Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Jun;11(6):3155–3162. doi: 10.1128/mcb.11.6.3155

Strand breaks without DNA rearrangement in V (D)J recombination.

E A Hendrickson 1, V F Liu 1, D T Weaver 1
PMCID: PMC360165  PMID: 2038323

Abstract

Somatic gene rearrangement of immunoglobulin and T-cell receptor genes [V(D)J recombination] is mediated by pairs of specific DNA sequence motifs termed signal sequences. In experiments described here, retroviral vectors containing V(D)J rearrangement cassettes in which the signal sequences had been altered were introduced into wild-type and scid (severe combined immune deficiency) pre-B cells and used to define intermediates in the V(D)J recombination pathway. The scid mutation has previously been shown to deleteriously affect the V(D)J recombination process. Cassettes containing a point mutation in one of the two signal sequences inhibited rearrangement in wild-type cells. In contrast, scid cells continued to rearrange these cassettes with the characteristic scid deletional phenotype. Using these mutated templates, we identified junctional modifications at the wild-type signal sequences that had arisen from strand breaks which were not associated with overall V(D)J rearrangements. Neither cell type was able to rearrange constructs which contained only a single, nonmutated, signal sequence. In addition, scid and wild-type cell lines harboring cassettes with mutations in both signal sequences did not undergo rearrangement, suggesting that at least one functional signal sequence was required for all types of V(D)J recombination events. Analysis of these signal sequence mutations has provided insights into intermediates in the V(D)J rearrangement pathway in wild-type and scid pre-B cells.

