CHARACTERIZATION AND EXPRESSION OF RECOMBINATION ACTIVATING GENES (RAG-1 AND RAG-2) IN XENOPUS-LAEVIS

The primary repertoire of B and T cells is established by V(D)J recomb ination. Two closely linked genes, RAG-1 and RAG-2, are essential for this process, and have been identified in mice, humans, and chickens. To study lymphocyte development in Xenopus laevis, we have characteriz ed RAG-1 and RAG-2 in this species and examined their patterns of expr ession. Degenerate oligonucleotides, based on the known highly conserv ed RAG-1 sequences, were used to amplify, by the polymerase chain reac tion, a segment of Xenopus RAG-1 from genomic DNA. A product of expect ed size was obtained and used to identify a genomic clone that contain ed the complete coding region of RAG-1 (1045 codons), and approximatel y the 3'- half of the coding region of RAG-2. The coding regions of RA G-1 and RAG-2 each lie on a single exon, are in opposite transcription al orientation, and are separated by approximately 6 kb. The sequence of the remainder of RAG-2 was determined by PCR amplification of genom ic DNA, with primers based on sequence analysis of RAG-2 cDNA clones. The predicted Xenopus RAG-1 protein is 71% identical in amino acid seq uence to the sequences of each of the mouse, human, and chicken protei ns; from position 392 to 1012 the identity is 88%. The coding region o f Xenopus RAG-2 (520 codons) is somewhat less conserved among the diff erent species. Tissue-specific expression of Xenopus RAG-1 and RAG-2 w as examined both by Northern blotting and by a reverse transcription-p olymerase chain reaction assay. In juvenile frogs, the highest levels of RAG-1 and RAG-2 expression were observed in the thymus, with lower levels in liver and spleen, and even lower levels in the kidneys. In a dults, the thymus and bone marrow were found to be the principal sites of expression of both genes. RAG-2, but not RAG-1, was expressed in o ocytes.