Tian et al. BMC Genomics 2010, 11:187 http://www.biomedcentral.com/1471-2164/11/187 Page 15 of 19 Figure 12 A proposed exon shuffling mechanism for origin of nabaecin. A. Sequence alignment of abaecin, nabaecin-3 and navitripenicin. Identical nucleotides and amino acids are shadowed in yellow and signal peptides are italized and underlined once. Alignable sequences are bolded and the corresponding nucleotides are boxed. Intron splice sites are showed in gray and omitted sequences are displayed as dots. Stop codons are shadowed in dark gray; B. The exon-shuffing model (for detailed description, see the text). Color codes are the same with those in Figure 12A. despoited in Protein Model DataBase [112] under id number of PM0075686-PM0075709. Maximum likelihood analysis Codon-substitution models were selected to estimate the nonsynonymous-to-synonymous rate ratio (ω = dN/dS) using the CODEML program of the PAML software package [113]. Four models recommended by Yang make two likelihood ratio tests (LRTs) by M1a/M2a and M7/M8: M1a (nearly neutral model) constraints a pro- portion p0 of conserved sites with 0 < ω < 1, while a proportion p1 = 1-p0 of neutral sites with ω1 = 1; M2a (positive selection model) adds an extra class of sites with the proportion p2 = 1-p0 -p1 and with ω estimated from the data. M7 (b distribution model) does not allow for positively selected sites and M8(b and ω model) adds an extra class of sites to M7, allowing for ω > 1, which means the presence of positively selected sites. In addition, Model 0 (M0) assuming one ω for all sites that does not allow the existence of positive selection was chosen for negative control [114,115]. Upon detection of the positively selected signals, the calculation of poster- ior probabilities was completed using the Bayes Empiri- cal Bayes (BEB) method [115,116]. Chemical synthesis and oxidative refolding Pronavicin and reduced nasonin-1 were chemically synthesized by Xi’an Huachen Bio-Technology Co., Ltd. (Xi’an, China). The cyclization reaction to form disulfide bridges in the reduced nasonin-1 molecule was carried