Culminating Project Title
Date of Award
Culminating Project Type
Biological Sciences - Cell and Molecular: M.S.
College of Science and Engineering
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.
Keywords and Subject Headings
Toxoplasma gondii, cell cycle mutant, RCC1, Yeast two-hybrid, S-phase
A Toxoplasma gondii cell cycle mutant was identified and linked to a temperature sensitive mutation, tsL137P, in ECR2 (TGME49_275430). This mutation manifests itself during S phase as chromosome irregularities and cell cycle arrest at the non-permissive temperature of 42°C. Using a GAL-4 based yeast two-hybrid system, cDNA libraries from asynchronous T. gondii were screened against 3 disordered fragments of ECR2 to search for protein partners. Fragment 1 (residues 1-500) wild type and temperature sensitive mutant alleles, fragment 2 (residues 1794-2200), and fragment 3 (residues 2192-2654) were used as baits to screen for interacting cDNA protein products in 5 screens. Fragments 2 and 3 yielded no interactors while wild type fragment 1 yielded 20 putative interactors out of 4.56 million diploids, and temperature sensitive fragment 1 yielded 19 putative interactors out of 2.68 million diploids. Sequencing of isolated prey plasmids yielded 3 hypothetical prey protein products (TgRCC1IV, APVIII-3, and Gra20) of interest that interacted with alleles of fragment 1. Interactions were recreated using each allele of fragment 1 ECR2 and the 3 prey proteins in Y2HGold and again in Y187 to quantitate the relative strength of interactions. The auto-activation and promiscuity of the interacting prey proteins was also assessed. β-galactosidase and growth assays at 30°C and 35°C suggest that the interactions are real, that the L137P mutation may form stronger or long interactions with protein partners, and that the interactions are specific. Interference of in vivo interactions between TgRCC1IV with ECR2 could explain the cell cycle defects and AP2VIII-3 was identified as having novel characteristics of a transcriptional factor. Gra20 is most likely a false positive. These discoveries give new insights into the interactome and DNA replication machinery of the T. gondii cell cycle.
Cash, Brock, "TgRCC1IV and AP2VIII-3 Are Putative Interactors of Organellar Division Protein ECR2 in Toxoplasma gondii" (2016). Culminating Projects in Biology. 15.