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Information to Users INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. in the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A NOVEL SECOND DOMAIN INVOLVED IN GEMINIVIRUS REP PROTEIN OLIGOMERIZATION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jared Quentin LeMaster * * * * The Ohio State University 2002 Dissertation Committee: Approved By Dr. David M. Bisaro, Advisor Dr. Mark Muller Dr. Michael Ostrowski ^ 2 y Advisor Dr. Deborah Parris Molecular Genetics Graduate Program Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3049067 _ ___ (8) UMI UMI Microform 3049067 Copyright 2002 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT The Geminiviridae is a growing family of small genome ssDNA viruses that causes significant disease in a variety of important commercial and staple crops throughout tropical and temperate regions of the world. Geminiviruses replicate their genomes by a rolling circle replication (RCR) mechanism similar to the bacteriophage 0X174. Their replication cycle depends heavily on host cellular replication machinery. Only one viral protein, Rep, is required for initiation and termination of RCR. Rep protein-protein complex formation, i.e. oligomerization, is a key event in geminivirus replication. Current evidence suggests that Rep forms several, most likely different, multi-protein complexes in infected cells. These complexes, which include Rep oligomers, Rep-REn (Viral Replication ENhancer protein) and Rep-pRBl (plant homologue of the mammalian retino­ blastoma related tumor suppressor protein, Rb) complexes, are all formed via protein-protein interactions with the same target region of Rep. Therefore, a mechanism must exist that regulates what kind of complexes Rep forms. Therefore, we hypothesized that multiple protein regions (domains) are involved in geminivirus Rep protein oligomerization. We supported this hypothesis by making use of the powerful yeast two-hybrid system. We constructed a variety of Rep deletions and examined their abilities to interact with one another. Our results indicate that the geminivirus, Tomato Golden Mosaic (TGMV),Virus Rep has two domains important for homo­ oligomerization. The first previously described region is located in the middle of TGMV Rep between residues 116 and 181 and is referred to as domain I in this work. The second, novel domain, domain II is located in the carboxy-terminal 52 amino acids of TGMV Rep and functions in a position-independent manner. Computer-aided analysis of the C-terminal interaction domain revealed the presence of a predicted a-helical region that is highly conserved throughout the Geminiviridae. Additionally, we demonstrated that the C-terminal region is involved in homo­ oligomerization of Rep proteins from three distinct geminiviruses. We also demonstrated that TGMV Rep is capable of forming oligomers with heterologous Rep proteins from two related geminiviruses, one of which belongs to a separate geminivirus genus. Lastly, geminivirus replication and the roles that Rep oligomerization may play in the replicative process are discussed in light of what is known about RCR and other relevant model replication systems. n Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. THIS DISSERTATION IS DEDICATED TO MY MOM AND DAD THANKS FOR EVERYTHING! iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS I would like to thank my advisor and committee member. Dr. David M. Bisaro for his patience, keen critical reviews and advice throughout my graduate education. I would also like to thank Dr. Garry Sunter, for advice on protocols and friendship. Janet Sunter and Dr. Kenn Buckley have also provided assistance with this work as well as many refreshing debates over the years. I would also like to thank my fellow graduate students for their help. To my good friends Linhui Hao and Hui Wang, thanks for all the times you looked alter my experiments and provided technical assistance. I am also indebted to graduate school veterans Marcus Hartitz and Dr. Fredrick Meyer for pointing out so many of the pitfalls graduate students can fail into along the way. I would also like to thank all the people that made everyday life bearable, the staff of the biotechnology center Melinda Parker, Dave Long, Mike Zianni, and Billy, the MCDB secretary, Jan Zinich, the Molecular Genetics office staff, especially Jessie, for all their hard work and patience. And Molecular Genetics graduate school committee chairman, Dr. Mike Ostrowski for his continued patience with me while I finished my dissertation. I would also like to thank the additional members of my committee: Dr. Mark Muller, Dr. Deborah Parris, and Dr. Mike Ostrowski. Their advice has helped me to become a more disciplined and focused scientist Additionally, I also owe much thanks to a great myriad of friends and family for all their help, thoughts and prayers. To my parents, James and Jean who have helped in every way imaginable and to whom I cannot say thank you enough. To my in-laws, Pete and Sharon, first of all, for letting me marry their wonderful daughter, and secondly, for providing so much support throughout the years. I must also thank the many friends I’ve been blessed with who have allowed me to live in their homes over the past two and a half years. Ed D’amato, Scott and Danielle Smith and Greg and Danielle Hartt. Although you never said it. I’m sure I was a nuisance, thank you very much for your hospitality. To my family. Dr. Elizabeth LeMaster (Betsy), Jacob Stanley (Jack) and Peter James (Pete) and little Isabelle, I’m sorry for all the inconvenience and irritation I have caused over the years and I am eternally in your debt for your constant love and support. Lastly, I would like to thank the Lord, Jesus Christ, whose constant presence has seen me through even the darkest times and to whom all thanks and credit is ultimately due. h r Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VITA Septembers, 1971 Bom - Ravenna, Ohio USA 1994, B.S. Biology, Kent State University, Ohio USA 1994-2001 Researcher, The Ohio State University, Columbus, Ohio Dept, of Molecular Genetics 1994-2002 Graduate Teaching Associate The Ohio State University, Columbus, Ohio USA Dept, of Molecular Genetics POSTERS, SEMINARS AND PUBLICATIONS Posters / Seminars: LeMaster, J.Q., Buckley, K., Sunter G., Davis, K.R., Bisaro, D.M. (Nov. 1996) Interactions Between Geminivirus Replication Proteins (REP and AL3). Plant Molecular Biology and Biotechnology 1996 Symposium- Plant Responses to the Environment. Museum of Biological Diversity, Columbus, Ohio. LeMaster, J.Q., Bisaro, D.M. (Sept. 1998) Protein Interactions of Tomato Golden Mosaic Virus Replication Proteins REP and REn. Stone Laboratory Research Center Symposium, Put-in-Bay, Ohio. LeMaster, J.Q., Bisaro, D.M. (Jan. 1999) In vitro Analysis of Recombinant TGMV Replication Proteins. Scott Falkenthal Memorial Graduate Student Colloquium, Columbus, Ohio. LeMaster, J.Q., Bisaro, D.M. (Jun. 1999) Interactions Between the Geminivirus Replication Proteins AL1 (Rep) and AL3 (REn). Keystone Symposium- Molecular Mechanisms in DNA Replication and Recombination, Taos, New Mexico. Research publications: LeMaster, J.Q., Bisaro, D.M. A Novel Second Domain Involved in Geminivirus Rep Protein Oligomerization. 2002. (manuscript in preparation) FIELDS OF STUDY Major Field: Molecular Genetics V Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Page Abstract.................................................................................................................
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