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Large Scale Investigation in Yeast, to Identify Novel Gene(S) Involved in Translation Pathway

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Large Scale Investigation in Yeast, to Identify Novel Gene(S) Involved in Translation Pathway Large scale investigation in yeast, to identify novel gene(s) involved in translation pathway by Bahram Samanfar A thesis submitted to the Faculty of Graduate and Postdoctoral Affairs in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Department of Biology, Ottawa-Carleton Institute of Biology Carleton University Ottawa, Ontario © 2014, Bahram Samanfar Abstract As a fundamental step in the gene expression pathway, protein synthesis (also known as translation) plays a crucial role in the biology of a cell. Although much has been discovered about the translation pathway over the last few decades, the list of novel factors affecting the translation pathway continues to grow indicating the presence of other novel players associated with the protein synthesis pathway which are yet to be discovered. This study aimed to identify novel genes involved in the translation pathway. To this end, we used a variety of large-scale screening techniques followed by low throughput experimental analyses designed for genetic studies in yeast, Saccharomyces cerevisiae. Using molecular biology techniques and bioinformatics, we systematically investigated the effect of specific gene deletions on translation fidelity and efficiency, Internal Ribosome Entry Sites (IRESs) functionality and translation-related helicase activities, for a total of ~ 70,000 strain analysis. We further studied the activity of ~15 genes in more details for their involvement in translation pathway. In light of the current study we propose that there remain other uncharacterized factors that influence translation regulation. Further investigations to characterize these novel factors are recommended. ii Acknowledgements First and foremost, I would like to express my sincere gratitude to Dr. Ashkan Golshani for his mentorship, perfect suggestions, encouragement, enthusiastic guidance, supervision and for providing me an inspiring environment to pursue and complete my research successfully. Without him, for sure, some parts of my dreams would not have become a reality. The members of my thesis committee (Dr. Myron Smith and Dr. Marie-Andree Akimenko) have also been extremely helpful in contributing their wide-ranging experience to my interdisciplinary project. I was extraordinarily fortunate in having Dr. M. Smith on my research committee. Besides his kindness and supports, he advised me on the primacy of using biological questions to motivate my research. I wish to extend my appreciation to the Department of Biology at Carleton University for their friendship, financial support through this project, and for the access granted to departmental facilities and equipments. I am grateful to Dr. Martin Holcik for the gift of plasmids HSP82-LacZ and HSC82-LacZ as well as his invaluable comments. My closest collaborators and fellow researchers have infused many enthusiastic insights into my research. I have benefited from collaborations with a very powerful bioinformatics team, including Dr. Frank Dehne, Dr. James R. Green, Dr. Sylvain Pitre and Andrew Schonrock for sharing their predicted data, PIPE, and their great help in computational analysis. I also would like to thank Dr. Andrew Emili, University of Toronto, and Dr. Mohan Babu, University of Regina, for providing research materials and for all their detailed and constructive comments. I am also grateful to many other people who have generously helped me during my PhD research. I want to thank Katayoun Omidi, Mohsen Hooshyar, Le Hoa Tan, Kristina Shostak, Daniel Burnside, Imelda Galván Márquez, Megan Sanders, Noor Sunba, Firoozeh Chalabiani, MD Alamgir, Matthew Jessulat, Kama Szereszewski, Anna York- Lyon, Rami Megarbane, Jennifer Yixin Jin, Houman Motesharei, Alex Jesso, Urvi Bhojoo and former colleagues/lab members from the Golshani Lab for their help, support and interest. My special thanks go to Anna York-Lyon, the best undergraduate research assistant in the world, for all her hard working in the lab and proofreading my thesis. iii Thanks to Laura Thomas, Lisa Chiarelli, Michelle O’Farrell, Caitlyn McKenzie and Ruth Hill-Lapensee who made all administrative issues very simple. Thanks to Ed Bruggink for his unconditional smile and support. I would like to thank all who were important contributors to the successful completion of this thesis. I apologize in advance to anyone whose name I may have forgotten to mention. It is difficult to overstate my appreciation and sincere thanks to Dr. Mansoor Omidi, my supervisor in Tehran University, who initiated my interest to continue my career in research. I am especially grateful to my parents, for their care, support and love. Simply, without