Date of Award

12-2016

Culminating Project Type

Thesis

Degree Name

Biological Sciences - Cell and Molecular: M.S.

Department

Biology

College

College of Science and Engineering

First Advisor

Christopher Kvaal

Second Advisor

Cassidy Dobson

Third Advisor

Timothy Schuh

Creative Commons License

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

Abstract

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.

Comments/Acknowledgements

First and foremost, I would like to thank my advisor Dr. Christopher Kvaal for his mentorship, unique insights, and the laughs we have had along the way. Additionally, I would like to thank him for the chance he took with me because of my background in chemistry and limited education in biology. His willingness to accept me as an MS student has given me one of the most positive educational and life experiences I can recall. The amount I have learned in these two years has been invaluable: both in the books, how to think about problems and research, and in the “real world”. His post-doc connections to Dr. Michael White, Dr. Elena Suvorova, and Dr. Radke (Jay or Josh or both-not sure which one) made this project possible so thanks there too. Thanks to the folks at USF for the opportunity to collaborate on this project and the NIH for funding. Thanks also go out to Dr. Timothy Schuh and Dr. Cassidy Dobson for their participation as committee members, their dedication of time, and their input into my project among other things. Special thanks goes out to Dr. Nathan Bruender for his outsider’s perspective on the allelic differences of ECR2 interactions that we observed in the ­Lac Z assays. Thanks also go out to Dr. Brian Olson and Dr. Michelle Wagner for their friendship and mentorship. I would also like to thank Saint Cloud State University, the Biology Department, and the faculty and staff who help provide and maintain the facilities and services needed to carry out this research. And last but not least thanks go to Blake Barnes, Sandra Hinz, Samuel Ellis, Emily Brown, and Logan Kludt for their time sacrificed helping me with the larger parts of my project. And to those I don’t have space for or cannot recall, thank you as well.

Available for download on Friday, December 15, 2017

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