The University of Maine DigitalCommons@UMaine Electronic Theses and Dissertations Fogler Library 2003 Effect on Heartbeat of Drosophila melanogaster of Mutations in the Calcium Channel Encoding cacophony Gene and its Interaction with the RNA Helicase Mutant maleless^napts Vanessa McGowan Follow this and additional works at: http://digitalcommons.library.umaine.edu/etd Part of the Medicine and Health Sciences Commons, and the Zoology Commons Recommended Citation McGowan, Vanessa, "Effect on Heartbeat of Drosophila melanogaster of Mutations in the Calcium Channel Encoding cacophony Gene and its Interaction with the RNA Helicase Mutant maleless^napts" (2003). Electronic Theses and Dissertations. 352. http://digitalcommons.library.umaine.edu/etd/352 This Open-Access Thesis is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of DigitalCommons@UMaine. EFFECT ON HEARTBEAT OF Drosophila melanogaster OF MUTATIONS IN THE CALCIUM CHANNEL ENCODING cacophony GENE AND ITS INTERACTION WITH THE RNA HELICASE MUTANT malelessnapts BY Vanessa McGowan B.S. University of Maine, 2000 A THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science (in Zoology) The Graduate School The University of Maine December, 2003 Advisory Committee: Harold B. Dowse, Professor of Zoology and Cooperating Professor of Mathematics, Advisor John M. Ringo, Professor of Biology Erik Johnson, Ph.D. 02003 Vanessa McGowan Ray All Rights Reserved EFFECT ON HEARTBEAT OF Drosophila melanogaster OF MUTATIONS IN THE CALCIUM CHANNEL ENCODING cacophony GENE AND ITS INTERACTION WITH THE RNA HELICASE MUTANT malelessnapts By Vanessa McGowan Ray Thesis Advisor: Dr. Harold B. Dowse An Abstract of the Thesis Presented in Partial Fulfillment of the Requirements for the Degree of Master of Science (in Zoology) December, 2003 There is a lack of information concerning the genetics of heart disease, especially of those due to aberrant pacemaker activity. Drosophila melanogaster is an ideal candidate for research in this area because of its suitability to genetic manipulation and its accessible genetic database. What is most compelling, however, is that the genesis of heartbeat is Drosophila is strikingly similar to that in many other organisms, including mammals. The myogenic heart ofDrosophiIa melanogaster is stimulated to contract by a caudal pacemaker, which is regulated by ion channel interactions. A voltage-gated calcium channel of PIQ- or N-type is implicated in the pacemaker by pharmacological studies. The voltage-gated calcium channel a,subunit encoding gene in Drosophila, cacophony (cac), was identified as a candidate for this role. After examining heartbeat of the cac alleles cacSand cacfi2I report that they have increased heart rates and rhythmicities, potentially due to altered inactivation of the calcium channels they encode affecting pacemaking. The gene with which the RNA helicase mutant no action potentialremperflrure-sens'r've (mlenflPrs)interacts, causing anhythmic heartbeat, has yet to be uncovered. mlenflP'5san allele of the dosage compensation gene maleless (mle). It is possible that mlenflPrsis interacting with cac to cause deviations in heartbeat phenotype fiom wild-type. By characterizing heartbeat of cac ;mlenflPrs double mutants I determine that the genes interact, but in an unresolved manner. ACKNOWLEDGMENTS I would like to thank the Association of Graduate Students for helping fund this project through research grant awards. I would also like to thank the members of my advisory committee for the time and energy they committed to helping me mold this project and thesis into its present form. I also appreciate the example they set for fulfilling the requirements placed on research scientists in academia. There are other people who deserve recognition for their contribution to my personal development throughout my time as a graduate student. During my time as a graduate student I gained valuable experience as a teachmg assistant. Because of this commitment, I had the privilege of working closely with the laboratory coordinator, Kevin Tracewski. I thank him not only for his professional guidance and steadfast support, but also for his insight on life planning. As an athlete on the University of Maine's cross country and track & field teams I forged a strong bond with my coach, Mark Lech. He has been one of my biggest advocates, and I have relied on him for advice and guidance in personal and athletic matters. Above all, my family has always supported me in my endeavors, personally, emotionally, and sometimes financially. My parents, sisters, and husband, Benjamin, have been temfic listeners and morale coaches. TABLE OF CONTENTS ... ACKNOWLEDGMENTS.......................................................................... ill ... LIST OF TABLES .................................................................................vm LIST OF FIGURES .................................................................................ix Chapter 1. EFFECT ON HEARTBEAT OF Drosophila melanogaster OF MUTATIONS IN THE CALCIUM CHANNEL ENCODING cacophony GENE ........... 