Abstract
The characterization of genes involved in the generation of the immune repertoire is an active area of research in lower vertebrate taxa. The recombination activating genes (RAG) have been shown to be essential for V (D) J recombination of T-cell antigen receptor (TCR) and immunoglobulin (Ig) genes, leading to the generation of the primary repertoire. As RAG1 is critical to the differentiation of pre-B and-T cells, its expression within an associated primary lymphoid organ can serve as a developmental marker. To examine the ontogeny of lymphocytes in Oncorhynchus mykiss, we cloned RAG1 from trout and examined its tissue-and lymphocyte-specific expression. The polymerase chain reaction, coupled with degenerate oligonucleotide primers, was used to amplify a homologous probe [(633 base pairs) (bp)] from rainbow trout genomic DNA, which in turn was used to isolate a lambda genomic clone. Sequence analysis of this genomic clone confirmed the RAG1 nature of this gene (3888 bp) and revealed an internal intron of 666 bp. When compared with other previously reported RAG1 sequences, the predicted amino acid translation (1073 aa) displayed a minimum of 78% similarity for the complete sequence and 89% similarity in the conserved region (aa 417-1042). Using northern blot analysis, we found the expression of RAG1 to be limited to surface Ig-n lymphocytes within the thymus. This data forms the basis for a proposal that the thymus of teleost species plays an essential developmental role in lymphopoiesis and thus can be regarded as a primary lymphoid organ.
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Aguilera, A. and Klein, H. L. HRP1, a novel yeast gene that prevents intrachromosomal excision recombination, shows carboxy-terminal homology to the Saccharomyces cerevisiae TOP1 gene. Mol Cell Biol 10: 1439–1451, 1990
Amemiya, C. T. and Litman, G. W. Complete nucleotide sequence of an immunoglobulin heavy-chain gene and analysis of immunoglobulin gene organization in a primitive teleost species. Proc Natl Acad Sci USA 87: 811–815, 1990
Bosma, G. C., Custer, R. P., and Bosma, M. J. A severe combined immunodeficiency mutation in the mouse. Nature 301: 527–530, 1983
Brunk, B. P., Martin, E. C., and Adler, P. N. Drosophila genes Posterior Sex Combs and Suppressor two of zeste encode proteins with homology to the murine bmi-1 oncogene. Nature 353: 351–353, 1991
Carlson, L. M., Oettinger, M. A., Schatz, D. G., Masteller, E. L., Hurley, E. A., McCormack, Baltimore, D., and Thompson, C. B. Selective expression of RAG-2 in chicken B cells undergoing immunoglobulin gene conversion. Cell 64: 201–208, 1991
Chomczynski, P. and Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987
Daggfeldt, A., Bengtén, E., and Pilström, L. A cluster type organization of the loci of the immunoglobulin light chain in Atlantic cod (Gadus morhua L.) and rainbow trout (Oncorhynchus mykiss Walbaum) indicated by nucleotide sequences of cDNAs and hybridization analysis. Immunogenetics 38: 199–209, 1993
DeLuca, D., Wilson, M., and Warr, G. Lymphocyte heterogeneity in the trout, Salmo gairdneri, defined with monoclonal antibodies to IgM. Eur J Immunol 13: 546–551, 1983
DeSoete, G. A least squares algorithm for fitting additive trees to proximity data. Psychometrika 48: 621–626, 1983
Devereux, J., Haeberli, P., and Smithies, O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12: 387–395, 1984
Dingwall, C. and Laskey, R. A. Nuclear targeting sequences — a consensus? Trends Biochem Sci 16: 478–481, 1991
Du Pasquier, L., Schwager, J., and Flajnik, M. F. The immune system of Xenopus. Annu Rev Immunol 7: 251–275, 1989
Earley, P., Rogers, J., Davies, M., Calame, K., and Hood, L. An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH. Cell 19: 981–992, 1980
Ellis, A. E. Ontogeny of the immune response in Salmo salar. Histogenesis of the lymphoid organs and appearance of membrane immunoglobulin and mixed leucocyte reactivity. In J. B. Solomon and J. D. Horton (eds.): Developmental Biology, pp 225–277, Elsevier North Holland Biomed Press, Amsterdam, 1977
Faisal, M. and Hetrick, F. Annual Review of Fish Diseases, Pergamon Press Ltd., New York, 1992
Feinberg, A. P. and Vogelstein, B. A. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13, 1983
Fellah, J. S., Kerfourn, F., Guillet, F., and Charlemagne, J. Conserved structure of amphibian T-cell antigen receptor B chain. Proc Natl Acad Sci 90: 6811–6814, 1993
Felsenstein, J. Phylip: phylogeny inference program (V 3.2). Cladistics 5: 164–166, 1989
