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Study of group II introns in Euglena chloroplast genomes: Structure, processing and evolution Item Type text; Dissertation-Reproduction (electronic) Authors Zhang, Liqun, 1969- Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 27/09/2021 14:23:33 Link to Item http://hdl.handle.net/10150/282618 INFORMATION TO USERS This manuscript has been reproduced from the microfihn master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter &ce, while others may be fi-om any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. 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Contact UMI directly to order. UMI A Bell & Howell Infomiation Company 300 North Zed> Road, Ann Aibor MI 48106-1346 USA 313/761-4700 800/521-0600 STUDY OF GROUP H INTRONS EST EUGLENA CHLOROPLAST GENOMES - STRUCTURE, PROCESSING AND EVOLUTION by Liqun Zhang A Dissertation Submitted to the Faculty of the DEPARTMENT OF BIOCHEMISTRY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 1998 UMl Nunber: 9829355 UMI Microform 9829355 Copyright 1998, by UMI Company. Ail rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 2 THE UNIVERSITY OF ARIZONA ® GRADUATE COLLEGE As members of the Final Examination Committee, we certify that we have read the dissertation prepared by LIQUN ZHANG entitled Study of Group II Introns in Euglena Chloroplast Genomes — Structure, Processing and Evolution and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philogophy 2/// Date ^ Date ""Tc*- Date Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. Dissertation Director Date 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in The University Library to be made available to borrowers under rules of the Library Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: 4 ACKNOWLEDGMENTS I would like to thank my advisor, Richard Hallick, for providing me the opportunity and the strict training to become a scientist. His strong encouragement and wise guidance have helped me tremendously during my research and study. I would also like to thank my committee members: Hans Bohnert, Don Bourque, Mark Dodson, William Montfort and Roy Parker, for their support and advice which helped me through so many research low tides. Thanks to my past and present lab members Donald Coppertino, Rober Drager, Jennifer Stevenson, Angie Boyer, Cathy Hibbert, Ling Hong, Kristin Jenkins, Mike Thompson, Mitch Favreau, Jane Huff and Natalie Doestch for their assistance and companionship. I'd like to specially thank Kristin and Natalie for their great effort helping me improve my technique writing skill. There are many friends outside my lab that have also given me strong support and advise. I would like to thank Wei Chen, Hua Sue, Li Zhang, Chunghong Mao, Nicoleta Constantin, David Whitacre and David Ascue. Thanks for helping me solve so many technical problems, providing me valuable information, editing my writings and, simply, for being great friends. They have made my six years of graduate school much more enjoyable. I would like to thank my family my father, Dongpei Chen, my mother, shouyu Zhang and my lovely sister, Cindy yao Chen, for being a strong support throughout my academic years, in China and in the States. Thanks for keeping home the warmest place on earth, thanks for telling me thousands of times that I can do this, thanks for inspiring me with your own achievements and persistent spirits. Specially, I would like to thank my dearly beloved husband, Charles Xiaobing Fang, thanks for being there for me all the time, thanks for hearing those endless complains and whining, and returning with encouragement and smart ideas. Without him, I would never pull it off. 5 DEDICATION I Would like to dedicate this thesis to my grandfather, Shouyi Zhang, my most respected person in the world, who has taught me, with his own words and deeds, how to