To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing ␣-and ␤-globin genes from the pufferfish Fugu rubripes. By DNA fluorescence in situ hybridization (FISH) analysis, we show that Fugu contains 2 distinct hemoglobin loci situated on separate chromosomes. One locus contains only ␣-globin genes (␣-locus), whereas the other also contains a ␤-globin gene (␣␤-locus). This is the first poikilothermic species analyzed in which the physical linkage of the ␣-and ␤-globin genes has been uncoupled, supporting a model in which the separation of the ␣-and ␤-globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of the Fugu ␣␤-locus suggest that a strong distal locus control region regulating the activity of the globin genes, as found in mammalian ␤-globin clusters, may not be present in the Fugu ␣␤-locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are proximal to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the 5 human globin loci known to date, each encoding proteins with specific functions in specific cell

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