The genomic DNA sequences of CXCL and
The genomic DNA sequences of CXCL8 and CXCRs were retrieved from the fugu genome database v4.0. The intron–exon boundaries were identified with the corresponding cDNA sequences. This comparison revealed that the fugu gene is about 1.13kb long and contains four exons separated by three introns (top panel, ). This genomic organization is similar to that seen in the gene in rainbow trout , carp CXCa and human CXCL8 . The intron sizes in fugu CXCL are far smaller than those found in primates and artiodactyl species (sheeps, goats), and are about one-third shorter than that found in rainbow trout. Fugu , which is ∼1.89kb in length, comprises two exons and the first exon codes only the 5'-URT region (mid panel, ). Notably, the transcription varieties were found only in cDNA sequences, not in the genome. Like , , which is ∼2.18kb in length, is also comprised of two exons, but the first exon encodes two amino ack1 inhibitor residues in addition to 5'-UTR (bottom panel, ). In human, is composed of two exons separated by one intron, while has three exons with two introns . However, in fugu, both receptor genes are organized similarly: two exons separated by one intron. These features indicate that IL8RA retained the ancestral gene organization, and that IL8RB evolved more recently. That is, the duplication of fugu CXCR1 occurred after the divergence of the teleost and mammalian lineages 400 million years ago. The detailed phylogenetic tree of CXC chemokine receptors from different taxa of vertebrates based on the cDNA- and predicted-gene sequences has been reported by Huising et al. . Nevertheless, we are presenting a simple tree, which was drawn from amino acid sequences, to show that teleost CXCR comprised a discrete cluster that is clearly divergent from mammalian CXCRs (). Neither fish CXCR associates with any of the mammalian CXCR. Homology searches revealed that CXCR2 is more similar to mammalian IL8RA than IL8RB. CXCRs are highly divergent even within the teleost species as indicated by the lower bootstrap values, suggesting that fish CXCR may have evolved rapidly. In human, IL8A and IL8RB are tandemly located on chromosome 2, suggesting that these genes may have arisen via a tandem duplication of an ancestral gene. To further examine the orthology of two human IL8Rs and two fugu CXCRs based on syntenic relationships, we determined the genomic location of fugu CXCR1 and CXCR2 by using a meiotic mapping panel . This analysis revealed that fugu CXCR1 and CXCR2 maps to distinct linkage groups, LG8 and LG1, respectively (). Comparative analysis of CXCRs and their neighboring genes on these fugu LGs and human chromosome 2 revealed conservative synteny between them. Therefore, it could be speculated that two fugu CXCRs might have arisen by the chromosomal duplication of an ancestral chromosome containing one CXCR or by the complementary silencing of CXCRs following the chromosomal duplication of an ancestral chromosome containing tandemly arrayed CXCRs. Given the fact that genomic organization of both fugu CXCRs are identical to human IL8RA, but not to human IL8RB, we favor the conclusion that both fugu CXCRs are orthologous to human IL8RA, and human IL8RB may have arisen after the divergence of the human and teleost lineages. This conclusion is also supported by phylogenetic analysis of fugu and human IL8Rs in which fugu CXCRs and human IL8Rs are clustered independently as mentioned in the above paragraph. The RT-PCR employed specific primer pairs (CXCL8F1 and CXCL8R2 for CXCL8, CXCR1FA2 and CXCR1RA for CXCR1 short transcript, CXCR1FA1 and CXCR1RB for CXCR1 long transcript, and CXCR2F1 and CXCR2R1 for CXCR2, ) and DNA polymerase for 30 cycles, except for the CXCR1 long transcript which was amplified for 40 cycles. Expression of CXCL8 in the fugu was primarily limited to PBL as well as the thymus, head kidney, trunk kidney and spleen (), tissues that normally contain large numbers of lymphocytes, neutrophils, and macrophages, implying the involvement of CXCL8 in the immune system of fugu. Expression of this chemokine was also seen in the heart and gill, though not in the other tissues examined. Though the expression of CXCR2 was similar to that of CXCL8, neither the short nor long transcripts of CXCR1 were observed in the thymus and gill. The results indicate tissue specific expression of CXCRs.