Sequence of the RAG1 and RAG2 Intergenic Region in Zebrafish (Danio rerio)

The recombination activating genes, rag1 and rag2 are essential for the rearrangement of antigen receptor V, D, and J gene segments (Oettinger et al. Shinkai et al., 1992). Both genes are found in all species that are known to rearrange their antigen-specific receptors. The coding regions as well as the genomic organization of the rag locus are highly conserved throughout evolution. Rag1 and rag2, which are convergently transcribed, are separated by an intergenic region of DNA that varies in size among species, being, for example, about 11 kb in the human Although ragl and rag2 have been intensively studied, little is known about specific transcriptional control mechanism(s) that regulate their expression. In general, they are coordinately expressed. Only in the chicken bursa, the mammalian brain, and in Xenopus oocytes is the expression of one gene found The coordinate expression of the rag genes may be mediated by common regulatory elements located in the 3' inter-genic region. Dobbeling and colleagues (1996) have recently provided evidence that elements regulating transcription of the murine rag locus may be contained within the 3' intergenic region. 3' regulatory elements are known to contribute to the regulation of several lymphoid specific genes, including the immunoglobulin and TCR genes (reviewed in Staudt and Lenardo, 1991; Leiden, 1993). Many of these regulatory elements are conserved among distantly related vertebrates (Magor et al., 1994). Because the rag intergenic region of teleosts is particularly small (Hansen and Kaattari, 1996; Willett et al., 1997), we sequenced this region from the zebrafish and analyzed the sequence for DNA elements likely to govern rag expression. The zebrafish intergenic region is part of a 6.3-kb Sac I fragment of the zebrafish rag locus, cloned into pBluescript, as previously described (Willett et al., 1997). A 2.9-kb section of this clone, from the 3' portion of ragl through the 3' portion of rag2, was sequenced on a Pharmacia A.L.F. automatic sequen-cer (HSC Biotech. Centre, University of Toronto). Sequences were assembled with DNA Strider. The zebrafish rag intergenic region of 2,625 bp (Genbank accession U69610) is shown schematically in Figure 1. This region contains no open reading frames longer than 65 amino-acid residues. Although the nucleotide sequence of enhancers in vertebrates may not be well conserved, studies with the immunoglobulin heavy-chain enhancer in the channel catfish (Ictalurus punctatus) and in the trout suggest that the function and regulatory mechanism(s) acting on enhancers are retained throughout vertebrate evolution. …