Making the most of phylogeny - OPUS - Universität Würzburg

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Bioinformatical methods Part II. Material and Methods 2.1.4. InParanoid clustering After all-against-all pairwise sequence comparisons between the EST sets of M. tardigradum and H. dujardini using TBLASTX, the results of this comparison were imported into InParanoid (Remm et al. 2001) for prediction of orthologs. As the EST sets cover a substantial fraction of the encoded proteins but do not yet represent the whole protein set of the compared tardigrades, we called the orthologs given by InParanoid shared candidate orthologous sequences (SCOSs) and the remaining sequences candidate single sequence ESTs. 2.1.5. Identification of regulatory elements The ESTs of H. dujardini and M. tardigradum were systematically screened using the software UTRscan (Grillo et al. 2010). This software screens 30 regulatory elements for RNA regulation with a focus on the 3 ′ untranslated region (UTR) elements and stability of messenger ribonucleic acid (mRNA). The default settings for batch mode were used and all reported elements were collected. 2.1.6. ITS2 work-flow description The ITS2 work-flow is described by Schultz and Wolf (2009) and consists of the following steps: HMM annotation, secondary-structure prediction, homology modelling, sequence-structure alignments, analysis of CBCs and finally the distinction of species. HMM annotation of the ITS2 The HMM annotation was performed according to Keller et al. (2009). I used the HMMs for metazoan 5.8S and 28S. The suggested e-value threshold of 0.001 was used. Secondary-structure prediction For the secondary-structure prediction I calculated the secondary structure for all tardigrade ITS2s using the software RNAstructure 3.46 (Mathews et al. 2004). RNAstructure implements a free energy minimisation algorithm and therefore calculate the secondary structure with the lowest free energy. The structure of P. ‘richtersi group 2’ was chosen as template for the homology modelling. Homology modelling The homology modelling (Wolf et al. 2005) was used to transfer the structure of P. ‘richtersi group 2’ to all other tardigrade ITS2s. It is based on semi-global alignments with the software needle and was completely rewritten by myself during our redesign of the ITS2 database. If not stated otherwise in the 18
Part II. Material and Methods Bioinformatical methods publication a threshold of 75 % was used for the helix transfer. For Schill et al. (2010) we used 66 %. Sequence-structure alignments All obtained sequence-structure pairs were multiple aligned with 4SALE (Seibel et al. 2006). The algorithm is based on the translation of sequence-structure pairs into pseudoproteins following the multiple alignment of the pseudoproteins with ClustalW (Thompson et al. 1994) and the retranslation into sequence-structure pairs. Tree reconstruction and CBC-analysis The CBC analysis was done with 4SALE using the obtained multiple sequence-structure alignment as input. Therefore, all possible sequence-structure pairs were scanned against each other for CBCs. The resulting CBC matrix was used to distinguish between species on appearance of a CBC with an accuracy of 93 % (Müller et al. 2007). Also a CBC tree was reconstructed with CBCAnalyzer (Wolf et al. 2005). Additionally, I reconstructed a neighbour-joining tree with ProfDistS (Wolf et al. 2008). 2.1.7. ITS2 database generation The ITS2 database was completely redesigned and new features were added to the pipeline process, e.g. HMM annotation. Therefore, it was necessary to create new generation scripts which also allow automated updates at regular intervals. The pipeline consists of the following steps: Retrieval of all NCBI sequences which match to a search string which is specific for the ITS2, a rescan of Genbank using the HMM annotation and a merge of all obtained sequences. Afterwards, the sequences which are annotated with a start and end are folded by UNAfold (Markham and Zuker 2005, 2008). The following iterative step tries to obtain homology modelled structures. All sequence-structure pair which were generated by the last step or the last iteration are used as input. The loop iterates as long as new sequence-structure pairs can be obtained. The last step searches through the remaining sequences using BLASTN, followed by a homology modelling step to get additional partial sequence-structure pairs. All sequence-structure pairs are stored in a Postgres database in our department and are publicly available via the web interface of the ITS2 database. 2.1.8. Simulation of ITS2 evolution We simulated 600, 000 sequence-structure pairs using SISSI v0.98 (Gesell and Haeseler 2006). The secondary structures were included in the simulation process of coevolution by application of two separate substitution models. Therefore it was necessary to estimate substitution models for the non-paired and the paired 19
Page 1: Making the most of phylogeny: Uniqu
Page 4 and 5: Eingereicht am: . . . . . . . . . .
