The evolution of mitochondrial genome structure and function in insects

208 Pages·2006·2.73 MB·English

by  Stewart, James Bruce

THE EVOLUTION OF MITOCHONDRIAL GENOME STRUCTURE
AND FUNCTION IN INSECTS
by
James Bruce Stewart
B.Sc.H, University of Waterloo, Waterloo, Ontario, 1997
M.Sc. Simon Fraser University, Burnaby, British Columbia, 2001
THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
in the
Department of Molecular Biology and Biochemistry
O James Bruce Stewart, 2005
SIMON FRASER UNIVERSITY
Summer 2005
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: James Bruce Stewart
Degree: Doctor of Philosophy
Title of thesis:
The Evolution of Mitochondria1 Genome Structure and Function in Insects
Examining Committee:
Chair: Dr. Nicholas Harden, Associate Professor, Department of Molecular Biology and Biochemistry
Dr. Andrew T. Beckenbach, Professor
Senior Supervisor
Department of Biological Sciences
Dr. David L. Baillie, Professor
Supervisory Committee Member
Department of Molecular Biology and Biochemistry
Dr. Peter J. Unrau, Assistant Professor
Supervisory Committee Member
Department of Molecular Biology and Biochemistry
Dr. Esther M. Verheyen, Associate Professor
Public Examiner
Department of Molecular Biology and Biochemstry
Dr. Laurie S. Kaguni, Professor
External Examiner
Department of Biochemistry and Molecular Biology
Michigan State University
Date Approved: August 19,2005
SIMON FRASER UNIVERSITY
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Abstract
The mitochondrial genomes (mt-genomes) of animals are very compact in
structure, encoding thirteen protein genes involved in the production of ATP, and the key
components of the translation system to express these proteins. The mitochondrial
expression system, which functions separately from that of the nucleus, shows
characteristics of both prokaryotic and eukaryotic expression systems, and has diverged
greatly from that currently observed in the closest living relatives of mitochondria, the
a-proteobacteria. Current understanding of transcript maturation is that large multi-gene
transcripts are processed by the removal of intervening tRNA genes, leaving behind RNA
templates to be matured into the functional mRNAs and rRNAs. One of the most striking
features of insect mt-genomes has been the apparent replacement of a start codon with a
stop codon for the essential mitochondrial gene cytochrome c oxidase subunit 1 (coxl).
When first observed in Drosophila, Clary and Wolstenholme proposed a highly unusual
four-base "ATAA" start codon. With the expanded sampling of mitochondrial sequence
across the various insect orders, the data does not support the use of this aberrant
initiation for coxl. At the initiation of this study, the diversity of insect groups
represented by complete mt-genome sequence was very poor. To address this deficit, I
undertook sequencing projects to increase the number of insect orders represented in the
mitochondrial sequence databases. I report the complete mt-genome sequences for two
insects, the spittlebug Philaenus spumarius, and the giant stonefly Pteronarcys princeps.
The sequences are annotated and compared to other insect mt-genomes in the sequence
databases. I report the cDNA sequences of Drosophila melanogaster mitochondrial
mRNAs, rRNA subunits, and a population of pre-mRNA molecules that are intermediates
of the RNA processing system. Models to explain mitochondrial transcript maturation in
light of these new observations are proposed. Comparative analyses were undertaken to
apply the information gained from the mitochondrial transcripts of D. melanogaster to
the mitochondrial structure and annotations of mt-genomes from the other insects. These
analyses suggest a 5' specific modification to the tRNA punctuation model for insect
mitochondria. This modification may represent a further evolutionary simplification of
the mitochondrial expression system.
To Laila, for everything,
And to You who we patiently await.
In memory:
Grandpa Bruce Stewart
December 6,1922 - February 27,200 1
and
Great-Grandpa Ted Baxter
October 20,1897 May 5,1990
-
Acknowledgements
To my senior supervisor Andy Beckenbach, for allowing me the chance to explore this
project and believing that I could get it done. The last few years have been a lot of fun,
and I will miss our lab chats.
A special thanks to my former lab-mate Russ Watkins for keeping in touch after moving
on, and for supplying a key tip for my early RNA struggles. To my lab-mates Andrea
Scouras, Dave Russell, Alison Cloutier, Karen Beckenbach, Denise Reichow, Cam Muir
and Mike Vankoeveringe. Thanks for all the tips, advice and silliness.
Thanks to the RNA-work troubleshooters Alex Ebhardt and Don Sinclair, and to the
computer guys Jeff Bryer and Duncan Napier. To Jillian Smith, Darrell Bessette and
Theresa Kitos for the help with cell culture.
To my committee members Peter Unrau and Dave Baillie for their time, effort and advice
over the course of my degree work. I would also like to thank Mike Smith for his role as
unofficial committee member. Thank-you to Jamie Cates for the very important
discussion of translation at a key time.
To Laurie Kaguni and Esther Verheyen, my external examining committee members, for
the useful, constructive and kind comments regarding my thesis.