them I would have not made it, at all. I wish to thank everyone with whom I have shared various unique experiences in life including Reza Samanfar, Mina Mahzooni and Kianoush Omidi; it is a pleasure to convey my gratitude to all of them in my humble acknowledgment. I would like to thank all my friends especially Alireza, Babak, Maryam, Shaghayegh, Mehdi, Ashkan, Eilnaz, Mohammad, Shabnam, Bardia, Parastoo and Mohsen for all their support and pure friendship. Last but not the least, I would like to give my special thanks to my beloved wife, Katayoun Omidi, whose patience, useful comments, friendship, support and love enabled me to complete this work. This work was partially funded by an Ontario Graduate Scholarship (OGS). iv Statement of contribution The thesis, “Large scale investigation in yeast to identify novel gene(s) involve in translation pathway” is composed of four studies. Chapter 2, “A global investigation of gene deletion strains that affect premature stop codon bypass in yeast, Saccharomyces cerevisiae”, is the result of experiments primarily designed and carried out by myself. A group of undergraduate and graduate students assisted me and worked under my supervision. Le Hoa Tan, Kristina Shostak and Firoozeh Chalabian helped me with β-galactosidase experiments. Mohsen Hooshyar and Katayoun Omidi helped me with SGA and PSA analysis. I wrote the manuscript. Chapter 3, “The identification and investigation of 7 novel genes involved in IRES- mediated translation in yeast, Saccharomyces cerevisiae”, is the result of experiments primarily designed and carried out by myself. A group of undergraduate students including Anna York-Lyon, Kama Szereszewski and Rami Megarbane helped me with β- galactosidase experiments. Kristina Shostak, helped me with SGA and PSA experiments. I wrote the first draft of the manuscript. Chapter 4, “Efficient prediction of human protein-protein interactions at a global scale”, is a collaborative effort mainly by our computer science collaborator and me. PIPE is designed and performed by Sylvain Pitre and Andrew Schoenrock. PIPE data analysis was performed by me, Sylvain Pitre and Andrew Schoenrock. Biology part of the paper is primarily designed and carried out by myself. Verification of MP-PIPE against experimental data was done by me and Andrew Schoenrock. Translation pathway related experiments were performed mainly by me, with the help of Yuan Gui and Katayoun Omidi in β-galactosidase assay. Confirmation of novel molecular markers for seasonal v allergic rhinitis was performed by our collaborator (Mikael Benson) and analyzed primarily by me. Network-wide analysis of hubs and betweenness centrality was carried out by me and Sylvain Pitre. Using the predicted human protein interaction network to assign breast cancer proteins was performed by me and Mohsen Hooshyar. Identification of protein complexes within the human interaction network was done by me and our collaborator, Charles Phillips. I wrote the initial biological related parts of the manuscript. Chapter 5, ‘Utilizing yeast, genetics to identify novel genes involved in translation- related helicase activity’’, is the result of experiments primarily designed and carried out by me. Anna Lyon-York (undergraduate student) helped me with β-galactosidase experiments. I wrote the initial manuscript for this project. During the course of my PhD studies at Carleton University I have been involved in a variety of interesting projects many of which have been published or submitted in form of a journal publication. The list of my current peer-reviewed publications is presented in Appendix B. vi Table of Contents Abstract ii Acknowledgements iii Statement of contribution v Table of contents vii List of tables xii List of figures xiii List of appendices xv List of abbreviations xvii 1 Chapter: Introduction 1 1.1 Systems biology 1 1.1.1 Functional genomics 5 1.1.2 Yeast functional genomics 5 1.2 Protein-protein and genetic interactions 8 1.3 Translation pathway 15 1.3.1 Ribosome 18 1.3.2 Translation activation 19 1.3.3 Translation initiation 21 1.3.4 Translation elongation 22 1.3.5 Translation termination 23 1.4 Internal Ribosome Entry Site (IRES) 24 1.5 Translation process and diseases 27 1.6 Objective 29 vii 2 Chapter: A global investigation of gene deletion strains that affect premature stop codon bypass in yeast, Saccharomyces cerevisiae 31 2.1 Abstract 31 2.2 Introduction 31 2.3 Materials and methods 33 2.3.1 Strains 33 2.3.2 Media and antibiotics 33 2.3.3 Plasmids 33 2.3.4 Gene knockout 34 2.3.5 Primers 34 2.3.6 qRT-PCR 34 2.3.7 Genetic materials extractions 34 2.3.8 β-galactosidase assay 35 2.3.9 Spot test (drug sensitivity analysis) 35 2.3.10 SGA and PSA analysis 35 2.4 Results and discussion 36 2.4.1 Identification of gene deletions that affect expression of β-galactosidase gene with premature stop codons 36 2.4.2 OLA1, BSC2
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