BMC Genomics 2005, 6:152 http://www.biomedcentral.com/1471-2164/6/152 human NOTCH4, found in the MHC, is absent in the fish whole-genome assembly, indicative that gene loss has fol- lowed the block duplication events in teleosts. This is also seen in Fugu [24] and may be due to it being the most divergent member of the Notch family [34]. Two dupli- cates of notch3 are found in scaffolds 1523 and 285. Until the assembly is complete, it is not clear whether there are two copies of this gene or if this is due to the status of the current assembly. Only one surrounding rdh8-like sequence is found in common between the two scaffolds. In addition, notch1 and notch2 have been mapped to chro- mosome 5 and contig NA15389. Retinoid receptors are soluble nuclear proteins belonging to the steroid/thyroid hormone receptor superfamily of transcriptional regulators. The RXR subfamily consists of three polypeptide chains, namely alpha, beta and gamma, encoded by separate loci. The human RXRB gene is found within the MHC, while the RXRA and RXRG paralogues are located on chromosomes 9 and 1, respectively. Five rxr loci have been identified in the zebrafish assembly in con- tig NA16779 and chromosomes 5, 9, 17 and 21. Two semi-orthologues of human rxrb appear to have arisen from a fish-specific duplication, and duplicates of rxra are also present in the zebrafish genome. Mapping data from the current assembly in comparison to the ZFIN linkage maps are contradictory and may highlight problems in both approaches to mapping genes. Interestingly, neither of the rxrb sequences map to chromosome 19 in the Zv4 whole-genome assembly as originally thought [35]. The human PBX2 gene encodes a homeodomain-contain- ing protein. Three paralogues are located within chromo- somes 1q23, 9q33 and 19p12, named PBX1, PBX3 and PBX4, respectively. Four PBX-like sequences were identi- fied in the zebrafish: pbx1a on scaffold NA14559 and chromosome 11; pbx3b on chromosome 18; and pbxy on scaffold NA11844. These are related to human PBX1, PBX3 and PBX4, respectively [36]. The orthologous sequence to human PBX2, which is present in the MHC, has not been identified in the current assembly. The complement component C4 gene encodes a protein that plays a central role in the innate immune response. Structurally, C4, C5 and C3 belong to the α2 macroglob- ulin (A2M) protein family and are derived from a single common ancestor. In human, they are found on four dif- ferent chromosomes: 6p21 (C4), 9q33 (C5), 19p13 (C3) and 12p13 (A2M), and a similar situation exists in all tetrapods studied to date. In zebrafish, the a2m and c4 genes are both found on chromosome 15 as previously shown [37]. Multiple copies of a2m have also been observed in zebrafish with a duplicate being present in NA17328. Two copies of the c3 gene were identified on chromosome 22 and NA14479. Six members of the chloride intracellular channel (CLIC) gene family (CLIC1–CLIC6) have been described in humans. They are involved in chloride ion transport within various subcellular compartments [38]. Four clic genes were identified in zebrafish on chromosomes 14, 16, 17 and 18. On phylogenetic analysis, they cluster with mammalian CLIC2, CLIC3, CLIC4 and CLIC5 genes, respectively. The orthologue of the human MHC-embed- ded CLIC1 was not found. Conclusion Comparative genomics reveals that the organisation of the MHC in more distantly-related organisms varies from the human model [39]. Teleost fish are particularly unusual in their organisation in comparison to mammals, chicken, Xenopus and shark in that their class I and class II loci are found on different chromosomes [14]. The core class I region in zebrafish, medaka, Fugu and rainbow trout [13,40-42] comprises genes involved in class I pep- tide presentation and processing: the classical class I mol- ecules, the immunoproteasome subunits, ATP-binding cassette transporters and tapasin. The whole-genome shotgun data for zebrafish has allowed a genome-wide analysis of major histocompatibility genes and their para- logues, and highlights that one or more copies exist of MHC-related genes in fish as in humans. The results obtained thus far extend the linkage information regard- ing major histocompatibility genes on chromosome 19 in zebrafish and the classical mammalian MHC, and further supports previous findings that the functional class II loci are found on a different chromosome. The distribution of the remaining MHC-related genes and their paralogues is genome-wide, confirming a fragmented MHC in fish. Methods The genome of the zebrafish is currently being sequenced at the Sanger Institute [43]. The analysis was carried out on the fourth Ensembl whole-genome assembly Zv4, released on September 2004 [32]. This assembly inte- grates the whole-genome shotgun assembly with data from the physical map [44]. Protein sequences encoded in 117 human MHC genes were chosen for BLAST sequence similarity searches against the zebrafish Ensembl assem- bly. Previously identified MHC-related zebrafish cDNAs [45] were also used in the analysis. Gene annotations were verified using VEGA [46]. A number of potentially dupli- cated genes were identified. Their predicted amino acid sequences were then aligned with other vertebrate gene family members present in the UniProt database [47] using CLUSTAL [48], and the multiple sequence align- ments were used for phylogenetic analysis by the neigh- bour joining method [49] via the PHYLO_WIN interface [50]. Official gene symbols assigned by the Zebrafish Nomenclature Committee (ZNC) and HUGO Nomencla- ture Committee (HGNC) have been used in the annota- Page 8 of 10 (page number not for citation purposes)