A sequence alignment (supplemental online material: Figure 1) was performed using PROMALS3D software which considers structural constraints for the divergent AQPs protein sequences [37].
From the alignment (available online at http://www.interscience.wiley.com/jpages/0265-9247/suppmat/2003/25/v25.1106.html), is is clear that the carboxyl-terminal half is more conserved than the amino-terminal part.
In brief, the automatic selection of parameters approximate optimal cutoffs by plotting, internally, the cumulative graphs of gap and similarity scores of the columns in the alignment (see online documentation).
However, multiple sequence alignments (see online supplementary appendix figure S4) clearly show that all detected peptide sequence results were uniquely specific to trypsins that is, no primary sequence identified was common to tryptases.
Both cysteine domains were aligned using the European Bioinformatics Institute ClustalW online alignment tool (http://www.ebi.ac.uk/Tools/msa/clustalw2/), and the TEXshade alignment shading software (Beitz, 2000) was used to create the graph.
PCR products were sequenced by de-deoxy sequencing and sequences were aligned using Multi-Align [37], a multiple sequence alignment software available online (http://bioinfo.genotoul.fr/multalin/multalin.html).html
The newly obtained sequences were deposited in the European Molecular Biology Laboratory nucleotide sequence database (accession numbers FR669194-FR669243 and FR675968-FR675974), aligned using the online Silva alignment tool (Pruesse et al. 2007), and merged with the ARB database using ARB software package version 5.1 (Ludwig et al. 2004).
Despite downloading pre-aligned LSU structural alignments from the online SILVA database, the LSU dataset was poorly aligned and it was difficult to infer homology amongst variable regions.
The amplified partial 18S rRNA gene was sequenced and compared with similar information available at the GenBank by an online alignment search.
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