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dc.contributor.advisorGoldie, Hughesen_US
dc.creatorPermala-Booth, Jasnehtaen_US
dc.date.accessioned2008-04-30T15:30:29Zen_US
dc.date.accessioned2013-01-04T04:30:21Z
dc.date.available2009-05-05T08:00:00Zen_US
dc.date.available2013-01-04T04:30:21Z
dc.date.created2008en_US
dc.date.issued2008en_US
dc.date.submitted2008en_US
dc.identifier.urihttp://hdl.handle.net/10388/etd-04302008-153029en_US
dc.description.abstractProkaryotic cells such as Escherichia coli use glucose as their preferred carbon source. In the absence of glucose, these cells resort to other sources to generate glucose and this process of de novo synthesis of glucose is termed gluconeogenesis. Phosphoenolpyruvate carboxykinase (Pck) is one of the three enzymes important in regulating gluconeogenesis. It converts oxaloacetic acid (OAA) from the Krebs cycle to phosphoenolpyruvate (PEP), a glycolytic intermediate. The Pck structural gene (pckA) is regulated by catabolite repression. There is a 100-fold induction of pckA-lacZ fusions at the onset of stationary phase concurrent with induction of glycogen synthesis. Mutants affecting the expression of pckA were analysed to shed some light on the mechanism of its genetic regulation.Spontaneous mutants isolated with Pck- (lack of PEP carboxykinase activity) and Suc- (inability to utilise succinate as carbon source) phenotypes were previously characterised as atpG mutants defective in the ã subunit of ATP synthase.In this work we find by reverse transcriptase and real time quantitative PCR that levels of pckA mRNA are normal in the atpG mutants and that the defects in expression of pckA are therefore likely at the level of translation, protein assembly and/or protein degradation. As expected, ATP synthase activity and proton pumping in inside-out membrane vesicles were defective in these atpG mutants. It is likely that one of these defects is affecting regulation or expression of the pckA gene. It was observed that atpG mutants were defective in calcium-dependent transformation although they could be made competent for electroporation. The atpG mutants were also defective for growth of P1 bacteriophage although they could serve as recipients for P1-dependent generalised transduction. These latter phenotypes are also likely due to defects in energy metabolism.en_US
dc.language.isoen_USen_US
dc.subjectfluorescence quenchingen_US
dc.subjectreal time reverse transcriptase PCRen_US
dc.subjectgene regulationen_US
dc.subjectPEP carboxykinaseen_US
dc.subjectatpG mutantsen_US
dc.subjectATP synthaseen_US
dc.subjectgluconeogenesisen_US
dc.subjectEscherichia colien_US
dc.subjectgeneticsen_US
dc.subjectacridine orangeen_US
dc.titleMutations in atpG affect postranscriptional expression of pckA in Escherichia colien_US
thesis.degree.departmentMicrobiology and Immunologyen_US
thesis.degree.disciplineMicrobiology and Immunologyen_US
thesis.degree.grantorUniversity of Saskatchewanen_US
thesis.degree.levelMastersen_US
thesis.degree.nameMaster of Science (M.Sc.)en_US
dc.type.materialtexten_US
dc.type.genreThesisen_US
dc.contributor.committeeMemberDmitriev, Olegen_US
dc.contributor.committeeMemberBull, Harolden_US
dc.contributor.committeeMemberHoward, S. Peteren_US


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