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Telomere Structure and Maintenance In Telomere structure and maintenance in Trypanosoma brucei Ranjodh Singh Sandhu Bachelor of Science Punjabi University, 2004 Master of Science (Biotechnology) Punjabi University, 2006 submitted in partial fulfillment of requirements for the degree Doctor of Philosophy in Regulatory Biology at the Cleveland State University October, 2014 © Copyright by Ranjodh Singh Sandhu, 2014 We hereby approve this dissertation for Ranjodh Singh Sandhu Candidate for the Doctor of Philosophy in Regulatory Biology degree for the Department of Biological, Geological and Environmental Sciences and the CLEVELAND STATE UNIVERSITY College of Graduate Studies ____________________________________ Date -_______________ Dr. Bibo Li, BGES, Cleveland State University Major Advisor ____________________________________ Date -_______________ Dr. Valentin Börner, BGES, Cleveland State University Advisory Committee Member ____________________________________ Date -_______________ Dr. Alexandru Almasan, Lerner Research Institute, Cleveland Clinic Advisory Committee Member ____________________________________ Date -_______________ Dr. Sailen Barik, BGES, Cleveland State University Advisory Committee Member ____________________________________ Date -_______________ Dr. Aaron Severson, BGES, Cleveland State University Internal Examiner ____________________________________ Date -_______________ Dr. Kausik Chakrabarti, Carnegie Mellon University External Examiner Student’s Date of Defense: October 28th 2014 Dedicated to my family Acknowledgements Working on my dissertation has been both challenging and interesting. The hurdles I met during this study were somehow made interesting by all those who inspired and guided me throughout this work. Most importantly, I would like to express my deepest gratitude to my advisor Dr. Bibo Li for her excellent guidance, patience, motivation and providing me the opportunity to work in her laboratory. She has been a great mentor, providing freedom that allowed me to think and work independently, yet always reachable and helpful when I needed guidance. I am grateful to my graduate advisory committee members Drs. Valentin Börner, Alexandru Almasan and Sailen Barik for their time, constructive criticism and invaluable suggestions. It has been a great honor to have leaders in their field show interest in my work. I would also like to thank members of my dissertation defense committee Dr. Aaron Severson and Dr. Kausik Chakrabarti for providing their valuable time and their willingness to be members of my dissertation defense committee. Ph. D. was impossible without guidance and persistent help from all the departmental faculty and staff members. I am thankful to my lab mates and friends Sanaa Jehi, Unnati Pandya, Vishal Nanavaty, Nicole Kresak, Imaan Benmerzouga and all the undergraduate students. Without their friendship, advice and encouragement I would not have succeeded in completing my work. I am deeply thankful to Jasvinder Singh Ahuja for his constant encouragement, help and invaluable friendship. I would like to thank my wife Rima for her support and patience at all times. This thesis would not have been possible without the love, support and constant encouragement of my family. They have always been there for me and gave me strength surpass difficult times. Telomere structure and maintenance in Trypanosoma brucei Ranjodh Singh Sandhu 1ABSTRACT Trypanosoma brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in animals. The main reason for persistent infection of T. brucei is that in mammalian hosts, T. brucei undergoes antigenic variation and regularly switches its major surface antigen, Variant Surface Glycoproteins (VSG), to evade the host's immune response. VSGs are exclusively expressed in a monoallelic manner from VSG expression sites (ESs) located at subtelomeric loci. We and others have shown that telomeres play important roles in VSG expression and switching regulation. In most eukaryotes, telomere maintenance mainly relies on telomerase, a specialized reverse transcriptase. In T. brucei, the protein component of telomerase (TbTERT) has been identified previously. In this study, we identified and characterized the RNA component of T. brucei telomerase (TbTR). We established that TbTR interacts with TbTERT and is critical for telomere maintenance. We also provided insights of biogenesis of RNA component and predicted its native folding. Telomere DNA consists of TG-rich sequences, and there is a single- stranded 3’ G-rich overhang at the very end of the telomere called G-overhang. The G-overhang serves as a primer for telomerase-mediated telomeric DNA synthesis and also participates in formation of the T-loop structure that helps protect telomere termini from illicit DNA repair activities. The