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Recoding of Translation in Turtle Mitochondrial Genomes: Programmed Frameshift Mutations and Evidence of a Modified Genetic Code

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Recoding of Translation in Turtle Mitochondrial Genomes: Programmed Frameshift Mutations and Evidence of a Modified Genetic Code RECODING OF TRANSLATION IN TURTLE MITOCHONDRIAL GENOMES: PROGRAMMED FRAMESHIFT MUTATIONS AND EVIDENCE OF A MODIFIED GENETIC CODE Robert David Russell B.Sc.H. (Biology), Queen's University, 2003 B.A. (Computer Science), Queen's University, 2003 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In the Department of Molecular Biology and Biochemistry O Robert David Russell 2006 SIMON FRASER UNIVERSITY Summer 2006 All rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without permission of the author. APPROVAL NAME: Robert David Russell DEGREE: Master of Science (Molecular Biology and Biochemistry) TITLE OF THESIS: Recoding of Translation in Turtle Mitochondria1 Genomes: Programmed Frameshift Mutations and Evidence of a Modified Genetic Code EXAMINING COMMITTEE: CHAIR: Dr. Melanie O'Neill Assistant Professor, Dept. of Chemistry Dr. Andrew T. Beckenbach Senior Supervisor Professor, Dept. of Biological Sciences Dr. David L. Baillie Supervisory Committee Member Professor, Dept. of Molecular Biology and Biochemistry Dr. Peter J. Unrau Supervisory Committee Member Assistant Professor, Dept. of Molecular Biology and Biochemistry Dr. Barry M. Honda Public Examiner Professor, Dept. of Molecular Biology and Biochemistry Date Approved: August 2,2006 u~lv~~srnlSIMON FRASER I' bra ry DECLARATION OF PARTIAL COPYRIGHT LICENCE The author, whose copyright is declared on the title page of this work, has granted to Simon Fraser University the right to lend this thesis, project or extended essay to users of the Simon Fraser University Library, and to make partial or single copies only for such users or in response to a request from the library of any other university, or other educational institution, on its own behalf or for one of its users. The author has further granted permission to Simon Fraser University to keep or make a digital copy for use in its circulating collection, and, without changing the content, to translate the thesislproject or extended essays, if technically possible, to any medium or format for the purpose of preservation of the digital work. The author has further agreed that permission for multiple copying of this work for scholarly purposes may be granted by either the author or the Dean of Graduate Studies. It is understood that copying or publication of this work for financial gain shall not be allowed without the author's written permission. Permission for public performance, or limited permission for private scholarly use, of any multimedia materials forming part of this work, may have been granted by the author. This information may be found on the separately catalogued multimedia material and in the signed Partial Copyright Licence. The original Partial Copyright Licence attesting to these terms, and signed by this author, may be found in the original bound copy of this work, retained in the Simon Fraser University Archive. Simon Fraser University Library Burnaby, BC, Canada ABSTRACT A +I frameshift insertion has been documented in the mitochondriai gene nad3 in some birds and reptiles. By sequencing the polyadenylated mRNA in the chicken (Gallus gallus), we have shown that the extra nucleotide is transcribed and is present in the mature mRNA. Evidence from other animal mitochondrial genomes has led us to hypothesize that certain mitochondria1 translation systems have the ability to tolerate frameshift insertions using programmed translationai frameshifting. To investigate this, we sequenced the mitochondrial genome of the red-eared slider turtle (Trachemys scripta), where both the common nad3 frameshift insertion and a novel site in nad4 were found. Sequencing the region surrounding the insertion in nad3 in a number of other turtles and tortoises revealed general mitochondrial +1 programmed frameshift site features as weil as the apparent redefinition of a stop codon in Parker's sideneck turtle (Macrochelodina parkeri), the first known example of this in vertebrate mitochondria. Keywords: programmed translational frameshifting, mitochondrial genetic code, Testudines, recoding, codon redefinition ACKNOWLEDGEMENTS I would like to thank everyone who has helped me to get to this point - if you are not listed below, I apologize, and I can only hope that you know I appreciate all you have done for me. First, I would like to thank my senior supervisor, Dr. Andy Beckenbach, for providing me with this opportunity, for helping develop what turned out to be a very interesting project, and for supporting me without concession throughout my research. My supervisory committee of Dr. Dave Baillie and Dr. Peter Unrau also deserve much credit for their guidance and valuable insights into the project. A very special thanks is due to Dr. Jim Stewart and Andrea Scouras, who not only welcomed me warmly into the lab, but also displayed an inexhaustible supply of patience in teaching me everything I needed to know. I would also like to thank my other labmates over the course of my studies, Mike Vankoeveringe and Jeremy Saunier, for their help and advice along the way. I owe much gratitude toward Eric Holt at the Empire of the Turtle and Paul Springate at the Rainforest Reptile Refuge for providing invaluable samples, without which this project would have never got off the ground. To my family and friends I would like to express my sincere thanks and appreciation for their unwavering support, especially to my parents, who are, in a word, amazing. Finally, to Poet, thank you, for everything. TABLE OF CONTENTS .. Approval ........................................................................................................................... 11 ... Abstract ....................................................................................................................... 111 Acknowledgements ............................................................................................................ iv Table of Contents ................................................................................................................. v ... List of Figures .............................................................................................................. vlll List of Tables ................................................................................................................ ix Introduction ........................................................................................................................1 Recoding .............................................................................................................................. 1 Normal translation ........................................................................................................ 1 Non-standard translation events ................................................................................... 2 Programmed translational frameshifting .............................................................................. 3 Insertion and deletion mutations and pseudogenes ...................................................... 3 Use of programmed translational frameshifting ........................................................... 4 General programmed translational frameshift characteristics ...................................... 5 Brief overview of relevant programmed translational frameshift examples ................6 . - 1 Frameshlfting ........................................................................................................... -6 . +1 Frameshlftlng ........................................................................................................... 8 Three major +1 programmed translational frameshift examples ................................ 10 Frameshifting in animal mitochondria1 genomes .............................................................. 16 Discovery of a frameshift mutation in the nad3 gene of birds and turtle ................... I6 + 1 Frameshift in a genus of ants and the eastern oyster ............................................. I8 Mitochondria1 genome as a model system for recoding of translation .............................. 19 Purpose of this study .......................................................................................................... 22 A note on the challenges of recoding research ...........................................................24 Materials and Methods ....................................................................................................25 nad3 DNA and mRNA in chickens ...................................................................................25 Chicken RNA and DNA samples ............................................................................... 25 Crude mitochondria isolated from fresh chicken liver ...............................................25 DNA extraction ...........................................................................................................26 RNA extraction ...........................................................................................................26 Reverse-transcription PCR ......................................................................................... 28 Control reactions to test RNA samples for DNA contamination ...............................28 DNA sequencing......................................................................................................... 29 Complete genome sequencing ..........................................................................................
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