Hence, to facilitate the identification of regions of the model expected to be of high quality (i.e. highly conserved sequence alignment with no or few insertions and deletions), the structurally derived target-template alignment is shown in colors, highlighting regions of conservation between the modeled protein and the structure template (41).
Regions of the P1 domain involved in dimerisation (aa positions 454 463) were found to be conserved in the alignment with VA387, and the variable regions identified in the P2 domain showed consistent variation when VA387 was aligned with samples representing clusters P2a, P2e and P2j (Figure 4).
The result at this stage is a sequence alignment (resulting from the Matt structural alignment) with conserved beta-strand pairs annotated according to the residue positions and conformation (buried or exposed to solvent).
While intron length varies among these five species (Fig. 2), intron position is conserved in the protein alignment, with the first intron found at QYQ|V(D/E E and the second at KLD|PNE (data not shown).
Since mammalian mitochondrial genome sequences are very similar, we designed a set of PCR primers based on highly conserved sequences of an alignment with full-length mitochondrial genomes from the available public data, including those of cow, deer, sheep, pig, and lama (Table 1).
The primer sequences used for the qPCR reactions are given in Table 2. Primers were designed on available Cx. pipiens sequences (partial or complete cDNAs, see GenBank references in Table 2) in conserved gene regions after alignment with several other sequences from closely related species.
We applied tests for signatures of positive selection using the models of codon substitution implemented in PAML (Yang 2007) to a subset of 124 genes (i.e., excluding conserved paralogs and alignments with multiple copies of one gene in one species).
We tested how many potential AuxREs were conserved, namely located in an alignment with a high score and had no substitutions in the TGTC core (See Methods).
For the remaining hits, we searched for the core in the alignment with highly conserved regions of at least the threshold percent identity and 100 bp length by using sliding windows.
The new change in TIMM44 C925A maps to exon 9, and corresponds to a nonconservative missense substitution, P308Q, in a highly conserved position, as indicated by clustal alignment with the orthologue proteins from different species.
The sequence alignment was limited to conserved domains, with ambiguously aligned regions removed using TrimAl (Capella-Gutierrez et al., 2009).
