ii Chemical-genetics profile and genetic interaction analyses for identification of novel genes involved in protein synthesis in yeast A thesis presented to The Faculty of Graduate Studies Carleton University by Md Alamgir In partial fulfillment of the requirements For the degree of Doctor of Philosophy (Ph.D.) 2010 © Copyright 2010, Md Alamgir Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 OttawaONK1A0N4 Canada Canada Your file Votre r6f6rence ISBN: 978-0-494-70540-7 Our file Notre r6ference ISBN: 978-0-494-70540-7 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Nnternet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1+1 Canada iv "I have, read about sixteen pages of your manuscript ...I suffered exactly the- same treatment at the hands of my teachers who disliked me for my independence and passed over me when they wanted assistants ... keep your manuscript for your sons and daughters, in order that they may derive consolation from it and not give a damn for what their teachers ted them or thinks of them. ... There is too much education altogether." 'Einstein, SllBert (1879-1955) V Chemical-genetics profile and genetic interaction analyses for identification of novel genes involved in protein synthesis in yeast As a major step in the gene expression pathway, protein synthesis, also known as translation plays an indispensable role in the survival of a cell. Much has been discovered about protein synthesis over the last few decades. However the list of novel factors that affect this process continues to grow, indicating the presence of other elements that are associated with protein synthesis. This thesis aimed to identify novel genes that affect the process of protein synthesis. For this purpose we employed large-scale screening methods developed for genetic studies in the yeast, Saccharomyces cerevisiae. Specifically, we used increased sensitivity to translation drugs (paromomycin, cycloheximide, 3-amino-l, 2, 4 triazole, streptomycin, neomycin) as a method to screen the entire set of yeast non­ essential gene knockout collection (-4700 strains). The chemical-genetics profiles identified in this manner were used to identify genes with possible links to protein synthesis. We further studied the activity of four genes that here we termed TAE1-A for translation associated elements 1-4, respectively; using various genetic assays including synthetic genetic interaction and phenotypic suppression analyses. vi AC%NpfWL'E<DQ'EM'E*£T First and foremost, I would like to express my sincere gratitude to Dr. Ashkan Golshani for his mentorship, stimulating suggestions, encouragement, enthusiastic guidance, supervision, continuous assistance and for providing me an inspiring environment to pursue and complete my research successfully. Without his assistance, this would not have become a reality. I have greatly appreciated his eagerness for discovery, thinking, elucidation of key questions, and they have been of great value for me. I also offer special thanks for his advice, accessibility and willingness to support the innovations which make research life smooth and rewarding for his students. This paper would not have been possible without his contribution and support. The members of my thesis committee have also been extremely helpful in contributing their wide-ranging expertise to my interdisciplinary project. I was extraordinarily fortunate in having Dr. Myron Smith on my research committee. Dr. Smith advised me on the primacy of using biological questions to motivate my research. I also wish to extend my gratitude to Dr. Prabhat Arya and Dr. Holcik for all of their detailed and constructive comments, criticisms, extensive discussions and support throughout my work. I wish to extend my appreciation to the Department of Biology, Carleton University for their financial support throughout this project, and for the access granted to department facilities and equipment. My closest collaborator and fellow, has infused many enthusiastic insights into our research. I am grateful to University of Toronto and, in particular, Dr. Charlie Boone, for providing the yeast strains that were used for SGA experiments in this thesis. Special thanks to Dr. Helena Friesen, from Dr. Brenda Andrews's lab at the University of Toronto, who graciously assisted during the initial steps of SGA experiments. Robert Smith from Myron Smith's lab shared reagents and advice that enabled my western blotting experiments as well as several troubleshooting tips. I have also benefited from collaborations with several bioinformaticians, including vii Dr. Frank Dehne, Sylvain Pitre, Dr. M. Dumontier for their permission to use the Yeast feature tool, and Negar Memarian for helping in colony size measurement using Growth Detector (GD) software. Dr. James R. Green helped to refine our computational analyses while eagerly learning chemical genetics. I have thoroughly enjoyed my discussions with all of them on a wide range of topics. I am also grateful to many other people who have generously furthered my research training. Veronika Eroukova, Matthew Jessulat and former colleagues/lab members from the Golshani lab shared practical suggestions from their inexhaustible knowledge of yeast genetics and molecular biology. I want to thank all of them for their help, support, and interest. I am especially obliged to Sunny, Maysoon, Steven and Ben. Thanks to Marija Gojmerac and the office staff who made all administrative issues very simple. Thanks to Ed Bruggink for his unconditional smile and support. It is difficult to overstate my appreciation and sincere thanks to Professor Willem F. Stevens, my supervisor from Asian Institute of Technology (AIT), who initiated my interest in research and influenced me to finish my PhD. Prof Sudip K. Rakshit, now vice president of AIT, inspired me to continue my career in research. They are not only great mentors and colleagues, but also the cornerstone of my professional development. Their ideas and concepts have had a remarkable influence in guiding me toward a life of research. I wish to thank everyone with whom I have shared various unique experiences in life, from my childhood to the discovery of what life is all about, and how to optimize every opportunity that presents itself. It is a pleasure to convey my gratitude to all of them in my humble acknowledgment. My deepest gratitude goes to my family members for their support throughout my life. I am grateful to my late father and mother, for their care and love. My father, AKM Kafiluddin, is the first person who instilled in me the fundamentals of a learning character, showing me the joy of intellectual pursuit ever since I was a child. My Mother, Faizunnessa Khatun, is the one who sincerely raised me with her caring and gentle love. Although they are no longer with us, they will be viii remembered forever. Habib, Kabir and Jahangir, thanks for being supportive and caring siblings. I remember their constant support. Without the support and encouragement of all of these caring individuals, this work could not have been possible. I would like to give my special thanks to my wife Zaheda, whose patience, support and love enabled me to complete this work. In addition, I express love and appreciation to Aymaan, my son, whose loving support help me to finish this task. Thank you, Zaheda and Aymaan! Finally, 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(s) I may have forgotten to mention. ix IMBUE 07C<y^TE9^TS XBSH9&CI. V !AC(K^(p(WL'L'Dg<EWE9^X VI LISTOfF'l&BL'ES XII LIS? OJ JIQIWtES XIII LIS1'OJ-ScU&E<yK$Ic\SEcI%!BJJES XV LIS?'05m<B<%EiViwnocH$ xvi 1.0 INTRODUCTION 1 1.1 Systems Molecular Biology 2 1.1.1 Functional Genomics 4 1.1.2 Yeast and Functional Genomics 5 1.2 Yeast 10 1.3 Genetic and Protein-Protein Interactions (PPIs) 12 1.4 Chemical-Genetics 19 1.5 Ribosome