Abstract
Rabbits are born with a limited VDJ gene repertoire formed primarily by rearrangement of one VH gene, VH1. The VDJ genes are undiversified at birth but become diversified by approximately 2 mo of age. To investigate more closely the time during which this diversity occurs, we determined the nucleotide sequences of VDJ genes from peripheral blood leukocytes taken from young rabbits at various time points, and we examined the extent of the diversification of the VDJ genes. At 4 wk of age there were, on average, 3 nucleotide changes per VH region, with approximately 75% of the genes showing some diversification. The number of nucleotide changes per VH region increased to 12 by 6-8 wk of age, and all but 1 of the 35 sequences analyzed were diversified. Because only a limited number of genes can be examined by nucleotide sequence analysis, we used an RNase protection assay to examine a large number of genes and we determined the level of undiversified VH1 mRNA in lymphoid organs of both young and adult rabbits. In young rabbits, we found a high level of undiversified VDJ genes, but the level was greatly reduced by 2 mo of age. By adulthood, essentially all VDJ genes of cells from appendix, peripheral blood, and bone marrow were diversified. Because we had expected B lymphopoiesis to be ongoing in the bone marrow of adult rabbits, we were surprised not to find undiversified VDJ genes from the newly generated B cells. Therefore, we searched for evidence of ongoing B lymphopoiesis in bone marrow by isolating and examining circular DNA for the presence of VD and DJ recombination signal joints. We found highly reduced levels of recombination signal joints in bone marrow of adult rabbits relative to the levels found in bone marrow of newborn rabbits. These data indicate that limited VD and DJ gene rearrangements occur in bone marrow of adult rabbits, and we therefore suggest that B lymphopoiesis is limited in adults.
Full Text
The Full Text of this article is available as a PDF (1.2 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Becker R. S., Knight K. L. Somatic diversification of immunoglobulin heavy chain VDJ genes: evidence for somatic gene conversion in rabbits. Cell. 1990 Nov 30;63(5):987–997. doi: 10.1016/0092-8674(90)90502-6. [DOI] [PubMed] [Google Scholar]
- Becker R. S., Zhai S. K., Currier S. J., Knight K. L. Ig VH, DH, and JH germ-line gene segments linked by overlapping cosmid clones of rabbit DNA. J Immunol. 1989 Feb 15;142(4):1351–1355. [PubMed] [Google Scholar]
- Berger C. N. In situ hybridization of immunoglobulin-specific RNA in single cells of the B lymphocyte lineage with radiolabelled DNA probes. EMBO J. 1986 Jan;5(1):85–93. doi: 10.1002/j.1460-2075.1986.tb04181.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carroll S. M., Gaudray P., De Rose M. L., Emery J. F., Meinkoth J. L., Nakkim E., Subler M., Von Hoff D. D., Wahl G. M. Characterization of an episome produced in hamster cells that amplify a transfected CAD gene at high frequency: functional evidence for a mammalian replication origin. Mol Cell Biol. 1987 May;7(5):1740–1750. doi: 10.1128/mcb.7.5.1740. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
- Friedman M. L., Tunyaplin C., Zhai S. K., Knight K. L. Neonatal VH, D, and JH gene usage in rabbit B lineage cells. J Immunol. 1994 Jan 15;152(2):632–641. [PubMed] [Google Scholar]
- Gu H., Tarlinton D., Müller W., Rajewsky K., Förster I. Most peripheral B cells in mice are ligand selected. J Exp Med. 1991 Jun 1;173(6):1357–1371. doi: 10.1084/jem.173.6.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klein U., Küppers R., Rajewsky K. Human IgM+IgD+ B cells, the major B cell subset in the peripheral blood, express V kappa genes with no or little somatic mutation throughout life. Eur J Immunol. 1993 Dec;23(12):3272–3277. doi: 10.1002/eji.1830231232. [DOI] [PubMed] [Google Scholar]
- Knight K. L., Becker R. S. Molecular basis of the allelic inheritance of rabbit immunoglobulin VH allotypes: implications for the generation of antibody diversity. Cell. 1990 Mar 23;60(6):963–970. doi: 10.1016/0092-8674(90)90344-e. [DOI] [PubMed] [Google Scholar]
