, 1998). The sequences
were analyzed for the presence of secretion signal sequences using SignalP (ver. 3.0; http://www.cbs.dtu.dk/services/SignalP/) with the hidden Markov model (Bendtsen et al., 2004) and for protein localization using Psort (ver. 1; http://psort.hgc.jp/form.html; Horton et al., 2007). Phylogenetic trees were constructed using mega (ver. 4; Tamura et al., 2007) with the neighbor-joining method and Poisson correction model. A comparison of synonymous and nonsynonymous nucleotide substitution rates is a http://www.selleckchem.com/products/abc294640.html useful approach for studying the mechanisms of DNA sequence evolution. The numbers of nonsynonymous and synonymous substitutions per site (dN and dS, respectively) and their ratio (dN/dS) are important indicators of selection pressure at the protein level; dN/dS values of <1, 1, and >1 imply stabilizing selection, neutral mutations, and diversifying LEE011 cost positive selection, respectively.
To examine positive selection pressure on dnaD, imp, and idpA of PoiBI, we used ClustalW (Thompson et al., 1994) to align the nucleotide sequences of dnaD, imp, and idpA of PoiBI and WX (Liefting & Kirkpatrick, 2003; GenBank Acc. No. AF533231). Alignments were adjusted manually, and dN/dS values were calculated as the overall average of the codon sites in each gene with Jukes-Cantor model of Nei-Gojobori method by mega (ver. 4; Tamura et al., 2007). A significant difference test for dN/dS was performed according to a previously described procedure (Messier & Stewart, 1997). For expression cloning, the entire imp gene from the Primelo Jingle Bells PoiBI isolate was PCR-amplified pheromone using primers impful-F and imp-R (Table S1), and a truncated form of the gene was PCR-amplified
using primers impout-F and imp-R (Table S1). The truncated gene was designed to encode an Imp derivative lacking the N-terminal transmembrane region. Similarly, the entire idpA gene from the Primelo Jingle Bells PoiBI isolate was PCR-amplified using primers idpAful-F and idpAful-R, and a truncated form of the gene idpA gene was PCR-amplified using primers idpAcent-F and idpAcent-R. The truncated gene was designed to encode an IdpA derivative lacking both transmembrane regions and containing only the hydrophilic domain. In addition, idpA gene fragments encoding the N- and C-terminal halves of the IdpA hydrophilic domain were amplified using the primer pairs idpAcent-F/idpA534-R and idpA532-F/idpAcent-R, respectively. A pET system (Novagen) was used to fuse histidine-tag (His-tag) and express the full-length and truncated Imp and IdpA proteins, as well as the IdpA hydrophilic fragments, in Escherichia coli. Each of the six PCR products described above was doubly digested with NdeI and XhoI and inserted into pET30a(+), thereby placing a His-tag at the C-terminus of the cloned fragments. The resulting constructs were transformed into E.