PhD thesis
PhD thesis
PhD thesis
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Chapter IV<br />
Submitted manuscript<br />
67<br />
Fig. 1 Characterization of the Not sequence of Terebratalia transversa. (A) Not<br />
homeodomain sequence alignment. The accession numbers for the EMBL/GenBank databases<br />
are given in brackets: Terebratalia transversa (Ttr, brachiopod, XXXXXXX), Nematostella<br />
vectensis (Nve, cnidarian, XP_001641364.1), Hydra vulgaris (Hvu, cnidarian, CAB88387.1),<br />
Trichoplax adhaerens (Tad, placozoan, AAQ82694.1), Drosophila melanogaster (Dme, fruit fly,<br />
NP_650701.1), Strongylocentrotus purpuratus (Spu, sea urchin, AAD20328.1), Hemicentrotus<br />
pulcherrimus (Hpu, sea urchin, BAD91047.1), Branchiostoma floridae (Bfl, Florida lancelet,<br />
XP_002601133.1), Danio rerio (Dre, zebrafish, NP_571130.1), Xenopus laevis (X, frog,<br />
NP_001081625.1). The following alternative species and Hox protein sequences were chosen<br />
as outgroups: Drosophila virilis, Antennapedia (DviAnt, fruit fly, AAQ67266.1), Drosophila<br />
melanogaster, Proboscipedia (DmePb, fruit fly, CAA45272), Neanthes virens, Hox7 and<br />
Engrailed (NviHox7 and NviEng, annelid, DQ366682 and DQ366680). Dots represent amino<br />
acid identity with the amino acid sequence of the T. transversa Not protein shown at the top of<br />
the alignment. (B) Alignment tree based on the 52 amino acid sequences shaded in Fig. 1A.<br />
Algorithm = UPGMA; Bootstrap = 10.000 replicates. Bootstrap values are given for each node.<br />
Due to the small number of residues for the analysis, the phylogenetic signal of the tree is<br />
limited. The tree shows, however, that TtrNot clusters with all other Not protein sequences and<br />
thus is a true Not protein.<br />
α-tubulin antibody. Subsequently,<br />
the larvae were washed four times<br />
over a period of 12h in PB containing<br />
0.2% Triton X-100 and an Alexa Fluor<br />
633-conjugated goat anti-rabbit as<br />
well as an Alexa Fluor 488-conjugated<br />
goat anti-mouse secondary antibody<br />
(Invitrogen) in a dilution of 1:400<br />
for 24h at 4ºC. Finally, the larvae<br />
were washed tree times for 15 min<br />
each in 0.1M PB and embedded in<br />
Flouromount G (Southern Biotech,<br />
Birmingham, AL, USA) on glass<br />
slides. The samples were analyzed<br />
with a Leica TCS SP5 II confocal<br />
system (Leica Microsystems, Wetzlar,<br />
Germany). The resulting image stacks<br />
were merged into maximum projection<br />
images and assembled using Adobe<br />
Photoshop CS3 software (Adobe, San<br />
Jose, CA, USA).