The recombination activating gene 2 (RAG2) of the rainbow trout Oncorhynchus mykiss

We have previously described the isolation and expression of RAG1 in trout to provide an initial understanding regarding the tissues involved in V(D)J recombination of antigen receptors in this teleost. Here we report that the recombination activating gene 2 (RAG2) of rainbow trout has now been cloned and characterized. The rainbow trout genomic RAG2 gene (1602 base pairs) displays an average of 60% and 75% similarity at the nucleotide and amino acid level when compared with clones from other species and was found to contain an acidic region in the carboxyl terminal end, which is typical of RAG2 sequences. The proximity of RAG1 and -2 within this teleost is similar to that found in other vertebrates. The genes are convergently transcribed and share a 3' untranslated (UT) region [2. 8 kilobases (kb)] which is much shorter than that found in higher vertebrates (6 - 8 kb). The entire 3' UT region was also sequenced and used in conjunction with cDNA clones to identify the polyadenylation sites for both RAG genes. Northern blot analysis of one-year-old trout demonstrated strong expression of RAG2 in the thymus, with a much weaker signal being detected in the pronephros. Using reverse transcriptase-polymerase chain reaction, we detected the highest expression of both RAG1 and -2 in the thymus followed by the pronephros, with much fainter signals being observed in the spleen, mesonephros, and liver. Finally, both genes are expressed in embryos beginning at approximately day 10 post-fertilization. Taken together, these findings indicate that the thymus and pronephros most likely serve as the primary lymphoid tissues in trout, based upon RAG expression. In addition, the trout sequences may provide further insight into the evolution and origins of the RAG genes as well as that of the immune system itself.