Cross-contamination levels were considered in filtering substitutions (see "Minor cross-contamination of DNA samples" section in Materials and Methods).
As an alternative means to filter synonymous substitutions, in some cases we also analyzed the noLRall1 + nt2 data set alone (see Discussion).
These results support our previous findings (e.g. [ 21]) that filtering synonymous substitutions (and thereby compositional heterogeneity) can result in more robust phylogenetic inference at deep levels.
It relates to a set of abatement strategies that can be applied to a chemical exposure problem, ranging from technical solutions to reduce emissions (end of pipe filters) to substitution and non-technical approaches such as the spatial planning of emissions vis a vis vulnerable receptors (e.g. protected ecosystems or drinking water production inlets).
The size of the protein, chemical nature of the residues that form the narrow aromatic/arginine selectivity filter and the substitutions found in the conserved NPA motifs are the common characteristics between the new family and the plant SIP channels.
The small size of the channel protein, the nature of selectivity filter residues and substitutions in the conserved NPA boxes indicate that the new fungal MIP cluster shares some characteristic features with plant SIPs.
Each mutation has been reported only once: a deletion mutation affecting isoleucine 157 (p.Ile157del), which is presumed to induce a conformational change near the selectivity filter; 28 two substitutions, both of which affect the same amino acid (glutamic acid 145), p.Glu145Gln (E145Q) 29 and p.Glu145Lys (E145K); 30 and a p.Trp126Arg (W126R) mutation.
Thus the newly identified fungal MIP group resembles more like the plant SIP subfamily in terms of the molecular weight, nature of selectivity filter residues and substitution in the highly conserved NPA motifs.
The evolutionarily shared gene pairs and the conserved regions between two planarians, D. japonica and S. mediterranea, were searched using the TBLASTX program against S. mediterranea unigenes (Build #4) with the following filter options: BLOSUM62 substitution matrix, sequence length of D. japonica unigene ≧600 bp, 1e-30 threshold and size of conserved region ≧80 bp.
Another principal TTX binding determinant is a DI aromatic phenylalanine or tyrosine residue, located adjacent to the aspartate of the selectivity filter [21], and substitution with a non-aromatic amino acid at this position accounts for TTX-insensitivity in the majority of tetrodotoxic animals [22 26].
