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Ribozymes Targeted to the Mitochondria Using the 5S Ribosomal Rna

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Ribozymes Targeted to the Mitochondria Using the 5S Ribosomal Rna RIBOZYMES TARGETED TO THE MITOCHONDRIA USING THE 5S RIBOSOMAL RNA By JENNIFER ANN BONGORNO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005 Copyright 2005 by Jennifer Bongorno To my grandmother, Hazel Traster Miller, whose interest in genealogy sparked my interest in genetics, and without whose mitochondria I would not be here ACKNOWLEDGMENTS I would like to thank all the members of the Lewin lab; especially my mentor, Al Lewin. Al was always there for me with suggestions and keeping me motivated. He and the other members of the lab were like my second family; I would not have had an enjoyable experience without them. Diana Levinson and Elizabeth Bongorno worked with me on the fourth and third mouse transfections respectively. Joe Hartwich and Al Lewin tested some of the ribozymes in vitro and cloned some of the constructs I used. James Thomas also helped with cloning and was an invaluable lab manager. Verline Justilien worked on a related project and was a productive person with whom to bounce ideas back and forth. Lourdes Andino taught me how to use the new phosphorimager for my SYBR Green-stained gels. Alan White was there through it all, like the older brother I never had. Mary Ann Checkley was with me even longer than Alan, since we both came to Florida from Ohio Wesleyan, although she did manage to graduate before me. Jia Liu and Frederic Manfredsson were there when I needed a beer. Marina Gorbatyuk was there when I just had to learn some Russian. From other labs, Dr. John Guy allowed me to use his equipment and taught me how to assay for ATP synthesis. Xioping Qi helped me section mouse eyes and, although technically part of the Guy lab, was part of the Lewin lab family in my eyes. Dr. Adrian Timmers skillfully injected my mouse eyes. Ian Elder taught me how to use the ERG machine. Vince Chiodo packaged my ribozymes in AAV. Sue Moyer, Tom Rowe and Nick Muzyczka gave continued support and valuble advice as my committee. The entire iv Molecular Genetics and Microbiology department and the IDP program were a pleasure to be a part of. I am sure I will miss everyone. v TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................................................................................. iv LIST OF TABLES........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix LIST OF ABREVIATIONS .............................................................................................. xi ABSTRACT..................................................................................................................... xiv CHAPTER 1 INTRODUCTION TO MITOCHONDRIA .................................................................1 Mitochondrial Components ..........................................................................................1 Genome..................................................................................................................1 Replication.............................................................................................................5 Transcription and Translation................................................................................7 Protein Import........................................................................................................9 RNA Import.........................................................................................................11 Mitochondrial Functions.............................................................................................19 Citric Acid Cycle.................................................................................................19 Respiratory Chain................................................................................................20 ATP Synthesis .....................................................................................................22 Reactive Oxygen Species ....................................................................................25 Mitochondrial Diseases ..............................................................................................25 Mitochondrial DNA Mutations ...........................................................................26 NARP and Leigh Syndrome................................................................................27 Mitochondrial Manipulations .....................................................................................31 Creating Heteroplasmy in Cells and Mice...........................................................31 Manipulating the Mitochondrial Genome ...........................................................33 RNA Import.........................................................................................................35 Altering Proteins..................................................................................................35 Ribozymes ...........................................................................................................38 Adeno-Associated Virus......................................................................................39 2 RESULTS...................................................................................................................41 vi Ribozyme Design and In Vitro Analysis ....................................................................41 Mouse Cell Culture Phenotypes .................................................................................47 Four Transfection Attempts.................................................................................48 Analysis of Clonal Isolates..................................................................................53 Localization of Ribozymes to the Mitochondria ........................................................61 Human Cell Culture Phenotypes ................................................................................65 HeLa Cells and a Toxic 5S Transcript ................................................................65 No Phenotype in 293 Cells..................................................................................67 Injection of Mice.........................................................................................................69 3 MATERIALS AND METHODS ...............................................................................75 In Vitro Ribozyme Analysis .......................................................................................75 Cloning .......................................................................................................................76 Cell Culture.................................................................................................................76 Transfections...............................................................................................................77 RNA Isolation.............................................................................................................78 RT-PCR ......................................................................................................................78 Growth in Galactose ...................................................................................................80 Cyanide Resistance Assay ..........................................................................................81 ATP Synthesis Assay..................................................................................................82 Mitochondrial RNA Isolation.....................................................................................83 Mouse Subretinal Injections .......................................................................................84 Electroretinograms......................................................................................................85 Sectioning of Mouse Eyes ..........................................................................................85 4 DISCUSSION.............................................................................................................87 In Vitro Ribozyme Assays..........................................................................................87 Studies on Total Transfected Mouse Cells .................................................................88 Cell Culture Studies on Mouse Clonal Isolates ..........................................................90 Measurements by RT-PCR..................................................................................90 Mitochondrial RNA Levels and Growth in Galactose ........................................91 ATP Synthesis and Cyanide Resistance..............................................................92 Human Cell Transfection Studies...............................................................................94 Transfection Toxicity ..........................................................................................94 5S-ATP8/6 Toxicity ............................................................................................95 Localization of Ribozymes to the Mitochondria ........................................................96 Injection of Mice.........................................................................................................96 Other Means to an End ...............................................................................................97
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