Freemont, P. S., Hanson, I. M., and Trowsdale, J. A novel cysteine-rich sequence motif. Cell 64: 483–484, 1991
Frohman, M. A., Dush, M. K., and Martin, G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85: 8998–9002, 1988
Fuschiotti, P., Nagaradona, H., Mage, R. G., McCormack, W. T., Dhanarajan, P., and Roux, K. H. Recombination activating genes-1 and-2 of the rabbit: cloning and characterization of germline and expressed genes. Mol Immunol 30: 1021–1032, 1993
Grace, M. F. and Manning, M. J. Histogenesis of the lymphoid organs in the rainbow trout, Salmo gairdneri Rich. Dev Comp Immunol 4: 255–264, 1980
Greenhalgh, P., Olesen, C. E., and Steiner, L. A. Characterization and expression of recombination activating genes (RAG-1 and RAG-2) in Xenopus laevis. J Immunol 151: 3100–3110, 1993
Greenhalgh, P. and Steiner, L. A. Recombination activating gene 1 (RAG1) in zebrafish and shark. Immunogenetics 41: 54–55, 1995
Hesse, J. E., Lieber, M. R., Mizuuchi, K., and Gellert, M. V (D) J recombination: a functional definition of the joining signals. Genes Dev 3: 1053–1061, 1989
Heinrich, G., Traunecker, A., and Tonegawa, S. Somatic mutation creates diversity in the major group of mouse immunoglobulin kappa-light chains. J Exp Med 159: 417–435, 1984
Higgins, D. G., Bleasby, A. J., and Fuch, R. Clustal V: improved software for multiple sequence alignments. Comput Appl Biosci 8: 189–191, 1992
Hohman, V. S., Schluter, S. F., and Marchalonis, J. J. Complete sequence of a cDNA clone specifying sandbar shark immunoglobulin light chain: gene organisation and implications for the evolution of light chains. Proc Natl Acad Sci USA 89: 276–280, 1992
Josefsson, S. and Tatner, M. F. Histogenesis of the lymphoid organs in sea bream (Sparus aurata L.) Fish and Shellfish Immunol 3: 35–49, 1993
Kaattari, S. L. and Irwin, M. J. Salmonid spleen and anterior kidney harbor populations of lymphocytes with different B cell repertoires. Dev Comp Immunol 9: 433–444, 1985
Kallenbach, S., Doyen, N., Fanton d'Andon, M., and Rougeon, F. Three lymphoid-specific factors account for all junctional diversity characteristic of somatic assembly of T-cell receptor and immunoglobulin genes. Proc Natl Acad Sci USA 89: 2799–2803, 1992
Kokubu, F., Hinds, K., Litman, R., Shamblott, M. J., and Litman, G. W. Complete structure and organization of immunoglobulin heavy chain constant region genes in a phylogenetically primitive vertebrate. EMBO J 7: 1979–1988, 1988
Kozak, M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44: 283–292, 1986
Landau, R. E., Schatz, D. G., Rosa, M., and Baltimore, D. Increased frequency of N-regional insertion in a murine pre-B cell line infected with a terminal deoxynucleotidyl transferase retroviral expression vector. Mol Cell Biol 7: 3237–3243, 1987
Li, W-H., Gouy, M., Sharp, P. M., O'hUigin, C., and Yang, Y-W. Molecular phylogeny of Rodentia, Lagomorpha, Primates, Artiodactyla, and Carnivora and molecular clocks. Proc Natl Acad Sci USA 87: 6703–6707, 1990
Li, W-H. and Graur, D. Fundamentals of Molecular Evolution, Sinauer Associates, Sunderland, 1991
Litman, G. W., Berger, L., Murphy, K., Litman, R., Hind, K., and Erickson, B. W. Immunoglobulin VH gene structure and diversity in Heterodontus, a phylogenetically primitive shark. Proc Natl Acad Sci USA 82: 2082–2086, 1985
Ma, A., Fisher, P., Dildrop, R., Oltz, E., Rathbun, G., Achacoso, P., Stall, A., and Alt, F. W. Surface IgM mediated regulation of RAG gene expression in Eμ-N-myc B cell lines. EMBO J 11: 2727–2734, 1992
Mombaerts, P., Lacomini, J., Johnson, R. S., Herrup, K., Tonegawa, S., and Papaiannou, V. E. RAG-1 deficient mice have no mature B and T lymphocytes. Cell 68: 869–877, 1992
Oettinger, M. A., Schatz, D. G., Gorka, C., and Baltimore, D. RAG-1 and RAG-2, adjacent genes that synergistically activate V (D) J recombination. Science 248: 1517–1523, 1990
Partula, S., Fellah, J. S., Deguerra, A., and Charlemagne, J. Identification of cDNA clones encoding the T-cell receptor beta chain in the rainbow trout (Oncorhynchus mykiss). Compt Rend de L Acad Des Sci Serie III 317: 765–770, 1994
Petrie, H. T., Livak, F., Schatz, D. G., Strasser, A., Crispe, N., and Shortman, K. Multiple rearrangements in T cell receptor alpha chain genes maximize the production of useful thymocytes. J Exp Med 178: 615–622, 1993
Pough, F. H., Beiser, J. B., and McFarland, W. N. Vertebrate Life, Macmillan Press, New York, 1989
