BMC Genomics 2009, 10(Suppl 1):S14 http://www.biomedcentral.com/1471-2164/10/S1/S14 There may also be a species-dependence influencing the preference of fixation of duplicated genes within GPCRs. This was reflected in the difference in the preference of retention of GPCRs of peptide ligands vs. non-peptide lig- ands in human vs. in fish [19]. In fish, the retention rate of GPCRs of non-peptide ligands was higher than that of GPCRs of peptide ligands after a whole genome duplica- tion 230 Myrs ago. In contrast, our results showed that, among human GPCRs, more GPCRs that bind peptide lig- ands were fixed after 400 Myrs than GPCRs of non-pep- tide ligands. In human, the subfamilies that contributed most to the continued expansion of GPCRs after 400 Myrs were those involved in immune responses and sensory responses. This may have reflected differences in environ- mental influences between human and fish on the selec- tive pressure that drove the fixation of GPCR duplicates. In this study, we have kept the model system simple by including only the downstream genes in the classical GPCR pathways. Many other genes that were indirectly influenced by GPCRs were not included in the study, which are mostly kinases and transcription factors in the signaling pathways. However, one of our previous studies [21] on the human tyrosin kinase super family found sim- ilar patterns with the "downstream genes". Conclusion In general, using the human GPCR model system, our results confirmed that the position of a gene in the gene networks has great influences on the likelihood of expan- sion of its gene family in evolution. However, we also found that the influence was asymmetric among sub- families of GPCRs. We found that the gene basis of expres- sion diversity of most GPCR pathways, which cover a substantial portion of the gene network, have been estab- lished at the time of early vertebrate emergence. Contin- ued expansions in human GPCR families were to a large extent contributed by several small subfamilies involved in immune responses and sensory responses in human. Exactly which subfamilies may see extra expansion may be contingent on environmental factors for different species at different evolutionary stages, as was exemplified by our comparison of the differences in retention preference of GPCRs binding peptide or non-peptide ligands between humans and fishes. In the future, we shall further investigate the association between gene family expansions and GPCR-related net- works [22-27]. For instance, we may study the functional divergence between the duplicated GPCR-related proteins [22,23], the role of alternative splicing isoforms [24], as well as the tissue-specific effects [25,26] on the gene evo- lution. Recently, Gu [27] proposed an evolutionary model for the origin of modularity in a complex gene network, suggesting that new (protein-protein) interactions after the gene duplication may be favored to link preexisted backbone of signaling pathway, while loss of interactions are at random. The vertebrate GPCR networks may be an ideal model to study the design principle of signaling net- works. Materials and methods Family and sequence data The GPCR gene families analyzed in this study were clas- sified based on the GRAFS classification system [14], which covers 342 human non-olfactory GPCRs in five major classes including Glutamate (15), Rhodopsin (241), Adhesion (24), Frizzled/taste2 (24), Secretin (15). The Rhodopsin class is further divided into four groups: alpha, beta, gamma and delta. The sequences of vertebrate GPCR genes were retrieved from the Hovergen database [28]. In cases where the GRAFS families were further split in Hovergen, the split families were used for age estima- tion separately. The invertebrate homologues, as well as some vertebrate homologues missed by Hovergen, were identified by extensive BLAST [29] searching of the Swiss- prot protein database [30]. Redundant sequences were removed either based on UniGene annotations when available, or based on chromosome positions otherwise. The "downstream genes" families were constructed simi- larly. Families were constructed for each entry in Figure 1. The vertebrate homologues in the gene families were retrieved from Hovergen [28], the invertebrate homo- logues and the vertebrate homolgues missed by Hovergen were identified by homology searching in the Swiss-prot database. Redundant sequences were removed similarly as for GPCRs. Inference of duplication events and estimating their ages In bifurcate trees, each new human homologue is created by a duplication event. As a result, for a gene family with n human genes, there will be n-1 duplication events. We used the "nearest-neighbor clock" approach [2] to date the duplication events. For a gene family, we first recon- structed the phylogenetic tree. We used Clustal W [31] to carry out multiple sequence alignment of the protein sequences of the genes in the family. Phylogenetic trees (Neighbour-joining) were reconstructed using the MEGA program [32]. Linearized trees [18] were also calculated using MEGA. Next, the age of a duplication event was esti- mated based on its distances to the nearest bracketing spe- cies-split times in the phylogenetic tree under the molecular clock hypothesis [17,18]. We used the widely accepted species split times for calibration, including pri- mate-rodent (80 Myrs ago), mammal-bird (310 Myrs ago), mammal-amphibian (350 Myr ago), tetrapod-tele- ost (430 Myr ago) and vertebrate-fly splits (830 Myr ago) [33]. In cases that a bracketing pair of species-split times can be found for a duplication event, the age of the event Page 8 of 10 (page number not for citation purposes)