To my fellow graduate students in Biology and the MBB for all the fun and for the shop
talk. I especially want to thank John Taylor, Ian Hamilton, Jeremy Mitchell, Tom
Chapman, Lynnette Kuervers, Nigel O'Neil, Greg Vatcher, Steve Springer, Michelle
Harrison, Susanne Kiihnholz, Richard Fahlman, Anat Feldman, Carrie Simms and
Daniela Ginta for important discussions over the course of my study. My personal thanks
to the Biergarten Entrepreneurs, past and present. Where else would the discussions take
place? To Carlo Artieri, Leslie Mitchell and Connie Roth for seminar practice sessions.
To our Vancouver friends; Annick, Kevin and Giselle; Cam; Cathy and Rasmus; David;
Kym, Dana and Tayme; Kat and Greg; Tina and Olie; Jennifer and Stevo; Markus, Lisa
and Anna; Cris and Diana; Anna, Jurgen, Max and Sophie; Nat, Dirk, Steffen and Gabby;
Leah, Omar and Arnira; Melissa. From Ontario, to Pete, Tina, Mason and Rowan; Jilly,
Bomina, Susan and Carole; Scott, Kurt and Karl and Christine for continued support and
friendship.
Thanks to Cathy, Don, Mike, Kelly Joel, Ardis, Fred, and to Norm, Joe, Vicky, Rob,
Tracy, Mary and all the rest of my family, for your patience, support, and well wishes
over the years. Thank-you to Agneta and Zubin for welcoming me.
Table of Contents
..
................................................................................................................................ ....
Approval
..............................................................................................................................1.1. .
Abstract
.........................................................................................................................1.1.1.. .
Dedication iv
................................................................................................................
Acknowledgements v
..................................................................................................................
Table of Contents vi
........................................................................................................................
List of Tables ix
.........................................................................................................................
List of Figures x
..................................................................................................
.
CHAPTER 1 Introduction 1
......................................................................................................
1.1. Mitochondria 1
...................................................................................
1.2. Mitochondria1 Evolution 2
.......................................................................
1.3. Animal Mitochondria1 Genomes 5
1.3.1. Animal mitochondria1 gene content ..................................................... 5
1.3.2. Animal mitochondria1 genome structure. ............................................. 9
1.3.3. Mitochondria1 genome sequencing ....................................................1 0
.............................................
1.4. Mitochondria1 Transcription And Replication 14
1.4.1. Mitochondria1 replication in animals ................................................ 14
1.4.2. Variation in transcriptional systems between eukaryotic
groups .......................................................................................................... 17
1.4.3. Mammalian mitochondria1 transcription ...........................................1 8
1.4.4. Transcript processing in mammalian mitochondria .......................... 19
1.4.5. Transcription in Drosophila mitochondria .......................................2 0
1.4.6. RNA editing and programmed frameshifting in animal
mitochondria .............................................................................................2 3
..................................................................................................
1.5. Thesis Outline 24
.
CHAPTER 2 The Complete Mitochondrial Genome Sequence of the Meadow
Spittlebug Philaenus spumarius (Hemiptera: Auchenorrhyncha:
....................................................................................................................
Cercopoidae) 26
....................................................................................................
2.1. Introduction 26
....................................................................................
2.2. Materials and Methods 28
2.2.1. DNA extraction and PCR amplification ...........................................2. 8
2.2.2. Sequence assembly, annotation and analysis .....................................3 2
.....................................................................................
2.3. Results and Discussion 33
2.3.1 . Genome content .................................................................................3 3
2.3.2. Protein coding genes ......................................................................3..9..
2.3.3. Genomic comparisons. ...................................................................4..3.
......................................................................................................
2.4. Conclusions 44
.
CHAPTER 3 The Complete Mitochondrial Genome Sequence of a Giant Stonefly.
............................................................
Pieronarcysprinceps (Plecoptera; Pteronarcyidae) 47
........................................................................................................
3.1. Introduction 47
......................................................................................
3.2. Materials and Methods 51
3.2.1. Specimen collection and identification. ...............................................5 1
3.2.2. DNA isolation ................................................................................5..1.. ..
3.2.3. PCR and Long-PCR amplification. ...................................................5..2.
3.2.4. Purification of PCR products. ...........................................................5 4
3.2.5. DNA sequencing. ..............................................................................5..6.
3.2.6. Sequence assembly, annotation and analysis ....................................... 56
3.2.7. Sequence comparisons. ........................................................................ 57
.......................................................................................
3.3. Results and Discussion 61
3.3.1 . Genome annotation. ..........................................................................6.1..
3.3.2. Genome architecture. ........................................................................... 65
3.3.3. Nucleotide composition. .................................................................6..5.. .
3.3.4. Stonefly A+T rich regions ...............................................................7..4..
..........................................................................................................
3.4 Conclusions 80
.
CHAPTER 4 Characterization Of Mitochondrial Transcripts In Drosophila
............................................................................................................................
melanogaster 82
....................................................................................................
4.1. Introduction 82
4.1 .1 . The cox1 initiation mystery in insect mitochondria. ............................ 82
4.1.2. Mitochondria1 transcription and RNA processing in Drosophila ........ 84
4.1.3. 5' amplification and sequencing. .......................................................... 86
......................................................................................