G-overhang vii structure in T. brucei was poorly understood. We employed various methods to characterize the structure of G-overhang in T. brucei. We show that the terminal nucleotides on both G-rich and C-rich strands are specific and are regulated by telomeric proteins. TbRAP1, an intrinsic component of the T. brucei telomere complex, is important for VSG silencing. However, the mechanism by which TbRAP1 regulates VSG silencing is unclear. We have established a number of TbRAP1 conditional knockout strains expressing various TbRAP1 mutants and provided preliminary data about functions of different TbRAP1 domains. We have provided new insights into telomere maintenance in T. brucei, which will help better understand how telomeres contribute to antigenic variation and develop T. brucei as a model system for telomere biology research. viii 2Table of Contents ABSTRACT ........................................................................................................ VII LIST OF FIGURES ............................................................................................. XII LIST OF TABLES ............................................................................................. XV CHAPTER I INTRODUCTION .............................................................................. 1 1.1 AFRICAN TRYPANOSOMIASIS ...................................................................... 1 1.2 HUMAN AFRICAN TRYPANOSOMIASIS .......................................................... 3 1.3 DISEASE DIAGNOSIS .................................................................................. 4 1.4 TREATMENT OF HUMAN AFRICAN TRYPANOSOMIASIS ................................... 6 1.5 THE LIFE CYCLE OF T. BRUCEI ................................................................... 8 1.6 THE T. BRUCEI GENOME .......................................................................... 10 1.7 GENE EXPRESSION AND GENOME ORGANIZATION IN T. BRUCEI .................... 12 1.8 SURFACE COAT PROTEINS AND THEIR EXPRESSION .................................... 15 1.9 ANTIGENIC VARIATION IN T. BRUCEI .......................................................... 18 1.9.1 VSG switching ............................................................................... 19 1.9.2 Monoallelic expression .................................................................. 22 1.10 TELOMERES ........................................................................................... 26 1.10.1 The telomere G-overhang structure .............................................. 28 1.10.2 The telomere protein complex ....................................................... 30 1.11 TELOMERASE ......................................................................................... 37 1.11.1 Telomerase reverse transcriptase (TERT) .................................... 38 1.11.2 Telomerase RNA (TR) .................................................................. 39 1.11.3 Recruitment of telomerase to telomeres ....................................... 42 1.12 TELOMERE AND TELOMERE MAINTENANCE IN T. BRUCEI .............................. 43 1.13 SIGNIFICANCE OF STUDY ......................................................................... 46 CHAPTER II MATERIALS AND METHODS ...................................................... 48 2.1 TRYPANOSOME STRAINS AND CULTURE CONDITIONS .................................. 48 2.2 TRANSFECTION ....................................................................................... 49 2.3 GENOMIC DNA ISOLATION ....................................................................... 49 2.4 ADAPTOR LIGATION ASSAY ...................................................................... 49 2.5 EXO-T TREATMENT OF GENOMIC DNA ..................................................... 51 2.6 SINGLE TELOMERE LENGTH ANALYSIS (STELA) ......................................... 51 2.7 T7 EXO NUCLEASE TREATMENT OF GENOMIC DNA ................................... 52 2.8 LIGATION MEDIATED PRIMER EXTENSION (LMPE) ...................................... 52 2.9 PREPARATION OF ARTIFICIAL TELOMERE SUBSTRATE FOR LMPE ................ 54 2.10 SOUTHERN BLOT ANALYSIS ...................................................................... 54 2.11 NORTHERN BLOT ANALYSIS ...................................................................... 55 2.12 REVERSE TRANSCRIPTION AND QUANTITATIVE REAL TIME PCR .................. 56 2.13 RNA IMMUNOPRECIPITATION (RNA IP) .................................................... 56 2.14 CLONING OF TERMINAL FRAGMENTS OF TELOMERES .................................. 57 2.15 TELOMERASE ACTIVITY ASSAY .................................................................. 58 2.16 PREPARING RADIOACTIVE PROBE ............................................................
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