be strong, confident, always decent and never, ever, give up. 6 TABLE OF CONTENTS LIST OFnCURES I 3 LIST OF TABLES 1 7 ABSTRACT I 8 CHAPTER I 2 0 INTRODUCTION 2 0 L The RNA world 2 0 Ribozyme 2 1 Retroelements 2 3 Introns 2 5 (1) Intron types, structures and splicing mechanisms 2 7 (2) Trans protein factors for intron splicing 3 2 II. Group II introns 3 4 The splicing process: the five steps 3 6 Strcutrual elements and functionalities 3 7 (1) Major ribozyme motifs 3 8 (2) Other tertiary intereaction elements 3 9 (3) Internally encoded proteins 4 2 Hydrolysis and trans-splicing of group II introns 4 3 Evolutionary relationship of group II introns and nuclear 7 spliceosome 4 4 III. Genus Euglena 4 6 The classification and taxonomy of Euglena 4 8 Phylogeny of Euglena 5 I IV Euglena gracilis chloroplast genome and the intron content.... 5 4 V. Thesis objectives 6 0 CHAPTER 2 6 3 THE EUGLENA GRACILIS INTRON-ENCODED MAT 2 LOCUS IS INTERRUPTED BY THREE ADDITIONAL GROUP H INTRONS 6 3 Introduction 6 3 Results 6 6 Identification of partially spliced psbC intron 2 pre- mRNAs by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis 6 6 Three group II introns are located in domain IV of psbC intron 2 6 9 Characterization of internal 5'- and -3' splice sites within psbC intron 2 7 0 Intron secondary models 7 5 psbC intron 2 splicing pathway is partially ordered 7 9 Internal introns of psbC interrupt an open reading frame encoding a putative maturase 8 4 Discussion 8 7 8 A new category of twintron 8 7 A partially ordered psbC intron 2 splicing pathway 8 7 mat 2 may be required for splicing 8 8 Possible functions of mat 2 8 9 Evolution of mat 2 9 I Materials and methods 9 2 RNA isolation 9 2 cDNA synthesis 9 2 PCR analysis, cloning and sequencing 9 3 Computer analysis of orf 758 9 4 CHAPTERS 9 5 PSBC INTRON 2 AND MAT 2 ARE DEEPLY ROOTED IN GENUS EUGLENA, WITH VARIOUS INTERNAL INTRON CONTENT 9 5 Introduction 9 5 Results 9 8 PCR amplification of ten Euglena species 9 8 Determination of processing sites of psbC intron 2 homologues in E. viridis, E. granulata, E. anabaena, and E. spirogyra 1 02 Complete sequencing and identification of the external intron and mat 2 gene of the psbC intron 2 homologues of E. viridis, E. granulata, E. anabaena, and E. spirogyra 105 9 RT-PCR analysis and identification of internal introns in mat 2 homologues of E. viridis, E. granulata, E. anabaena and E. spirogyra 105 Processing of the internal introns in E. granulata is partially ordered 112 Comparative analysis of mat 2 sequences, and identification of conserved domains 115 Discussion I 1 8 psbC intron 2 and mat 2 are deeply rooted in Euglena species 1 1 8 High local conservation of mat 2 suggests a conserved function as an intron maturase 119 psbC2 intron 2 as the Euglena group II introns founder1 2 1 Internal introns and their evolutionary implications ....12 2 Materials and methods 124 Euglena cultures 124 Nucleic acid isolation 125 PCR and RT-PCR primers 125 PCR amplification, cloning and sequencing 126 RT-PCR amplification, cloning and analysis 127 CHAPTER 4 130 COMPARATIVE ANALYSIS AND SECODNARY STRUCTURE MODEL STUDY OF PSBC INTRON 2 EXTERNAL AND INTERNAL INTRONS 13 0 10 Introduction 1 30 Results I 3 2 Primary sequence comparison of psbC intron 2a 132 Comparative analysis of external intron 2as, establishing the secondary structure models 137 Analysis of domains I and II of psbC intron 2a 140 Sequence analysis and secondary structure model establishment of the internal introns, characterization of a mini-group II intron and identification of homologous intron 2ds 142 Discussion 1 49 The idenfitication of psbC intron 2 as a group II intron has been confirmed 149 The Euglena group II intron "microcosm" 150 The internal introns seem to have a much higher mutation rate 15 1 CHAPTER 5 153 EXPRESSION OF PARTIAL MAT 2 GENE OF EUGLENA GRACILIS IN E.