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 recombination. 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 characterized RAG-1 and RAG-2 in this species and examined their patterns of expression. Degenerate oligonucleotides, based on the known highly conserved RAG-1 sequences, were used to amplify, by the polymerase chain reaction, a segment of Xenopus RAG-1 from genomic DNA. A product of expected size was obtained and used to identify a genomic clone that contained the complete coding region of RAG-1 (1045 codons), and approximately the 3'-half of the coding region of RAG-2. The coding regions of RAG-1 and RAG-2 each lie on a single exon, are in opposite transcriptional orientation, and are separated by approximately 6 kb. The sequence of the remainder of RAG-2 was determined by PCR amplification of genomic DNA, with primers based on sequence analysis of RAG-2 cDNA clones. The predicted Xenopus RAG-1 protein is 71% identical in amino acid sequence to the sequences of each of the mouse, human, and chicken proteins; from position 392 to 1012 the identity is 88%. The coding region of Xenopus RAG-2 (520 codons) is somewhat less conserved among the different species. Tissue-specific expression of Xenopus RAG-1 and RAG-2 was examined both by Northern blotting and by a reverse transcription-polymerase 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 adults, 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 oocytes.
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
P
P
Greenhalgh
C E
CE
Olesen
L A
LA
Steiner
D000367:Age Factors; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins; D015870:Gene Expression; D005796:Genes; D018398:Homeodomain Proteins; D007814:Larva; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D011995:Recombination, Genetic; D015183:Restriction Mapping; D016415:Sequence Alignment; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000235:genetics*
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009838:Oligodeoxyribonucleotides; D011506:Proteins; C499310:RAG2 protein, Xenopus; D012333:RNA, Messenger; C064655:V(D)J recombination activating protein 2; D029867:Xenopus Proteins; C064658:RAG-1 protein; D004247:DNA