Neoteny in lymphocytes: Rag1 and Rag2 expression in germinal center B cells

Science

Volumn 274, Issue 5295, 1996, Pages 2094-2097

  Han, Shuhua ^a   Zheng, Biao ^a   Schatz, David G ^b   Spanopoulou, Eugenia ^c   Kelsoe, Garnett ^a  

^a UNIVERSITY OF MARYLAND SCHOOL OF MEDICINE   (United States)

^b YALE UNIVERSITY SCHOOL OF MEDICINE   (United States)

^c Mount Sinai School of Medicne   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ANIMAL CELL; ANTIBODY RESPONSE; APOPTOSIS; ARTICLE; CELL COMPARTMENTALIZATION; GENE ACTIVATION; GENE EXPRESSION REGULATION; GENE REARRANGEMENT; GERMINAL CENTER; LYMPHOCYTE DIFFERENTIATION; LYMPHOCYTE SUBPOPULATION; NONHUMAN; PRIORITY JOURNAL; PROTEIN ASSEMBLY; RECEPTOR GENE;

ANIMALS; B-LYMPHOCYTES; DNA NUCLEOTIDYLTRANSFERASES; DNA-BINDING PROTEINS; FEMALE; GENE EXPRESSION; GENE REARRANGEMENT; GENES, IMMUNOGLOBULIN; GENES, RAG-1; GERMINAL CENTER; HOMEODOMAIN PROTEINS; IMMUNIZATION; IMMUNOGLOBULIN CLASS SWITCHING; LYMPHOCYTE ACTIVATION; MICE; MICE, INBRED C57BL; POLYMERASE CHAIN REACTION; PROTEIN BIOSYNTHESIS; PROTEINS; VDJ RECOMBINASES;

ANIMALIA; MURINAE;

EID: 0030460833     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5295.2094     Document Type: Article

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4. For example, murine GC B cells, like developing B cells in the bone marrow, avidly bind the lectin peanut agglutinin (PNA), express large amounts of heat-stable antigen (HSA), and are labeled by monoclonal antibodies recognizing the GL-7 and LIP-6 determinants [Y. Reisner et al., Proc. Natl. Acad. Sci. U.S.A. 76, 447 (1979); M. L. Rose et al., Nature 284, 364 (1980); G. Lazlo, K. S. Hathcock, H. B. Dickler, R. J. Hodes, J. Immunol. 150, 5252 (1993); C. Miller, G. Kelsoe, S. Han, Aging Immunol. Infect. Dis. 5, 249 (1994); K. L. Holmes et al., Cell, Immunol. 166, 131 (1995); G. Kelsoe, Immunity 4, 107 (1996); R. Hodes and K. L. Holmes, personal communication].
5. For example, murine GC B cells, like developing B cells in the bone marrow, avidly bind the lectin peanut agglutinin (PNA), express large amounts of heat-stable antigen (HSA), and are labeled by monoclonal antibodies recognizing the GL-7 and LIP-6 determinants [Y. Reisner et al., Proc. Natl. Acad. Sci. U.S.A. 76, 447 (1979); M. L. Rose et al., Nature 284, 364 (1980); G. Lazlo, K. S. Hathcock, H. B. Dickler, R. J. Hodes, J. Immunol. 150, 5252 (1993); C. Miller, G. Kelsoe, S. Han, Aging Immunol. Infect. Dis. 5, 249 (1994); K. L. Holmes et al., Cell, Immunol. 166, 131 (1995); G. Kelsoe, Immunity 4, 107 (1996); R. Hodes and K. L. Holmes, personal communication].
6. For example, murine GC B cells, like developing B cells in the bone marrow, avidly bind the lectin peanut agglutinin (PNA), express large amounts of heat-stable antigen (HSA), and are labeled by monoclonal antibodies recognizing the GL-7 and LIP-6 determinants [Y. Reisner et al., Proc. Natl. Acad. Sci. U.S.A. 76, 447 (1979); M. L. Rose et al., Nature 284, 364 (1980); G. Lazlo, K. S. Hathcock, H. B. Dickler, R. J. Hodes, J. Immunol. 150, 5252 (1993); C. Miller, G. Kelsoe, S. Han, Aging Immunol. Infect. Dis. 5, 249 (1994); K. L. Holmes et al., Cell, Immunol. 166, 131 (1995); G. Kelsoe, Immunity 4, 107 (1996); R. Hodes and K. L. Holmes, personal communication].
7. For example, murine GC B cells, like developing B cells in the bone marrow, avidly bind the lectin peanut agglutinin (PNA), express large amounts of heat-stable antigen (HSA), and are labeled by monoclonal antibodies recognizing the GL-7 and LIP-6 determinants [Y. Reisner et al., Proc. Natl. Acad. Sci. U.S.A. 76, 447 (1979); M. L. Rose et al., Nature 284, 364 (1980); G. Lazlo, K. S. Hathcock, H. B. Dickler, R. J. Hodes, J. Immunol. 150, 5252 (1993); C. Miller, G. Kelsoe, S. Han, Aging Immunol. Infect. Dis. 5, 249 (1994); K. L. Holmes et al., Cell, Immunol. 166, 131 (1995); G. Kelsoe, Immunity 4, 107 (1996); R. Hodes and K. L. Holmes, personal communication].
8. For example, murine GC B cells, like developing B cells in the bone marrow, avidly bind the lectin peanut agglutinin (PNA), express large amounts of heat-stable antigen (HSA), and are labeled by monoclonal antibodies recognizing the GL-7 and LIP-6 determinants [Y. Reisner et al., Proc. Natl. Acad. Sci. U.S.A. 76, 447 (1979); M. L. Rose et al., Nature 284, 364 (1980); G. Lazlo, K. S. Hathcock, H. B. Dickler, R. J. Hodes, J. Immunol. 150, 5252 (1993); C. Miller, G. Kelsoe, S. Han, Aging Immunol. Infect. Dis. 5, 249 (1994); K. L. Holmes et al., Cell, Immunol. 166, 131 (1995); G. Kelsoe, Immunity 4, 107 (1996); R. Hodes and K. L. Holmes, personal communication].
9. For example, murine GC B cells, like developing B cells in the bone marrow, avidly bind the lectin peanut agglutinin (PNA), express large amounts of heat-stable antigen (HSA), and are labeled by monoclonal antibodies recognizing the GL-7 and LIP-6 determinants [Y. Reisner et al., Proc. Natl. Acad. Sci. U.S.A. 76, 447 (1979); M. L. Rose et al., Nature 284, 364 (1980); G. Lazlo, K. S. Hathcock, H. B. Dickler, R. J. Hodes, J. Immunol. 150, 5252 (1993); C. Miller, G. Kelsoe, S. Han, Aging Immunol. Infect. Dis. 5, 249 (1994); K. L. Holmes et al., Cell, Immunol. 166, 131 (1995); G. Kelsoe, Immunity 4, 107 (1996); R. Hodes and K. L. Holmes, personal communication].
10. + (double-positive) cortical thymocytes; soluble antigen induces apoptosis in immature and GC B cells, and agents such as antibody to CD3, bacterial superantigens, and glucocorticoids that readily kill double-positive thymocytes are equally toxic for GC T cells [F. Pezzella et al., Am. J. Pathol. 137, 225 (1990); D. M. Hockenbery et al., Proc. Natl. Acad. Sci. U.S.A. 88, 6961 (1991); B. Pulendran et al., Nature 375, 331 (1995); K. M. Shokat and C. C. Goodnow, ibid., p. 334; S. Han, B. Zheng, J. Dal Porto, G. Kelsoe, J. Exp. Med. 182, 1635 (1995); B. Zheng, S. Han, G. Kelsoe, ibid. 184, 1083 (1996); B. Zheng, S. Han, Q. Zhu, R. Goldsby, G. Kelsoe, Nature, In press).
11. + (double-positive) cortical thymocytes; soluble antigen induces apoptosis in immature and GC B cells, and agents such as antibody to CD3, bacterial superantigens, and glucocorticoids that readily kill double-positive thymocytes are equally toxic for GC T cells [F. Pezzella et al., Am. J. Pathol. 137, 225 (1990); D. M. Hockenbery et al., Proc. Natl. Acad. Sci. U.S.A. 88, 6961 (1991); B. Pulendran et al., Nature 375, 331 (1995); K. M. Shokat and C. C. Goodnow, ibid., p. 334; S. Han, B. Zheng, J. Dal Porto, G. Kelsoe, J. Exp. Med. 182, 1635 (1995); B. Zheng, S. Han, G. Kelsoe, ibid. 184, 1083 (1996); B. Zheng, S. Han, Q. Zhu, R. Goldsby, G. Kelsoe, Nature, In press).
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32. Two microliters of cDNA from each 20-μl sample reverse transcribed from extracted RNA was amplified in RT-PCR assays which used the following primer pairs: Rlag1, 5′-CCAAGCTGCAGACATTCTAGCACTC-3′, 5′-CTGGATCCGGAAAATCCTGGCAATG-3′; Rag2, 5′-CACATCCACAAGCAGGAAGTACAC-3′, 5′-GGTTCAGGGACATCTCCTACTAAG-3′; and HPRT, 5′-GCTGGTGAAAAGGACCTCT-3′, 5′-CACAGGACTAGAACACCTGC-3′. Tag polymerase (Gibco, Gaithersburg, MD) was used in all amplifications. PCR was carried out as follows: for Rag1 - at 94°C for 2 min followed by eight cycles at 94°C for 1 min, 54°C for 1 min, and 72°C for 2 min, and 22 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min; for Rag2 - at 94°C for 2 min followed by 35 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min; for HPRT - at 94°C for 2 min followed by 35 cycles at 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min. The amplified products (6 μl) were loaded onto a 1% agarose gel and visualized with ethidium bromide. To ensure that RT-PCR products were not derived from contaminating genomic DNA, the primer pairs for Rag1 and Rag2 each span an intron and primers for HPHT span two introns. The molecular sizes of the PCR products were determined by comparison to standards of 50, 100, 200, 300, 400, 500, 700, and 1000 base pairs (bp) (Marker Xl, Boehringer-Mannheim).
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62. We thank J. Cerny and M. Schlissel for their review of this manuscript; S. V. Desiderio for RAG2-specific antibody; R. Hodes, K. Holmes, and A. Lanzavecchia for describing unpublished observations; and F. W. Alt for the communication of unpublished results confirming Ig class switching in the absence of RAG proteins. Supported in part by USPHS grant AI32524 to D.G.S. and grants AI24335, AG10207, and AG13789 to G.K. E.S. is the recipient of a grant from the Leukemia Research Foundation. D.G.S. is an assistant investigator of the Howard Hughes Medical Institute.