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I. Examination of Four Trypanosomal 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase Paralogs by RNA Interference Studies. II. Trypanosoma brucei Essential Nuclear Factor DNA Binding, Transcription and Interacting Protein Studies

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Description: Trypanosoma brucei, the unicellular parasites responsible for causing African sleeping sickness, possesses four phosphofructokinase-2/fructose-2, 6-bisphosphatase (PFK-2/FBPase-2) monofunctional enzymes. In contrast, humans, who also posses four paralogs of the enzyme, have bifunctional tissue specific paralogs. Unusually, trypanosomes, which are unicellular, still possess all four paralogs, each being monofunctional. Truncated recombinant his-tagged forms of each paralog were successfully cloned into E.coli, purified to approximate homogeneity and were used as antigen to obtain custom polyclonal antibodies against each paralog. RNA interference (RNAi), verified by western blot, was performed for each paralog to knock down the protein and measure cell viability. However, the results proved to be inconclusive. A second trypanosomal essential nuclear factor, Tb TAF49, was studied to determine its role, if any, in trypanosome transcription. DNA pulldown studies, verified by western blot, were performed using biotinylated promoters to capture the TAF49 interaction with DNA in the absence and presence of exogenous DNA. TbTAF49 was successfully shown to bind non-specifically to all tested forms of DNA. RNAi was shown to be reproducible and confirmed that Tb TAF49 is essential to all stages of parasite life. RNA was isolated from the induced and non-induced Tb TAF49 RNAi parasites. Semi-quantitative PCR, verified by agarose and polyacrylamide gel electrophoresis, was used to measure various transcript levels of other factors associate with transcription by comparing cDNA generated through reverse transcription of the isolated RNA. Thus far, no change is seen in any of the transcripts tested. Finally, Tandem Affinity Purification (TAP), verified by western blot, was attempted to identify interacting proteins with Tb TAF49. The first TAP-tagged construct was an SBP-tagged Tb TAF49 and showed that the tag was not binding to the column likely because it was not accessible. At the time of this thesis, new TAP-tagged constructs were being generated to find interacting proteins and aid in determining the role of Tb TAF49 in trypanosomes.
Language: English
Format: Degree Work