BMC Genomics 2005, 6:152 http://www.biomedcentral.com/1471-2164/6/152 searches. For eight human genes, ATP6V1G2, AIF1, CLIC1, CREBL1, DDAH2, AGPAT1, PBX2 and NOTCH4, the par- alogues but not the orthologues of the genes in the human MHC class III region have been identified by sequence similarity in zebrafish. This observation extends to IER3, RING1 and B3GALT4 found in the class I and extended class II regions. The presence or absence of other genes may be attributed to lineage-specific evolution. For exam- ple, lamprey [27], zebrafish [26] and medaka [28] possess genes equally similar to both mammalian BF and C2, while a Xenopus clone has clearly been identified as encompassing BF. This BF/C2 ancestral gene has further duplicated in zebrafish [29], copies of which were identi- fied on chromosome 21 and contig NA17761. Similarly, a recent survey of hsp70 genes in Xiphophorus maculates (platyfish) [30] has revealed that a single HSP70 gene gave rise to four distinct groups of genes: mammalian testis- specific HST70, mammalian MHC-linked HSP70, mam- malian HSP70B' and the fish HSP70. Human class III HSPA1A, HSPA1B and HSPA1L genes are intronless and encode identical or near-identical proteins. Intronless zebrafish hsp70 genes were identified on chromosome 8 and scaffold NA17761. Three additional copies of HSP70- like genes were identified on chromosome 3, although these contained one, two or three introns in the 3' end of their sequence. The identified zebrafish genes cluster phy- logenetically within the fish subgroup, apart from the sequence on scaffold NA17761, which appears to be sim- ilar to the mammalian MHC-linked heat shock protein sequences (data not shown). Construction of in-silico gene maps The zebrafish MHC gene map (Figure 1) was constructed using primarily mapping information from the whole- genome assembly displayed in the Ensembl platform. The position of at least 49 genes mapped in this survey could be verified by comparison to experimental data stored in the ZFIN database, which also yielded information for genes residing in unmapped contigs. Mapping data from these two sources did not coincide for several genes, including bat3, clica, vars2, c3, pou5f1, rgl2, tubb5, ddx39, col11a1, pbx1a, and stk19-like. These discre- pencies may be attributed to the high levels of polymor- phisms and regions of misassembly caused by the source DNA used for the whole-genome assembly being col- lected from over 1000 embryos. Until the genome sequence is complete, it will not be possible to accurately predict the position and number of all human MHC orthologues in zebrafish. There are also a number of genes, notably CD1, MOG and HFE, that could not be identified in the draft assembly used here (these are listed as 'not found' in Tables 1, 2, 3). There were a number of difficulties associated with assess- ing the degree of synteny between the zebrafish whole- genome assembly and the human MHC. Although BLAST is a heavily used analysis tool for identifying related sequences, it does not discriminate between large gene family members. To maximise the identification of MHC- related loci in zebrafish, only the highest BLAST scoring sequences were chosen for further analysis (many of which were unique BLAST reciprocal hits), and searches were conducted using blastp or tblastx. Orthology can be confirmed by obtaining mapping data of surrounding genes, with the assumption that groups of syntenic genes would remain in close proximity through evolution, and therefore be maintained in similar segments of DNA. The physical linkage between numerous MHC-related loci is apparent on zebrafish chromosome 19. Genes mapping to zebrafish chromosome 5 have also been observed to be syntenic with human chromosome 9 [31]. Gene blocks, in particular present in contig NA17761 and NA17767, map closely together in the human MHC class III region. This criterion is more difficult to apply when genes are dis- persed, as seen in Figure 1. The Ensembl platform [32] used to map the MHC in zebrafish provides a combina- tion of alignment data, genomic location, detailed tran- script structures to compare functional domains of orthologous proteins, in conjunction with multi-species comparisons. In combination with further phylogenetic analyses when duplicates of one gene were identified, the distinction between orthologues and paralogues was ascertained. Nevertheless, it is very difficult to assign orthologous comparative relationships with multigene families until all members of the family have been sequenced in both organisms. This might be the case in human, but the zebrafish genome is yet to be completed. Duplicated genes and MHC paralagous regions In human, it was observed that four regions, the MHC on 6p21.3 as well as 9q33–q34, 1q21–q25/1p11–p32 and 19p13.1–p13.4 are paralogous regions [33] that share members of the same gene family. Further analysis has shown that paralogues of human MHC genes are also scat- tered essentially over all chromosomes [18]. Likewise, genome-wide duplications have recently been examined in zebrafish [31], confirming that an extra round of a whole-genome duplication event occurred early in the tel- eost lineage after it split from the tetrapod lineage. Evi- dence that paralogous genes exist in zebrafish became apparent when many duplicates associated with the MHC, and mainly the class III region, were identified (Figure 1). Here we discuss five MHC-encoded gene families in more detail. The Notch gene family members encode evolutionary transmembrane receptors that regulate cell fate determi- nation. Four Notch paralogues (NOTCH1-4) have been identified in human, and only three of their orthologues are found in zebrafish (Table 3). The orthologue of Page 7 of 10 (page number not for citation purposes)