Page 7 and 8: Contents Acronyms xi I. Introductio
Page 9: List of Figures Contents List of Fi
Page 12 and 13: Acronyms Acronyms xii ITS2 internal
Page 15 and 16: Part I. Introduction Thesis outline
Page 17 and 18: Part I. Introduction The phylum Tar
Page 19 and 20: Part I. Introduction Internal trans
Page 21 and 22: Part I. Introduction Internal trans
Page 23: Part II. Material and Methods 11
Page 27 and 28: Chapter 2. Methods 2.1. Bioinformat
Page 29: Part II. Material and Methods Bioin
Page 33: Part III. Results 21
Page 36 and 37: BMC Genomics BioMed Central Researc
Page 38 and 39: BMC Genomics 2009, 10:469 http://ww
Page 47 and 48: Chapter 4. Transcriptome survey of
Page 49 and 50: Mali et al. BMC Genomics 2010, 11:1
Page 59 and 60: Chapter 5. Transcriptome analyzed a
Page 61 and 62: Abstract Tardigrades are unique met
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We used primer3 90 for the design o
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cathode buffer (25 mM Tris-HCl pH 9
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24. Ostareck-Lederer, A., Ostareck,
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59. Gaudermann, P., Vogl, I., Zient
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91. Bork, P. & Gibson, T. J. (1996)
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Fig. 2. PCR validation of heat shoc
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Fig. 7. Comparison of COGs/KOGs spe
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Table IIa. Subset of important COGs
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KOG0660:[T]Mitogen-activated protei
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Table III. Enzymes in metabolic pat
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ec:5.3.3.2 isopentenyl-diphosphate
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Table IVa. Specific stress pathway
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Chapter 6. Proteomic analysis of ta
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Figure 1. SEM images of M. tardigra
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listed in Table 2. The individual i
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Table 1. Overview of identified pro
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Table 2. Cont. Sequence coverage No
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Table 3. Identified proteins withou
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Table 3. Cont. Spot no. Accession n
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Table 3. Cont. Spot no. Accession n
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Figure 8. Detection of hsp60 and hs
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Unique proteins, when analyzed on t
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(version 56.1, September 2008, The
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Chapter 7. Stress response in tardi
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Due to the remarkable ability of ta
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Fig. 1 Alignment of two α-crystall
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active state. This leads to the ass
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Pfaffl MW, Horgan GW, Dempfle L (20
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Tardigrade bioinformatics: Molecula
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searched against the next more exte
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Concerted changes in tardigrade ada
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mit dynamischen Modellierungstechni
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Tables and Figures Table 1: Bioinfo
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Figure 1: Maximum likelihood tree f
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Chapter 9. The ITS2 Database III—
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D276 Nucleic Acids Research, 2010,
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D278 Nucleic Acids Research, 2010,
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Chapter 10. Including RNA Secondary
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Keller et al. Biology Direct 2010,
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BMC Evolutionary Biology Research a
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BMC Evolutionary Biology 2008, 8:21
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Chapter 12. Distinguishing species
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Abstract Distinguishing species in
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Distinguishing species in Paramacro
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ITS 2 gene amplification and sequen
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References Distinguishing species i
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Figure legends Distinguishing speci
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221
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Part IV. General Discussion and Con
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Summary succeeded to show the benef
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Zusammenfassung der internal transc
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Bibliography Bibliography Bavan, S.
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Bibliography Bibliography 146.4 (Ap
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Bibliography Bibliography Horikawa,
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Bibliography Bibliography Letunic,
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Bibliography Bibliography Pertea, G
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Bibliography Bibliography Schultz,
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Bibliography Bibliography Westh, P.
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Contributions Markus Grohme wrote t
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Contributions model and the ITS2 se
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List of Publications Publications a
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List of Publications Förster, F.,
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