the activity of PR1-4 can be co-ordinately reduced by disrupted PR1-1.Different minimal PR1-lacZ promoter fusions were also transformed into strains with mutations in the genes that encode these regulatory proteins (IHF, SlyA and H-NS) and the transcription activity was examined by β-galactosidase assay at both 37 0C and 20 0C. IHF is required indirectly for maximum transcription at PR1-1 promoter but directly represses transcription from PR1-4 due to binding at +160 region at 37 0C. Global regulator H-NS represses the transcription at both 37 0C and 20 0C at PR1 and plays an important role for transcriptional temperature regulation at PR1 region. The anti-repressor SlyA activates transcription at PR1-1 at 37 0C. This study identified for the first time growth phase dependent expression from the PR1 promoter. Also, this study discovered different temporal patterns of promoter PR1-1 and PR1-4 transcription was coordinated with bacterial growth cycle. Overall this study will be helpful to decipher the complex regulation of capsule gene expression in E. coli.

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