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Proquest Dissertations Isolation and molecular characterization of a gene from Drosophila melanogaster encoding a predicted Rho guanine nucleotide exchange factor Item Type text; Dissertation-Reproduction (electronic) Authors Werner, Lisa Anne, 1958- 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 30/09/2021 09:01:22 Link to Item http://hdl.handle.net/10150/282483 INFORMATION TO USERS This manuscript has been reproduced from the microfihn master. UMI fihns 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 indistina print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely afreet 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. Each original is also photographed in one exposure and is included in reduced form at the back of the book. 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 m this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Aibor MI 48106-1346 USA 313/761-4700 800/521-0600 I ISOLATION AND MOLECULAR CHARACTERIZATION OF A GENE FROM DROSOPHILA MELANOGASTER ENCODING A PREDICTED RHO GUANINE NUCLEOTIDE EXCHANGE FACTOR by Lisa Anne Werner A Dissertation Submitted to the Faculty of the DEPARTMENT OF MOLECULAR AND CELLULAR BIOLOGY In Partial Falfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 19 9 7 UMX Number: 9814358 UMI Microform 9814358 Copyright 1998, by UMI Company. All 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 THE UNIVERSITY OF ARIZONA ® GRADUATE COLLEGE As members of the Final Examination Committeet we certify that we have read the dissertation prepared fay Lisa Anne Werner entitled ISOLATION AND MOLECULAR CHARACTERIZATION OF A GENE FROM DROSOPHILA MELANOGASTER ENCODING A PREDICTED RHO GUANINE NUCLEOTIDE EXCHANGE FACTOR and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy leau Date. ///2s/^ J- Date 1)1^4hi David Mount Date Date 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. ^ J ^ 2-/^/ f f- Diss:fer tat ion/ Director Lynn Manseau Date i r i 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 am very grateful to a number of people who provided me with the guidance and support that enabled me to complete this work. I would like to thank my dissertation advisor, Lynn Manseau, for giving me the opportunity to do this research, and for helping me to develop as a scientist. I especially appreciate the enthusiasm and passion that she had for my projects. I would like to thank the other members of my committee, Danny Brower, Carol Dieckmann, Roger Miesfeld. and David Mount for their advice and encouragement over the years. My friend, Leona Mukai, has helped me tremendously with the completion of this dissertation and its submission to the graduate college. I accepted a lecturer position with the University of Maryland in Korea before defending my dissertation. Leona did all the leg work and more to distribute my dissertation to my committee, schedule my defense and submit my dissertation to the graduate college. That was a hell of a lot of work! Very special thanks to you Leona. there is no way that I could have finished graduate school without you. The Manseau Lab was a very exciting and fun place to do research. Again, thank you Lynn for creating such a synergistic environment. I am indebted to John Calley for patiently sharing his computer expertise with me on many, many occasions. John's wife, Cindy, and daughter Amelia drove me to the hospital in the wee hours of the morning when 1 had an attack of appendicitis. Steve Emmons conducted work to show that DrtGEF was not spire. Anne LaPorte assisted me with a portion of the chromosomal walk. Wenliang Chen, Huy Phan, Brian James, Lin Hollis, Andrea Wellington and the other members of the Manseau lab were also super to work with. The fifth floor, as well as some of the lower echelons of the Life Sciences South building was populated by a number of individuals who brightened my life. Thank you to my friends Tanya Sandrock, Dave Lydall, Chris Byrd, Rachel Hughes, Julie Mustard, Robin Staples, Chunhong Mao, Tracy Futch. Mike Graner, and Marc Brabant, as well as non-Life Science South people Herman Gordon and Anne Pollack for interesting discussions on science and philosophy, hikes and outdoors fun. parlour games such as poker, and your ability to get silly. I would like to thank Thomas Lindell, Marty Hewlett and Vas Aposhian for allowing me to be a Teaching Assistant for their courses and encouraging and supporting my growth as a teacher. You have helped guide me to a profession 1 truly love. A personal source of inspiration comes from my friend, Nicole Lohr Smith who has conquered a brain tumor, twice, all the while keeping her sunny disposition (and her desire to play hide and seek). Also thanks to Nic's mother, Margie Smith who has been a terrific friend and supporter since my first year in graduate school. My family has been behind me all the way. I thank my father, Pete Werner; my mother. Hazel Werner, my siblings. Eve, Tom and Tim: and my dog Margie. My husband. Hubert Meitz, has provided unconditional love, support, and proofreading. Thank you Hubert. This dissertation is dedicated to those who wonder. 6 TABLE OF CONTENTS LIST OF RGURES 7 LIST OF TABLES 9 LIST OF ABBREVIATIONS 10 ABSTRACT 12 CHAPTER 1: THE MOLECULAR MAPPING OF38DI TO 38C2 ON THE DROSOPHILA SECOND CHROMOSOME 13 Summary 14 Introduction to Drosophila Oogenesis and Establishment of the Body Axes... 15 Oogenesis 15 The Ovarian Cytoskeleton 18 The Posterior Group Genes 21 The Role of giirken and the Oocyte Nucleus in Establishing A/P and D/V polarity 23 spire and cappuccino 24 Results 27 Cytogenetic Mapping of spir and Initiation of a Chromosomal Walk . .27 Chromosomal Walk in the 38C Region 30 Identification of Transcription Units in the spir Region 31 RFLP Analysis 31 Ovarian Expression of the Candidate cDNAs 40 Expression of the Candidate Genes in spir Mutant Ovaries 41 Sequence Analysis of Mutant Alleles 41 Discussion 43 CHAPTER 2: THE MOLECULAR CHARACTERIZATION OF A PREDICTED GUANINE NUCLEOTIDE EXCHANGE FACTOR FOR RHO GTPASES 46 Summary 47 The Importance of Rho Guanine Nucleotide Exchange Factors as Activators of the Rho Signaling Cascade and Regulators of Cellular Events. .48 Rho Guanine Nucleotide Exchange Factors 50 Rho Signaling Pathways 56 Molecular Mechanisms for Rho Pathway Regulation of the Cytoskeleton 58 Rho Pathways Control Cytoskeletal Changes in Morphogenesis 61 Results 70 Isolation and Identification of a Drosophila RhoGEF Gene 70 DrtGEF mRNA is Abundant in Cells Undergoing Morphogenic Movements 99 Discussion 123 General Discussion and Future Directions 133 CHAPTER 3: MATERIALS AND METHODS 139 REFERENCES 145 7 LIST OF FIGURES RGURE I: The Stages of Oogenesis in Drosophilamelanogaster 16 nOURE 2: Organization Of Microtubules During Oogenesis 19 FIGURE 3: Deficiency Map of the Location of spir 28 FIGURE 4: Genomic Map of the Chromosomal Walk 32 RGURE 5: Genomic Map of the Region Containing DrtGEF 73 RGURE 6: DNA Sequence of DrtGEF 72 FIGURE 7: Genomic Organization of DrtGEF 82 FIGURE 8: Domain Structure of DrtGEF and Other Putative RhoGEFs 84 RGURE 9: Sequence Alignment of the DrtGEF RhoGEF Domain with RhoGEF Domains from Other Proteins
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