Thesis advisor: Marc Jan Gubbels / Thesis advisor: Thomas Chiles / The length of the tachyzoite cell cycle, in particular G1, is an important virulence factor in Toxoplasma gondii. Cdk and Cyclin activities ultimately control the cell cycle; however, the checkpoint control mechanisms diverge from higher eukaryotes and are poorly understood. In order to elucidate these mechanisms, temperature sensitive (ts) mutants were generated by chemical mutagenesis. One of these mutants, called FV-P6, dies within one cell cycle in the G1 phase upon transfer from the permissive (35°C) to the restrictive temperature (40°C). Cosmid complementation identified the gene responsible for this G1 arrest as a `Cactin' ortholog. A single point mutation in this gene that resulted in an amino acid substitution from Tyrosine to Histidine at position 661 in the highly conserved C-terminus was shown to underlay the temperature sensitive effect. Cactin is highly conserved across eukaryotes and plays a role in embryonic development of metazoa although its mechanism of action is poorly understood. In agreement with the predicted nuclear localization signal in the N-terminus, expression of a fluorescent reporter gene fusion resulted in nuclear localization. Genome-wide expression profiling analysis of mutant and wild type at the permissive and restrictive temperatures confirmed the G1 arrest and furthermore demonstrated up-regulation of bradyzoite and Toxoplasma cat life cycle stage genes, hinting at TgCactin's role as a repressor. Since DNA binding domains or enzymatic domains are absent in TgCactin, TgCactin must act in a complex. Native blue gel electrophoresis demonstrated that TgCactin is present in large complexes of 720 and 800 kDa. A yeast two-hybrid screen (YTH) identified 40 potential TgCactin-interacting proteins of which 10 were selected for further validation. Eight out of these ten candidates are involved in DNA/RNA processes pertaining to transcription and translation, respectively. One-on-one YTH interactions between mutated and N-terminal deletion mutants of TgCactin and the above 10 interactors were abolished except for a single RNA helicase. Studies in Toxoplasma of four of these interactors demonstrated that only the RNA helicase localized to the nucleus; however, co-immunoprecipitation experiments to demonstrate that this protein is present in a complex with TgCactin were inconclusive. Furthermore, TgCactin self interactions identified domains necessary for TgCactin-TgCactin binding. Taken together, these findings indicate that TgCactin likely functions as a repressor of gene expression, possibly through an epigenetic mechanism reminiscent of an RNA/DNA helicase- based system in plants. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.
| Identifer | oai:union.ndltd.org:BOSTON/oai:dlib.bc.edu:bc-ir_101644 |
| Date | January 2010 |
| Creators | Szatanek, Tomasz Artur |
| Publisher | Boston College |
| Source Sets | Boston College |
| Language | English |
| Detected Language | English |
| Type | Text, thesis |
| Format | electronic, application/pdf |
| Rights | Copyright is held by the author, with all rights reserved, unless otherwise noted. |
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