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Chapter IV Submitted manuscript 67 Fig. 1 Characterization of the Not sequence of Terebratalia transversa. (A) Not homeodomain sequence alignment. The accession numbers for the EMBL/GenBank databases are given in brackets: Terebratalia transversa (Ttr, brachiopod, XXXXXXX), Nematostella vectensis (Nve, cnidarian, XP_001641364.1), Hydra vulgaris (Hvu, cnidarian, CAB88387.1), Trichoplax adhaerens (Tad, placozoan, AAQ82694.1), Drosophila melanogaster (Dme, fruit fly, NP_650701.1), Strongylocentrotus purpuratus (Spu, sea urchin, AAD20328.1), Hemicentrotus pulcherrimus (Hpu, sea urchin, BAD91047.1), Branchiostoma floridae (Bfl, Florida lancelet, XP_002601133.1), Danio rerio (Dre, zebrafish, NP_571130.1), Xenopus laevis (X, frog, NP_001081625.1). The following alternative species and Hox protein sequences were chosen as outgroups: Drosophila virilis, Antennapedia (DviAnt, fruit fly, AAQ67266.1), Drosophila melanogaster, Proboscipedia (DmePb, fruit fly, CAA45272), Neanthes virens, Hox7 and Engrailed (NviHox7 and NviEng, annelid, DQ366682 and DQ366680). Dots represent amino acid identity with the amino acid sequence of the T. transversa Not protein shown at the top of the alignment. (B) Alignment tree based on the 52 amino acid sequences shaded in Fig. 1A. Algorithm = UPGMA; Bootstrap = 10.000 replicates. Bootstrap values are given for each node. Due to the small number of residues for the analysis, the phylogenetic signal of the tree is limited. The tree shows, however, that TtrNot clusters with all other Not protein sequences and thus is a true Not protein. α-tubulin antibody. Subsequently, the larvae were washed four times over a period of 12h in PB containing 0.2% Triton X-100 and an Alexa Fluor 633-conjugated goat anti-rabbit as well as an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Invitrogen) in a dilution of 1:400 for 24h at 4ºC. Finally, the larvae were washed tree times for 15 min each in 0.1M PB and embedded in Flouromount G (Southern Biotech, Birmingham, AL, USA) on glass slides. The samples were analyzed with a Leica TCS SP5 II confocal system (Leica Microsystems, Wetzlar, Germany). The resulting image stacks were merged into maximum projection images and assembled using Adobe Photoshop CS3 software (Adobe, San Jose, CA, USA).
68 Submitted manuscript Chapter IV Fig. 2 Characterisation of the Cdx sequence of Terebratalia transversa. (A) Cdx homeodomain sequence alignment. The accession numbers for the EMBL/GenBank databases are given in brackets: Terebratalia transversa (Ttr, brachiopod, XXXXXXX), Nematostella vectensis (Nve, cnidarian, DQ500749), Patella vulgata (Pvu, gastropod, AJ518062.1), Capitella teleta (Cte, annelid, AAZ95508.1), Drosophila melanogaster (Dme, fruit fly, NM_057606.4), Tribolium castaneum (Tca, flour beetle, NM_001039409.1), Ciona intestinalis (Cin, tunicate, NP_001071669), Saccoglossus kowalevskii (Sko, hemichordate, NP_001158415), and Mus musculus (Mmu, mouse, NM_009880.3). The following alternative species and Hox protein sequences were chosen as outgroups: Drosophila simulans, Abdominal B (DsiAbdB, fruit fly, XP_002103136), Drosophila melanogaster, Proboscipedia (DmePb, fruit fly, CAA45272), Drosophila virilis, Antennapedia (DviAnt, fruit fly, AAQ67266.1), Mus musculus, HoxA9 (MmuHoxA9, mouse, NP_034586.1), Neanthes virens, Engrailed (NviEng, annelid, DQ366680). Dots represent amino acid identity with the amino acid sequence of the T. transversa Cdx protein shown at the top of the alignment. (B) Alignment tree based on the 49 amino acid sequences is shaded in Fig. 2A. Algorithm = UPGMA; Bootstrap = 10.000 replicates. Bootstrap values are given for each node. Due to the small number of residues used in the analysis, the phylogenetic signal of the tree is limited. The tree shows, however, that TtrCdx clusters with all other Cdx/caudal protein sequences and thus is a true Cdx protein. RESULTS Characterization of the Terebratalia Not and Cdx genes The PCR-amplified region of the TtrNot homeobox encodes for a 45 amino acids long peptide which is largely similar to the NveNot sequence of the cnidarian Nematostella vectensis (82% sequence identity). This peptide has clear affinities to the HvuNot protein of the cnidarian Hydra vulgaris (69% sequence identity, Fig. 1). The TtrNot segment cloned by RACE-PCR was 667 base pairs long and is deposited in GenBank under the accession number XXXXXX. The transcribed region downstream of the homeobox ends with a poly-A stretch and shows no similarity to other known gene sequences. The conserved protein sequence of the TtrCdx homeobox identified in this study
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Comparative neurogenesis, muscle de
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2 Principal supervisor Assoc. P
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4 Preface This thesis presents
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6 Abstract Brachiopods are a sm
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8 Acknowledgements The endeavou
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10 Introduction Nervous system Micr
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12 Material and methods Material an
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14 Material and methods purpuratus,
Page 17 and 18: 16 Results and discussion Results a
Page 19 and 20: 18 Results and discussion posterior
Page 21 and 22: 20 Results and discussion Myogenesi
Page 23 and 24: 22 Results and discussion Wanninger
Page 25 and 26: 24 Results and discussion Growth pa
Page 27 and 28: 26 Results and discussion argument
Page 29 and 30: 28 References References Abdelkhale
Page 31 and 32: 30 References priapulids and protos
Page 33 and 34: 32 References James, M. A., Ansell,
Page 35 and 36: 34 References regionalization, prol
Page 37 and 38: 36 References 211. Williams, A., Ca
Page 39 and 40: 38 Chapter II Frontiers in Zoology
Page 53 and 54: 52 Chapter III Chapter III Altenbur
Page 55 and 56: 54 Chapter III Altenburger and Wann
Page 63 and 64: 62 Chapter IV Chapter IV Altenburge
Page 65 and 66: 64 Submitted manuscript Chapter IV
Page 67: 66 Submitted manuscript Chapter IV
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