Making the most of phylogeny: Unique adaptations in tardigrades and 216374 internal transcribed spacer 2 structures Universität Würzburg Kumulative Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg Making the most of phylogeny: Unique adaptations in tardigrades and 216374 internal transcribed spacer 2 structures vorgelegt von Frank Förster aus Hohenmölsen Würzburg, 2010 Eingereicht am: . Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Thomas Dandekar 1. Gutachter: Prof. Dr. Thomas Dandekar 2. Gutachter: Prof. Dr. Markus Engstler Tag des Promotionskolloquiums: . Doktorurkunde ausgehändigt am: . Acknowledgements I would like to express my gratitude to my academic advisor Prof. Dr. Thomas Dandekar for his guidance and constant support in helping me to conduct and complete this work. In addition, I want to thank Prof. Dr. Markus Engstler for serving on my advisory committee. I am thankful to the BMBF project FUNCRYPTA (FKZ 0313838B) for funding. Thanks to my collaborators at the University of Stuttgart, the German Cancer Research Center in Heidelberg, the University of Applied Sciences in Wildau and the company Oncoscience in Wedel who have contributed experimental work, advice, fruitful discussions, nice meetings and a wonderful 11th International Symposium on Tardigrada 2009 in Tübingen. I am also grateful to the journal ‘Cell Stress and Chaperones’ for the permission to print my publication ‘Stress response in tardigrades: differential gene expression of molecular chaperones’ in this thesis. Many thanks to all the people I have met in the Department of Bioinformatics at the University of Würzburg. Special thanks to all of my roommates Dr. Roland Schwarz, Dr. Dr. Marcus Diettrich, Dr. Armin Robubi, Nicole Philippi, Michael Seidl, Gaby Wangorsch and Felix Bemm for their help and their exercised patience. I must not forget to thank our coffee machine, which always provided me with the fuel I needed ;). Thanks to Sabine and Alexander Keller for proof-reading this thesis. A big thank you goes to Prof. Dr. Jörg Schultz, Dr. Matthias Wolf, Dr. Tobias Müller, Gaby Wangorsch, and Christian Koetschan for the helpful comments during the creation of this thesis. I owe everything to my family who has always supported and encouraged me over the years. I especially want to thank Sabine for her inspiration, continuous encouragement and patience (“I will leave the office in 5 minutes”) during my studies. Finally, I want to express my deepest appreciation to my beloved parents for their love, affection, and unlimited support during my life and studies. v Contents Acronyms xi I. Introduction1 II. Material and Methods 11 1. Material 13 1.1. Hardware requirements............................ 13 1.2. Databases.................................... 13 2. Methods 15 2.1. Bioinformatical methods............................ 15 2.1.1. Sequence alignment.............................. 15 2.1.2. Expressed sequence tags............................ 15 2.1.3. CLANS clustering............................... 16 2.1.4.InParanoid clustering........................... 18 2.1.5. Identification of regulatory elements.................... 18 2.1.6. ITS2 work-flow description.......................... 18 2.1.7. ITS2 database generation........................... 19 2.1.8. Simulation of ITS2 evolution......................... 19 2.1.9. Reconstruction of LEA and DnaJ protein trees............... 20 2.2. Biochemical methods............................. 20 III. Results 21 3. Tardigrade workbench: comparing stress-related proteins, sequence-similar and functional protein clusters as well as RNA elements in tardigrades 23 4. Transcriptome survey of the anhydrobiotic tardigrade Milnesium tardi- gradum in comparison with Hypsibius dujardini and Richtersius coronifer 35 vii Contents Contents 5. Transcriptome analyzed at different levels in Hypsibius dujardini and Milnesium tardigradum: Specific adaptations, motifs and clusters as well as general protective pathways 47 6. Proteomic analysis of tardigrades: towards a better understanding of molecular mechanisms by anhydrobiotic organisms 99 7. Stress response in tardigrades: differential gene expression of molecular chaperones 137 8. Tardigrade bioinformatics: Molecular adapations, DNA j family and dy- namical modelling 147 9. The ITS2 Database III—sequences and structures for phylogeny 161 10. Including RNA Secondary Structures improves Accuracy and Robustness in Reconstruction of Phylogenetic Trees 167 11. ITS2 data corroborate a monophyletic chlorophycean DO-group (Sphaero- pleales) 181 12. Distinguishing species in Paramacrobiotus (Tardigrada) via compensatory base change analysis of internal transcribed spacer 2 secondary struc- tures, with the description of three new species 195 IV. General Discussion and Conclusion 223 Summary 233 Zusammenfassung 235 Bibliography 237 Contributions 251 Curriculum Vitae 255 List of Publications 257 Erklärung 261 viii List of Figures Contents List of Figures 1. First drawing of a ‘waterbear’....................... 