Full text

PDF
3162

Images in this article

3157Image
on p.3157
3158Image
on p.3158

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akira S., Okazaki K., Sakano H. Two pairs of recombination signals are sufficient to cause immunoglobulin V-(D)-J joining. Science. 1987 Nov 20;238(4830):1134–1138. doi: 10.1126/science.3120312. [DOI] [PubMed] [Google Scholar]
  2. Alt F. W., Baltimore D. Joining of immunoglobulin heavy chain gene segments: implications from a chromosome with evidence of three D-JH fusions. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4118–4122. doi: 10.1073/pnas.79.13.4118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alt F., Rosenberg N., Lewis S., Thomas E., Baltimore D. Organization and reorganization of immunoglobulin genes in A-MULV-transformed cells: rearrangement of heavy but not light chain genes. Cell. 1981 Dec;27(2 Pt 1):381–390. doi: 10.1016/0092-8674(81)90421-9. [DOI] [PubMed] [Google Scholar]
  4. Bender M. A., Palmer T. D., Gelinas R. E., Miller A. D. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J Virol. 1987 May;61(5):1639–1646. doi: 10.1128/jvi.61.5.1639-1646.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blackwell T. K., Malynn B. A., Pollock R. R., Ferrier P., Covey L. R., Fulop G. M., Phillips R. A., Yancopoulos G. D., Alt F. W. Isolation of scid pre-B cells that rearrange kappa light chain genes: formation of normal signal and abnormal coding joins. EMBO J. 1989 Mar;8(3):735–742. doi: 10.1002/j.1460-2075.1989.tb03433.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bosma G. C., Custer R. P., Bosma M. J. A severe combined immunodeficiency mutation in the mouse. Nature. 1983 Feb 10;301(5900):527–530. doi: 10.1038/301527a0. [DOI] [PubMed] [Google Scholar]
  7. Early P., Huang H., Davis M., Calame K., Hood L. An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH. Cell. 1980 Apr;19(4):981–992. doi: 10.1016/0092-8674(80)90089-6. [DOI] [PubMed] [Google Scholar]
  8. Ferrier P., Covey L. R., Li S. C., Suh H., Malynn B. A., Blackwell T. K., Morrow M. A., Alt F. W. Normal recombination substrate VH to DJH rearrangements in pre-B cell lines from scid mice. J Exp Med. 1990 Jun 1;171(6):1909–1918. doi: 10.1084/jem.171.6.1909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Foss K., McClain W. H. Rapid site-specific mutagenesis in plasmids. Gene. 1987;59(2-3):285–290. doi: 10.1016/0378-1119(87)90336-2. [DOI] [PubMed] [Google Scholar]
  10. Fulop G. M., Phillips R. A. The scid mutation in mice causes a general defect in DNA repair. Nature. 1990 Oct 4;347(6292):479–482. doi: 10.1038/347479a0. [DOI] [PubMed] [Google Scholar]
  11. Hendrickson E. A., Schatz D. G., Weaver D. T. The scid gene encodes a trans-acting factor that mediates the rejoining event of Ig gene rearrangement. Genes Dev. 1988 Jul;2(7):817–829. doi: 10.1101/gad.2.7.817. [DOI] [PubMed] [Google Scholar]
  12. Hendrickson E. A., Schlissel M. S., Weaver D. T. Wild-type V(D)J recombination in scid pre-B cells. Mol Cell Biol. 1990 Oct;10(10):5397–5407. doi: 10.1128/mcb.10.10.5397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hesse J. E., Lieber M. R., Mizuuchi K., Gellert M. V(D)J recombination: a functional definition of the joining signals. Genes Dev. 1989 Jul;3(7):1053–1061. doi: 10.1101/gad.3.7.1053. [DOI] [PubMed] [Google Scholar]
  14. Johnson R. C., Simon M. I. Hin-mediated site-specific recombination requires two 26 bp recombination sites and a 60 bp recombinational enhancer. Cell. 1985 Jul;41(3):781–791. doi: 10.1016/s0092-8674(85)80059-3. [DOI] [PubMed] [Google Scholar]
  15. Kim M. G., Schuler W., Bosma M. J., Marcu K. B. Abnormal recombination of Igh D and J gene segments in transformed pre-B cells of scid mice. J Immunol. 1988 Aug 15;141(4):1341–1347. [PubMed] [Google Scholar]
  16. Korman A. J., Frantz J. D., Strominger J. L., Mulligan R. C. Expression of human class II major histocompatibility complex antigens using retrovirus vectors. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2150–2154. doi: 10.1073/pnas.84.8.2150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lafaille J. J., DeCloux A., Bonneville M., Takagaki Y., Tonegawa S. Junctional sequences of T cell receptor gamma delta genes: implications for gamma delta T cell lineages and for a novel intermediate of V-(D)-J joining. Cell. 1989 Dec 1;59(5):859–870. doi: 10.1016/0092-8674(89)90609-0. [DOI] [PubMed] [Google Scholar]
  18. Landy A. Dynamic, structural, and regulatory aspects of lambda site-specific recombination. Annu Rev Biochem. 1989;58:913–949. doi: 10.1146/annurev.bi.58.070189.004405. [DOI] [PubMed] [Google Scholar]
  19. Lewis S. M., Hesse J. E., Mizuuchi K., Gellert M. Novel strand exchanges in V(D)J recombination. Cell. 1988 Dec 23;55(6):1099–1107. doi: 10.1016/0092-8674(88)90254-1. [DOI] [PubMed] [Google Scholar]
  20. Lewis S., Gellert M. The mechanism of antigen receptor gene assembly. Cell. 1989 Nov 17;59(4):585–588. doi: 10.1016/0092-8674(89)90002-0. [DOI] [PubMed] [Google Scholar]
  21. Lieber M. R., Hesse J. E., Lewis S., Bosma G. C., Rosenberg N., Mizuuchi K., Bosma M. J., Gellert M. The defect in murine severe combined immune deficiency: joining of signal sequences but not coding segments in V(D)J recombination. Cell. 1988 Oct 7;55(1):7–16. doi: 10.1016/0092-8674(88)90004-9. [DOI] [PubMed] [Google Scholar]
  22. Malynn B. A., Blackwell T. K., Fulop G. M., Rathbun G. A., Furley A. J., Ferrier P., Heinke L. B., Phillips R. A., Yancopoulos G. D., Alt F. W. The scid defect affects the final step of the immunoglobulin VDJ recombinase mechanism. Cell. 1988 Aug 12;54(4):453–460. doi: 10.1016/0092-8674(88)90066-9. [DOI] [PubMed] [Google Scholar]
  23. Morzycka-Wroblewska E., Lee F. E., Desiderio S. V. Unusual immunoglobulin gene rearrangement leads to replacement of recombinational signal sequences. Science. 1988 Oct 14;242(4876):261–263. doi: 10.1126/science.3140378. [DOI] [PubMed] [Google Scholar]
  24. Okazaki K., Nishikawa S., Sakano H. Aberrant immunoglobulin gene rearrangement in scid mouse bone marrow cells. J Immunol. 1988 Aug 15;141(4):1348–1352. [PubMed] [Google Scholar]
  25. Prasad P. V., Horensky D., Young L. J., Jayaram M. Substrate recognition by the 2 micron circle site-specific recombinase: effect of mutations within the symmetry elements of the minimal substrate. Mol Cell Biol. 1986 Dec;6(12):4329–4334. doi: 10.1128/mcb.6.12.4329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sakano H., Hüppi K., Heinrich G., Tonegawa S. Sequences at the somatic recombination sites of immunoglobulin light-chain genes. Nature. 1979 Jul 26;280(5720):288–294. doi: 10.1038/280288a0. [DOI] [PubMed] [Google Scholar]
  27. Schuler W., Weiler I. J., Schuler A., Phillips R. A., Rosenberg N., Mak T. W., Kearney J. F., Perry R. P., Bosma M. J. Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency. Cell. 1986 Sep 26;46(7):963–972. doi: 10.1016/0092-8674(86)90695-1. [DOI] [PubMed] [Google Scholar]
  28. Senecoff J. F., Cox M. M. Directionality in FLP protein-promoted site-specific recombination is mediated by DNA-DNA pairing. J Biol Chem. 1986 Jun 5;261(16):7380–7386. [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES