View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by University of Liverpool Repository Comparative genomics approaches to study species divergence in ageing Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Master in Philosophy by Yang Li September 2010 ABSTRACT Life, ageing and death have been concepts known to man since time immemorial. To this day, human beings have been fascinated by death and focused on stipulating what occurs after the life ends. In comparison, little attention has been given to ageing, which all experience daily and many take for granted. It is only the recent scientific movement that enabled mankind to shift its attention to studying ageing through the field of biology and gerontology. The observation that different species show different ageing phenotypes has intrigued many biologists. Comparative biologists, who study genetic, anatomy and behaviour of different species in order to understand the diversity of life, have been trying to explain the different ageing phenotypes through their divergence in their anatomy, natural habitat and behaviour. And since the expansion of the field of genetics, comparative biologists have been employing genomics to study differences in ageing phenotypes at the genome level. In the last few years, the number of species with their genome sequenced has increased at an impressive rate. However, the number of studies exploiting this wealth of data to study specific phenotypes has remained surprisingly small. Here, we present our work on comparative genomics to study species differences in i ageing, that is, why many species age at different rates. We describe three projects which helped us 1) find a correlation between amino acid usage in mitochondrial proteins and maximal lifespan, 2) detect patterns of selection associated with longevity increases in proteins during mammalian evolution, and 3) compare the genes expression level in two closely related mammalian organisms, the naked mole-rat and the wild-type mouse. These projects led to several insights about ageing in mammals. We argue that the lack of detection of proteins with anti-oxidant properties in our analyses, coupled with a similar absence observed in other mammalian studies, suggest that contrary to some lower organisms, reactive oxygen species (ROS) may have a lesser impact on ageing in mammals. However, we found evidence that mammalian species may regulate ROS levels through the optimisation of pathways involved, for instance, in the actin cytoskeleton and lipid peroxidation, as well as through the optimisation of amino acid usage in mitochondrial proteins. Additionally, we discovered that genes involved in two pathways, namely lipid metabolism genes and proteasome-related genes, were associated to ageing in both the protein evolution project and the genes expression analysis, suggesting that these two pathways may be important regulators of mammalian ageing. We also discovered a few interesting genes. For instance, the DNA damage-binding protein 1, DBB1, which has a strong selection pattern related to longevity evolution in mammals. Additionally, we found that alpha-2-macroblobulin, A2M, is over-expressed at more ii than few hundreds fold in the long-lived naked mole-rat and has previously been associated to ageing. All in all, we present work that is interesting not only because of the original approaches taken to study mammalian ageing, but also because of the significance of the biological implications obtained. We hope to convince the readers that some of the discoveries made are good candidates for further studies by giving ideas of possible follow up experiments. iii ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor, Dr. João Pedro de Magalhães, who has been of invaluable help. Indeed, Pedro has not only been of great help in the several research projects that I have been working on, but he gave me many sound advices about how to become a good researcher of international calibre. Pedro has been extremely generous with his time and I praise him for that. He has let me grow and learn at my own pace by trusting me and making research feel like a hobby rather than a chore. I look forward to continue working with him in hope that one day, our research will contribute to the invention of interventions that can slow or even halt human ageing. Next, I would like to thank my parents, who have been supportive, from the very beginning, of my switch from mathematics and theoretical computer science to the much more applied field of comparative genomics. Switching out from a field in which I was comfortable was a difficult and risky choice, and having to move to Liverpool from Montreal for the switch certainly did not help. However, no one rebuked me for my decisions (perhaps not to crush a young man's dream) and I decided to follow my heart. I'd like to thank Dr. Mathieu Blanchette who has been a great mentor ever since I have met him. His passion in all questions related to biology and computer science is extremely contagious. In every meeting I have had with him, he showed iv interested in what I was doing, helped me think through ideas that I had and gave constructive criticisms. Mathieu is a remarkable researcher and teacher, and I believe myself to be incredibly lucky to have met him. I'd also like to thank Dr. Pietro Lió, who though I have only met briefly, acted like an excellent role model and gave me useful advices on being a young researcher. Last but not least, I would like to thank my friends which have been both motivating and supportive all through my studies. I have yet to meet a group of friends more motivating than the ones I have met at McGill. It was an incredible time, guys. I would like to thank the lab members at the University of Liverpool which made the office life much more bearable and sometimes enjoyable as well as the few friends that I have met in Liverpool. I thank Dr. Chuanfei Yu to have been both a friend and a good colleague to work with in the Church lab where I have learnt a lot about cutting-edge research in biotechnology research. Thanks to Yeting (no, I have not forgotten). Thanks to Andrew for reading my thesis and complaining about my “incorrect” American way of spelling words. Thanks to Louise for reading over my thesis. And lastly, Sipko for the entertainment and for his hospitality. v CONTENTS INTRODUCTION......................................................................................................1 Chapter 1: Biology of ageing.................................................................................3 1.1 Free radical theory of ageing.......................................................................3 1.2 Damage-linked theories of ageing...............................................................5 1.3 Evolutionary theories of ageing...................................................................6 1.4 Hypotheses on divergence in species ageing...............................................7 Chapter 2: Comparative Biology............................................................................9 2.1 Comparative biology and definition............................................................9 2.2 Selection of model organisms in ageing studies........................................10 2.3 Species divergence studies in ageing and the naked mole-rats.................13 2.4 Importance of the mitochondria in ageing.................................................16 2.5 Correlation of different traits with maximal longevity and phylogenetic dependence......................................................................................................17 Chapter 3: Computational biology.......................................................................18 3.1 Computational biology is an emerging field.............................................18 3.2 The multiple subfields of computational biology .....................................20 3.3 Comparative genomics and how it can help research in ageing................22 3.4 Genes expression profiling........................................................................24 Chapter 4: Principles of comparative genomics ..................................................26 4.1 Substitution matrices.................................................................................26 4.2 Sequence alignment tools..........................................................................27 4.3 Ancestral genome reconstruction...............................................................30 4.4 Alignments of short reads..........................................................................32 4.5 Phylogenetic independence contrasts and other corrections.....................34 AIMS.........................................................................................................................35 METHODS AND RESULTS....................................................................................36 Chapter 5: Residue frequencies in mammalian mitochondrial proteins..............37 Chapter 6: Accelerated protein evolution analysis reveals genes and pathways associated with the evolution of mammalian longevity.......................................40 6.1 Detecting longevity specific selection in proteins.....................................42 6.2 Normalisation of evolutionary scores........................................................44 6.3 Selectivity criteria and longevity selectivity