EVOLUTION OF CYCLOOXYGENASE IN THE CHORDATES By JUSTIN CHASE HAVIRD A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008 1 © 2008 Justin Chase Havird 2 To the teachers who inspired, directed, and challenged me. My high school marine biology teacher, Larry Joye, first introduced me to the fascinating world of life in the water and since then my endeavors have never strayed far from it. Dr. David Evans enthusiastically welcomed me into his lab as an undergraduate, although I had no former research experience. What started as a part-time project soon evolved into an academic career as I was given the opportunity to conduct original research. During this time, Dr. Keith Choe was an invaluable asset and inspiration. Growing from this original partnership, I continued working with David on my master’s research and he has always supported my academic and research endeavors; I cannot envision a better advisor. 3 ACKNOWLEDGMENTS I most of all thank my supervisory committee chair, Dr. David H. Evans. Although David was nearing retirement and had resolved to not accept any new students, he welcomed me as a graduate student without any hesitation. He has guided me in developing research strategies, implementing those strategies, and interpreting the results. He has always supported my projects and none of them would have been accomplished without his enthusiasm. I also thank my supervisory committee members for their commitment to my research and their willingness to share their expertise. Dr. Michael Miyamoto contributed heavily to the phylogenetic aspects of my project and encouraged me to be thorough in my analysis. When I started this project I knew very little about generating meaningful evolutionary relationships. Michael selflessly took the time necessary to make sure I understood how to develop a biologically relevant reconstruction of evolutionary changes that took place over 600 million years ago. He also put his own projects aside while I used his computers for months at a time. Finally, his guidance was absolutely necessary in interpreting the conflicting results of my analyses. Dr. Marty Cohn was a great influence on my research and prompted me to think in an integrative and comparative framework. He generously allowed me to use his lab space. His expertise working with evolutionarily interesting chordates was invaluable. His students also graciously helped me learn many of the techniques used in this research. I am also indebted to several colleagues for their assistance. Dr. Keith Choe pioneered and taught me many of the molecular techniques now commonly used in the Evans’ lab that were critical to this research. He spent countless hours with me in multiple laboratories troubleshooting problems, explaining theory, and developing this research. He also encouraged me to work with the hagfish, lamprey, and amphioxus and continually helped me develop new ideas and strategies. Dr. Kelly Hyndman was also constantly supportive of this research. She 4 taught me the localization techniques used here and also helped with many aspects of retrieving and analyzing genetic data. Finally, she was always receptive to critical discussions of my research and tackled every circumstance in a friendly, often contagious manner. I am very thankful to my family and friends for their continued support of my education and research. My entire family has been very interested in the research I have undertaken and when they were not interested they feigned interest in order to make me happy. My parents, Kurt and Dawn Havird, have encouraged my desire to be a scientist, and have been overwhelmingly supportive during my academic career; often in the form of food, clean laundry, or money. My friends have also been very supportive of my research interests: fellow scientists have empathized with the typical problems associated with research and non-scientists have given me the confidence to persevere. I also thank the following people for providing help with this project in particular, other related projects, or the daily support needed to become a successful student: Dr. James B. Claiborne, Rachel Rose, Dr. GuangJun Zhang, Donovan German, Justin Catches, Andrew Diamanduros, Sara Takeuchi, Jim Stidham, Curtis Lanier, Julia Curtis-Burnes, Oriana Galardi- Este, Rebecca Kreh, Dr. Susan Edwards, Gini Luchini, Makesha Foster, Emily Cornwell, Dr. Larry Page, Pete Ryschkewitsch, Frank Davis, Diana Davis, Cathy Moore, Karen Pallone, Vitrell Sherif, Dr. David Reed, Dr. Paul Lewis, and the laboratories of Dr. Lou Guillette and Dr. David Julian. My research was primarily funded by National Science Foundation grants IBN-0089943 and IOB-0519579 to David Evans. A grant-in-aid of research to Justin Havird from Sigma Xi also supported a portion of this research. Finally, initial support was provided in part by a University Scholars award to Justin Havird. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 ABSTRACT...................................................................................................................................11 CHAPTER 1 GENERAL INTRODUCTION..............................................................................................13 Introduction.............................................................................................................................13 Cyclooxygenase Evolution .....................................................................................................16 Mammals .........................................................................................................................17 Fishes...............................................................................................................................18 Other Vertebrates.............................................................................................................19 Other Chordates...............................................................................................................19 Cyclooxygenase Function in Mammals..................................................................................20 Gastrointestinal Tract ......................................................................................................20 The Kidney ......................................................................................................................21 Platelets............................................................................................................................23 Reproduction ...................................................................................................................23 Nervous System...............................................................................................................25 Cyclooxygenase Function in Non-Mammals .........................................................................27 Fishes...............................................................................................................................27 Osmoregulation ........................................................................................................27 Reproduction ............................................................................................................29 Differential functions of novel forms.......................................................................30 Other functions.........................................................................................................30 Amphibians......................................................................................................................31 Reptiles............................................................................................................................31 Birds ................................................................................................................................32 Study Overview ......................................................................................................................33 2 CYCLOOXYGENASES IN THE ANCESTRAL CHORDATES: SEQUENCE AND PHYLOGENETIC ANALYSES............................................................................................39 Introduction.............................................................................................................................39 Materials and Methods ...........................................................................................................41 Animals and Holding Conditions ....................................................................................41 Tissue Collection.............................................................................................................42 Reverse Transcription, Primer Design, PCR, Cloning, and Sequencing.........................43 5’ and 3’ Rapid Amplification of cDNA Ends (RACE)..................................................45 6 Sequence Analysis...........................................................................................................45 Phylogenetic Analysis