4 2. Ranges for often used phylogenetic markers.............. 8 3. Organisation of a typical eukaryotic rRNA cistron........... 8 4. Secondary structure of the ITS2 of the Dahlia brevis .......... 9 5. Annotation pipeline............................ 17 ix Acronyms 15-LOX-DICE 15-lipoxygenase differentiation control element AQP Aquaporin BLAST basic local alignment search tool CBC compensatory base change cDNA complementary desoxyribonucleic acid CLANS cluster analysis of sequences COG cluster of orthologous group COI Cytochrome c oxidase subunit I CPU central processing unit DBMS database management system DDBJ DNA Data Bank of Japan DNA desoxyribonucleic acid EMBL European Molecular Biology Laboratory EST expressed sequence tag ETS external transcribed spacer FABP fatty acid binding protein gDNA genomic desoxyribonucleic acid GO Gene Ontology GST Gluthathione S-transferase GTR generalised time reversible HMM hidden markov model HPC high performance computing HSP heat shock protein ITS internal transcribed spacer xi Acronyms Acronyms ITS2 internal transcribed spacer 2 LEA late embryogenesis abundant LSU large subunit mRNA messenger ribonucleic acid mtDNA non-coding regions of animal mitochondrial DNA NAS network attached storage NCBI National Center for Biotechnology Information NRdb non-redundant protein database NTS non transcribed spacer ORF open reading frame PCR polymerase chain reaction PFAM Protein Families rbcL the large subunit of the chloroplast gene encoding ribulose bisphosphate carboxylase rDNA ribosomal desoxyribonucleic acid RNA ribonucleic acid rRNA ribosomal ribonucleic acid SCOS shared candidate orthologous sequence SMART Simple Modular Architecture Research Tool SOD Superoxide dismutase SSU small subunit TSP tardigrade-specific protein UniProt Universal Protein Resource UTR untranslated region xii Part I. Introduction 1 Part I. Introduction Thesis outline Thesis outline The members of the phylum Tardigrada are outstanding animals with the capability to resist environmental stress like low and high temperatures or pressure, the lack of water and radiation. In this thesis I describe bioinformatical approaches to investigate such capabilities and their impact on tardigrade physiology. Further the taxonomic relationship between several tardigrade species are of interest in this thesis which are currently unknown. Thus the second part of the thesis gives attention to phylogenetic methods used to distinguish species and concerning their systematics. The first part of the thesis is an introduction into the phylum Tardigrada and the internal transcribed spacer 2 (ITS2) as a phylogenetic marker. The second part describes the methods I used, which cover bioinformatical meth- ods such as sequence alignment, database searches as well as database generation and laboratory work. The third part, as the main part and results section of this thesis, is composed of the scientific articles as individual chapters we published or submitted to several journals: Chapter 3 (pages 23–34) describes the creation of a web-based workbench for our Funcrypta project. It covers the presentation of the sequencing results and their annotation and additionally offers some tools like motif search or BLAST in our sequence database. The next article (chapter 4 (pages 34–47)) gives an overview about the still ongoing nucleotide sequencing project for Milnesium tardigradum. I was involved in the cleaning, assembly and annotation of the sequences and contributed to further steps of data analysis. The third publication (chapter 5 (pages 47–98)) covers the survey and translation of the complete available transcriptome sequence information from different tardigrades. We compare this information to other organisms including Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens. The chapter 6 (pages 98–137) shows the first results of the proteomic approach of the Funcrypta-Project for which I prepared the hypothetical pro- teins to allow the identification of the picked protein spots. The next publication (chapter 7 (pages 137–146)) shows an investigation of several heat shock proteins (HSPs) in M. tardigradum. Chapter 8 (pages 146–160) shows several examples and techniques of bioinformatical analyses applied to the available tardigrade data, e.g. the identification of ribonucleic acid (RNA) stability motifs and clustering of protein families. We showed the diversity of DNA-j like proteins which maybe a reason for the