The Phylogenetic Analysis of Cytochrome C to Gain an Insight into Mitochondrial Evolution and Endosymbiotic Gene Transfer By Rhys M. Thomas Word Count-20,614 Abstract Word Count-377 Submitted in partial fulfilment of the requirement for the degree of Master of Science in Molecular Biology School of Life Sciences and Education Staffordshire University Submitted August 2020 Student Number-18022710 Contents Table of Figures ............................................................................................................................. 4 Abstract ........................................................................................................................................ 5 1.0 Introduction ............................................................................................................................ 6 1.1 The Mitochondria ................................................................................................................ 6 1.1.1 Mitochondrial Function................................................................................................. 8 1.1.2 Mitochondrial Origins ................................................................................................. 10 1.1.3 Mitochondrial Genome ............................................................................................... 14 1.1.4 Mitochondrial Gene Transfer ...................................................................................... 18 1.2 Cytochrome C .................................................................................................................... 22 1.2.1 Cytochrome C Structure .............................................................................................. 22 1.2.2 Eukaryotic Function .................................................................................................... 23 1.2.3 Prokaryotic Function ................................................................................................... 26 1.2.4 Cytochrome C Similarity .............................................................................................. 27 1.2.5 Cytochrome C’s Molecular Clock ................................................................................. 28 1.3 Phylogenetics .................................................................................................................... 29 1.3.1 Cytochrome C in Phylogenetics ................................................................................... 30 1.3.2 How Phylogenetic Trees Work .................................................................................... 31 1.3.3 Maximum Likelihood Phylogenetic Trees .................................................................... 32 1.4 Projects Aims, Objectives and Hypotheses ......................................................................... 33 2.0 Methods and Materials ......................................................................................................... 35 2.1 How the Sequences Were Chosen ..................................................................................... 35 2.2 Databases Utilised ............................................................................................................. 37 2.3 Software and Tools Utilised ............................................................................................... 37 2.4 Statistical Analysis ............................................................................................................. 39 3.0 Results .................................................................................................................................. 40 3.1 Cytochrome C Structure and Function ............................................................................... 42 3.2 Cytochrome C Sequence Alignments ................................................................................. 44 3.3 Cytochrome C Sequence Variation ..................................................................................... 46 3.4 Mitochondrial and Nuclear Cytochrome C Sequence Comparison ...................................... 48 3.5 Cytochrome C Phylogenetic Tree ....................................................................................... 50 3.6 Cytochrome C Syntenic Analysis ........................................................................................ 52 3.7 Estimated Time of Divergence ........................................................................................... 55 4.0 Discussion ............................................................................................................................. 56 4.1 Origins of the Mitochondria ............................................................................................... 57 4.2 Cytochrome C Structure and Function ............................................................................... 59 4.3 Cytochrome C Sequence Variations ................................................................................... 62 2 Student Number-18022710 4.4 Mitochondrial and Nuclear Cytochrome C Variations ......................................................... 64 4.5 Cytochrome C Phylogenetic Tree ....................................................................................... 68 4.6 Cytochrome C Syntenic Analysis ........................................................................................ 72 4.7 Limitations of the Study ..................................................................................................... 76 4.8 Further Work ..................................................................................................................... 77 5.0 Conclusion............................................................................................................................. 79 References .................................................................................................................................. 80 Acknowledgements ..................................................................................................................... 94 Appendix 1 – Project Declaration Form ....................................................................................... 95 Appendix 2 – Raw Entropy Data .................................................................................................. 96 Appendix 3 – Prokaryotic and Eukaryotic Maximum Likelihood Tree ......................................... 102 Appendix 4 – Abbreviation List .................................................................................................. 103 3 Student Number-18022710 Table of Figures Figure 1. Diagrammatic Mitochondria.. ......................................................................................... 6 Figure 2. A Tree of Life.. .............................................................................................................. 12 Figure 3. Cytochrome C Variation in 3D Structure.. ...................................................................... 43 Figure 4. MUSCLE Sequence Alignment of Eukaryotic and Prokaryotic Cytochrome C.. ................ 45 Figure 5. Cytochrome C Eukaryotic and Prokaryotic Entropy Plot.. ............................................... 47 Figure 6. Comparison of D. variabilis Cytochrome C under Different Genetic Codes..................... 49 Figure 7. Evolutionary analysis of Cytochrome C by Maximum Likelihood method....................... 51 Figure 8. Syntenic Analysis of Various Species Cytochrome C Genomic Regions.. ......................... 54 Figure 9. Electron Microscope of Parakaryon myojinensis.. ......................................................... 58 4 Student Number-18022710 Abstract 4.6 billion years ago life began at submarine volcanic vents referred to as ‘black smokers’ common during this period. These supplied thermal energy and a constant supplied of inorganic molecules separated by a pH gradient similar to what seen in mitochondria. However, it wasn’t until the ‘great oxidation event’ approximately 2.4 million years ago, the environment began to oxidise in cycles powered by cyanobacteria’s oxygen production. This would lead to an eventual endosymbiotic event. In which the uptake of a α-proteobacterium by an archaeon predating 1.45 billion years ago, dated via the earliest eukaryotic microfossils. Then came the ‘Cambrian explosion’ 540 million years ago which gave rise to the huge diversity witnessed today. However, since the endosymbiotic event almost 98% of genes that are required for mitochondrial function have undergone endosymbiotic gene transfer to the nuclear genome. Using cytochrome c as an example due to it being nearly entirely encoded on the nuclear genome, an essential protein with a small conserved size making it a prime example to be utilised in phylogenetic analysis. The method used bioinformatics software to create a maximum likelihood tree to infer evolution relationships and online resources to identify syntenic regions around the cytochrome c gene to infer a common endosymbiotic gene transfer event. Including times of divergence between species inferring a possible time of endosymbiotic gene transfer. The results biased in Chordata data due to the availably of the genetic information accessible via online databases, indicate that the endosymbiotic event occurred prior to the divergence of mammals and reptiles, 312 million years ago. Inferred by the use of TimeTree to estimate the time of divergence of two given species that CoGe was