Type of Document Dissertation Author Wiles, Natasha Shawn URN etd-05202005-114507 Title Identification of Regulatory Binding Sites and Corresponding Transcription Factors Involved in the Developmental Control of 5'-nucleotidase Expression in Dictyostelium discoideum Degree PhD Department Biology Advisory Committee
Advisor Name Title Rutherford, Charles L. Committee Chair Cowles, Joseph R. Committee Member Sible, Jill C. Committee Member Winkel, Brenda S. J. Committee Member Wong, Eric A. Committee Member Keywords
- promoter analysis
- protein purification
Date of Defense 2005-05-06 Availability unrestricted AbstractGene 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 5’-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, 5’ 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.
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