- Knight K. L., Crane M. A. Generating the antibody repertoire in rabbit. Adv Immunol. 1994;56:179–218. doi: 10.1016/s0065-2776(08)60452-6. [DOI] [PubMed] [Google Scholar]
- Knight K. L. Restricted VH gene usage and generation of antibody diversity in rabbit. Annu Rev Immunol. 1992;10:593–616. doi: 10.1146/annurev.iy.10.040192.003113. [DOI] [PubMed] [Google Scholar]
- Krug M. S., Berger S. L. First-strand cDNA synthesis primed with oligo(dT). Methods Enzymol. 1987;152:316–325. doi: 10.1016/0076-6879(87)52036-5. [DOI] [PubMed] [Google Scholar]
- McCormack W. T., Thompson C. B. Somatic diversification of the chicken immunoglobulin light-chain gene. Adv Immunol. 1990;48:41–67. doi: 10.1016/s0065-2776(08)60751-8. [DOI] [PubMed] [Google Scholar]
- Pascual V., Liu Y. J., Magalski A., de Bouteiller O., Banchereau J., Capra J. D. Analysis of somatic mutation in five B cell subsets of human tonsil. J Exp Med. 1994 Jul 1;180(1):329–339. doi: 10.1084/jem.180.1.329. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pospisil R., Young-Cooper G. O., Mage R. G. Preferential expansion and survival of B lymphocytes based on VH framework 1 and framework 3 expression: "positive" selection in appendix of normal and VH-mutant rabbits. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):6961–6965. doi: 10.1073/pnas.92.15.6961. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Raman C., Spieker-Polet H., Yam P. C., Knight K. L. Preferential VH gene usage in rabbit Ig-secreting heterohybridomas. J Immunol. 1994 Apr 15;152(8):3935–3945. [PubMed] [Google Scholar]
- Reynaud C. A., Anquez V., Dahan A., Weill J. C. A single rearrangement event generates most of the chicken immunoglobulin light chain diversity. Cell. 1985 Feb;40(2):283–291. doi: 10.1016/0092-8674(85)90142-4. [DOI] [PubMed] [Google Scholar]
- Reynaud C. A., Anquez V., Grimal H., Weill J. C. A hyperconversion mechanism generates the chicken light chain preimmune repertoire. Cell. 1987 Feb 13;48(3):379–388. doi: 10.1016/0092-8674(87)90189-9. [DOI] [PubMed] [Google Scholar]
- Reynaud C. A., Dahan A., Anquez V., Weill J. C. Somatic hyperconversion diversifies the single Vh gene of the chicken with a high incidence in the D region. Cell. 1989 Oct 6;59(1):171–183. doi: 10.1016/0092-8674(89)90879-9. [DOI] [PubMed] [Google Scholar]
- Reynaud C. A., Garcia C., Hein W. R., Weill J. C. Hypermutation generating the sheep immunoglobulin repertoire is an antigen-independent process. Cell. 1995 Jan 13;80(1):115–125. doi: 10.1016/0092-8674(95)90456-5. [DOI] [PubMed] [Google Scholar]
- Reynaud C. A., Mackay C. R., Müller R. G., Weill J. C. Somatic generation of diversity in a mammalian primary lymphoid organ: the sheep ileal Peyer's patches. Cell. 1991 Mar 8;64(5):995–1005. doi: 10.1016/0092-8674(91)90323-q. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spieker-Polet H., Yam P. C., Knight K. L. Differential expression of 13 IgA-heavy chain genes in rabbit lymphoid tissues. J Immunol. 1993 Jun 15;150(12):5457–5465. [PubMed] [Google Scholar]
- Thompson C. B., Neiman P. E. Somatic diversification of the chicken immunoglobulin light chain gene is limited to the rearranged variable gene segment. Cell. 1987 Feb 13;48(3):369–378. doi: 10.1016/0092-8674(87)90188-7. [DOI] [PubMed] [Google Scholar]
- Tlaskalová-Hogenová H., Stépánková R. Development of antibody formation in germ-free and conventionally reared rabbits: the role of intestinal lymphoid tissue in antibody formation to E. coli antigens. Folia Biol (Praha) 1980;26(2):81–93. [PubMed] [Google Scholar]
- Weinstein P. D., Anderson A. O., Mage R. G. Rabbit IgH sequences in appendix germinal centers: VH diversification by gene conversion-like and hypermutation mechanisms. Immunity. 1994 Nov;1(8):647–659. doi: 10.1016/1074-7613(94)90036-1. [DOI] [PubMed] [Google Scholar]
- Wu G. E., Atkinson M. J., Ramsden D. A., Paige C. J. VH gene repertoire. Semin Immunol. 1990 May;2(3):207–216. [PubMed] [Google Scholar]
- Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