4.2. Materials and methods 89
4.2.1. Blast searches for nuclear copies of the cox1 gene in Drosophila. ......8 9
4.2.2. Maintenance of Drosophila fly stocks. ................................................8 9
4.2.3. Maintenence of Drosophila Schneider-2 cell lines ..............................9 0
4.2.4. Mitochondria1 isolation protocols. ....................................................9..1.
4.2.5. RNA extraction protocol .................................................................9..3.. .
4.2.6. Reverse-transcription pcr protocols. ..................................................9.6.
4.2.7. Protocol for 5'-RACE amplification. ................................................1. 00
4.2.8. Protocol for 3'-RACE amplicication. ...............................................1.0. 1
4.2.9. RNA circularization for RT-PCR ....................................................1..0 1
4.2.10. Cloning of amplification products. .................................................. 102
4.2.1 1. Sequencing of cDNAs and sequence alignment. ............................. 104
...............................................................................................................
4.3. Results 104
4.3.1. Search for a nuclear copy of cox1. ...................................................1..0 4
4.3.2. RNA extraction from mitochondria. ................................................1.0. 6
4.3.3. Detection of unprocessed pre-mRNA molecules in flies ..................1 07
4.3.4. Mapping of mRNA and rRNA boundaries by RACE .......................1 07
4.3.5. Detection of RACE amplified RNAs from Schneider-2 cells ........... 111
4.3.6. Characterization of RNAs by CR-RT-PCR ......................................1. 11
vii
.........................................................................................................
4.4. Discussion 117
4.4.1. Schnieder-2 cells versus adult fly data .............................................1. 17
4.4.2. Detection of pre-mRNA molecules. .................................................1. 18
4.4.3. Description of mitochondria1 mRNAs .............................................1..1 9
4.4.4. Intitiation of the cox1 gene ..............................................................1..2 7
4.4.5. Characterization of the ribosomal RNAs ....................................1 29
..............................................................
4.4.6. Polyadenylated pre-mRNAs 130
. .......................................................................
CHAPTER 5 Conclusions And Discussion 136
...........................
5.1. Transcript Characterization in Drosophila Mitochondria 136
5.1.1. Characterization of mt-rRNAs. .......................................................... 136
5.1.2. Characterization of mt-mRNAs .......................................................1..3 8
5.1.3. Processing of tRNAs versus mRNAs in insect mitochondria ............1 41
......................................................................
5.2. Insect Mitochondria1 Genomics 143
5.2.1. Two insect mitochondria1 genomes ................................................... 143
5.2.2. Annotation of mitochondria1 genes ..................................................1..4 3
5.2.3. Genome structure and rearrangements. ............................................1.4 5
.............................................................................................
5.3. Future Directions 148
5.3.1. Modification of the CR-RT-PCR protocol. ....................................... 148
5.3.2. Transcrption of A+T rich region sequence. .......................................1 49
5.3.3. Further characterization of nuclear transcription and processing
factors .......................................................................................................... 151
. ..............................................
APPENDIX A Genbank Flatfile for Philaenus spumarius 155
. ..............................................
APPENDIX B Genbank Flatfile for Pteronarcys princeps 165
....................................................................................................................
REFERENCES 174
...
Vlll
List of Tables
Table 2.1. List of Primer Pairs Utilized in the Amplification of the Mitochondrial
..........................................................................
Genome of Philaenus spumarius 30
Table 2.2. Insect Mitochondrial Genomes Utilized in Alignments and Comparative
Analyses. .............................................................................................................-.3 5
Table 2.3. Nucleotide Composition of Meadow Spittlebug (P. spumarius) Genome
Features. ...........................................................................................................4..0.. .
Table 2.4. Relative Synonymous Codon Usage for Meadow Spittlebug. the Kissing
Bug and the Migratory Locust Mitochondria1 Genomes ......................................4 5
Table 3.1 Primers Used in the Amplification of the Mitochondrial Genome of the
Giant Stonefly Pteronarcys princeps .................................................................... 53
Table 3.2. Complete and Near-Complete Hexapod Mitochondrial Genome Sequences
Utilized in this Study. with their GenBank Accession Numbers .........................5. 9
Table 3.3. Nucleotide Composition of Stonefly Mitochondria] Genome Features. ............... 66
Table 3.4. Comparison of Relative Synonymous Codon Usage for the Giant Stonefly.
a Silverfish and a Locust. .....................................................................................6. 8
Table 3.5. Positional Nucleotide Bias in the Mt-Genome of the Giant Stonefly
and Other Representative Insects. ......................................................................7..3.
Table 4.1. Primers Used in 5' and 3' RACE Amplification .................................................9..7
Table 4.2. Primers Utilized in CR-RT-PCR Reactions. ......................................................9..8.
Table 4.3. Results of tBlastn Searches for a Nuclear cod Gene .......................................... 105
Table 4.4. Positional Annotation of Transcribed RNA Nucleotides as Determined
Through cDNA Sequencing ...........................................................................1.. ..1 5