1 Introduction ...................................................................................1 Background Information on cacophony: Alleles and their Phenotypes ......1 The Discovery of the cacophony Gene: Courtship Song ............1 cac Alleles: Visual and Lethal Mutants ................................. 3 Pleiotropic Effects of cac Mutations: Courtship Defects ............ 5 Pleiotropic Effects of cac Mutations: Motor Defects .................6 Behavioral Exclusions of cac Mutants ..................................7 Calcium Channel Information .....................................................8 Composition of Voltage-Gated Calcium Channels ....................8 Types of Voltage-Gated Calcium Channels .......................... 16 Diversity of Voltage-Gated Calcium Channels ......................21 Roles of Voltage-Gated Calcium Channels ...........................22 Molecular Characterization of cacophony ..................................... 23 The cac Gene Transcript - Dmcal A ..................................23 cac Transcript Diversity ................................................. 25 Molecular Lesions of cac Mutants ..................................... 29 Heartbeat of Drosophila melanogaster .........................................31 Synaptic Physiology of cac Mutants ...................................31 The Heart of Drosophila melanogaster ...............................33 Ion Channels of the Drosophila Cardiac Pacemaker ................35 Perspectives: Importance of Drosophila Research ...........................40 Similarities Between Cardiac Physiologies of Homo sapiens and Drosophila ...........................................................41 Importance of Pacemaker Research ....................................42 Importance of Studying cac Heartbeat Mutants ..................... 46 Materials and Methods .....................................................................48 Fly Culture and Experimental Strains ..........................................48 Control Strains.... :................................................................. 49 Heartbeat Recordings andTemperature Step Protocols ......... ; ...........50 Data Analysis .....................................................................-52 Results ......................................................................................-54 Wild-type Heart Rate Phenotype ................................................54 Effect of cackn Heartbeat Frequency ......................................... 54 Effect of cacIs2 on Heartbeat Frequency .......................................79 Wild-type Rhythmicity Index Phenotype ......................................80 Effect of cac Mutations on Heartbeat Rhythmicity Index ................... 84 Discussion ................................................................................... 86 The cac Gene's Participation in Heartbeat Pacemaking ..................... 86 Aberrant Calcium Channel Inactivation in cac Mutants .....................88 Alternate Explanation for cac Mutants' Heartbeat Phenotypes .............95 Differences in Calcium Channel Defects of cac Mutants ....................97 Ion Channels of the Drosophila Cardiac Pacemaker ........................ 100 2 . EFFECT ON HEARTBEAT OF Drosophila melanogaster OF AN INTERACTION BET WEEN THE RNA HELIC A SE MUTANT malelessnflptS AND THE VOLTAGE-GATED CALCIUM CHANNEL MUTANT cacophony...................................................................................... 103 Introduction................................................................................ 103 he malelessnflP?Mutant ........................................................ 103 Interaction between mlenflPtsand para ......................................... 105 Interaction between . cbc and mlenflPts........................................... 108 Materials and Methods .......... :.......................................................... -111 Fly Culture, Experimental and Control Strains ..............................111 Double Mutant Mating Schemes .............................................. 112 Heartbeat Recordings, Temperature Step Protocols, and Data Analysis ...........................................................................115 Results ......................................................................................116