Rast, J. P. and Litman, G. W. T-cell receptor gene homologs are present in the most primitive jawed vertebrates. Proc Natl Acad Sci USA 91: 9248–9252, 1994
Rast, J. P., Anderson, M. K., Ota, T., Litman, R. T., Margittai, M., Shamblott, M. J., and Litman, G. W. Immunoglobulin light chain class multiplicity and alternative organizational forms in early vertebrate phylogeny. Immunogenetics 40: 83–90, 1994
Razquin, B. E., Castillo, A., Lopez-Fierro, P., Alvarez, F., Zapata, A., and Villena, A. J. Ontogeny of IgM-producing cells in the lymphoid organs of rainbow trout, Salmo gairdneri Richardson: an immuno- and enzyme-histochemical study. J Fish Biol 36: 159–173, 1990
Renu, J., Gomer, H., and Murtagh, J. J. Increasing specificity from the PCR-RACE technique. Biotechniques 12: 58–59, 1992
Sakano, H., Maki, R., Kurosawa, Y., Roeder, W., and Tonegawa, S. Two types of somatic recombination are necessary for the generation of complete immunoglobulin heavy chain genes. Nature 286: 676–683, 1980
Sanger, F., Nicklen, S., and Coulson, A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467, 1977
Schatz, D. G. and Baltimore, D. Stable expression of immunoglobulin gene V (D) J recombinase activity by gene transfer into 3T3 fibroblasts. Cell 53: 107–115, 1988
Schatz, D. G., Oettinger, M. A., and Baltimore, D. The V (D) J recombination activating gene, RAG-1. Cell 59: 1035–1048, 1989
Schatz, D. G., Oettinger, M. A., and Schissel, M. S. V (D) J recombination. Molecular biology and regulation. Annu Rev Immunol 10: 359–383, 1992
Schuler, W., Weiler, I. J., Schuler, A., Phillips, R. A., Rosenberg, N., Mak, T. W., Kearney, J. F., Perry, R. P., and Bosma, M. J. Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency. Cell 46: 963–972, 1986
Schwager, J., Grossberger, D., and Du Pasquier, L. Organization and rearrangement of immunoglobulin M genes in the amphibian Xenopus. EMBO J 7: 2409–2415, 1988
Schwager, J., Burckert, N., Schwager, M., and Wilson, M. Evolution of immunoglobulin light chain genes: analysis of Xenopus IgL isotypes and their contribution to antibody diversity. EMBO J 10: 505–511, 1991
Shinkai, Y. G., Rathbun, G., Lam, K. P., Oetz, E. M., Stewart, V., Mendelson, M., Charron, J., Datta, M., Young, F., Stall, A. M., and Alt, F. W. RAG-2 deficient mice lack mature lymphocytes owing to inability to initiate V (D) J rearrangement. Cell 68: 855–867, 1992
Silver, D. P., Spanopoulou, E., Mulligan, R. C., and Baltimore, D. Dispensable sequence motifs in the RAG-1 and RAG-2 genes for plasmid V (D) J recombination. Proc Natl Acad Sci USA 90: 6100–6104, 1993
Strauss, W. M. Preparation of genomic DNA from mammalian tissue. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (eds): Current Protocols in Molecular Biology, Vol 1, pp 2.2.1–2.2.3, Greene Publishing Associates and Wiley-Interscience, New York, 1989
Takeda, S., Masteller, E. L., Thompson, C. B., and Buerstedde, J. M. RAG-2 expression is not essential for chicken immunoglobulin gene conversion. Proc Natl Acad Sci USA 89: 4023–4027, 1992
Tatner, M. F. The migration of labeled thymocytes to the peripheral lymphoid organs in the rainbow trout, Salmo gairdneri Richardson. Dev Comp Immunol 9: 85–91, 1985
Tonegawa, S. Somatic generation of antibody diversity. Nature 302: 575–581, 1983
Wang, J. C., Caron, P. R., and Kim, R. A. The role of DNA topoisomerase in recombination and genome stability: a double edged sword? Cell 62: 403–406, 1990
Wilson, M. R., Marcus, A., van Ginkel, F., Miller, N. W., Clem, L. W., Middleton, D., and Warr, G. W. The immunoglobulin M heavy chain constant region of the channel catfish, Ictalurus punctatus: an unsual mRNA splice pattern produces the membrane form of the molecule. Nucleic Acid Res 18: 5227–5233, 1990
Yancopoulos, G., Blackwell, T. K., Suh, H., Hood, L., and Alt, F. W. Introduced T-cell receptor variable region gene segments recombine in pre-B cells: evidence that B and T cells use a common recombinase. Cell 44: 251–259, 1986
Zachau, H. Immunoglobulin light-chain genes of the K type in man and mouse. In T. Honjo, F. W. Alt, and T. H. Rabbitts (eds.): Immunoglobulin Genes, pp. 92–109, Academic Press, London, 1989
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Hansen, J.D., Kaattari, S.L. The recombination activating gene 1 (RAG1) of rainbow trout (Oncorhynchus mykiss): cloning, expression, and phylogenetic analysis. Immunogenetics 42, 188–195 (1995). https://doi.org/10.1007/BF00191224
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DOI: https://doi.org/10.1007/BF00191224